WO2020093574A1 - Tumor-related sequence, long-chain non-coding rna and use thereof - Google Patents

Tumor-related sequence, long-chain non-coding rna and use thereof Download PDF

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WO2020093574A1
WO2020093574A1 PCT/CN2018/125262 CN2018125262W WO2020093574A1 WO 2020093574 A1 WO2020093574 A1 WO 2020093574A1 CN 2018125262 W CN2018125262 W CN 2018125262W WO 2020093574 A1 WO2020093574 A1 WO 2020093574A1
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gman
mfr
gastric cancer
cancer
sequence
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周天华
卓巍
刘易曼
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浙江大学
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  • the invention relates to a tumor-related sequence, long-chain non-coding RNA and its application.
  • RNA RNA with a transcription length of more than 200 nucleotides and no apparent coding ability.
  • LncRNA long non-coding RNA
  • RNAs have important biological functions, such as gene transcription, post-transcriptional regulation, RNA splicing, translation, epigenetic regulation, genetic imprinting, X chromosome inactivation, and disease development. They all play an important role in the process.
  • LncRNA LncRNA plays an important role in the occurrence, invasion and metastasis of various tumors, including melanoma, colon cancer, prostate cancer, leukemia, liver cancer, breast cancer, etc. However, there are few reports on its specific mechanism of action. Rajnish et al.
  • LncRNA HOTAIR is located in the intron of the HOX family gene, which is highly expressed in breast cancer and highly correlated with tumor metastasis; further research shows that HOTAIR overexpression can promote tumor invasion and metastasis ability.
  • some LncRNAs also play an important role in inhibiting tumorigenesis.
  • LncRNAs that are down-regulated in breast cancer GAS5 growth-arrest-specific5 can play a role in tumor suppression by inhibiting cell growth and inducing apoptosis Gene function.
  • LncRNA MAYA plays an important role in promoting bone metastasis of breast cancer.
  • ROR1 phosphorylates HER3, recruits LLGL2-MAYA-NSUN6RNA protein complex, promotes the methylation of Hippo / MST1, leads to inactivation of MST1 and activates the YAP targeting gene, thereby promoting bone metastasis of breast cancer.
  • MAYA's treatment experiments show that in mice that produce bone metastases from breast cancer cells, LNA injected with MAYA can significantly inhibit bone metastases from breast cancer cells.
  • LncRNA not only plays an important regulatory role in tumors but also has great potential for effective treatment of tumors, indicating that lncRNA can be used as a new drug for tumor therapy Target.
  • LncRNA BC032469 acts as a sponge for miR-1207-5P, regulates hTERT expression, and promotes the proliferation of gastric cancer cells.
  • LncRNA HOXA11-AS serves as a scaffold for the chromosomal regulatory factors PRC2, LSD1, DNMT1, and promotes the proliferation and invasion of gastric cancer cells.
  • gastric cancer is one of the important factors leading to cancer death.
  • gastric cancer is the malignant tumor with the fifth highest morbidity and third highest mortality, while China is a country with a high incidence of gastric cancer.
  • the occurrence and development of gastric cancer have seriously endangered human health.
  • the onset of gastric cancer metastasis is hidden, and most patients are difficult to remove when found, so the prognosis is extremely poor, and its 5-year survival rate is less than 10%, which is one of the main causes of death in patients with advanced gastric cancer. Therefore, we urgently need to deeply understand the pathogenesis of gastric cancer organ metastasis and look for key factors that regulate the metastasis process of gastric cancer cells.
  • LncRNA plays an important role in a variety of tumors, suggesting that the function of LncRNA may provide new targets and new ideas for the diagnosis, prognosis and treatment of gastric cancer metastasis.
  • the purpose of the present invention is to provide a tumor-related sequence, long-chain non-coding RNA and its application in view of the deficiencies of the prior art.
  • the object of the present invention is achieved by the following technical solution: a tumor-related sequence whose sequence is the MFR sequence shown in SEQ ID NO.1.
  • the tumors include malignant tumors of the nasal cavity and sinuses, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardia cancer, breast cancer, and mediastinum Tumor, stomach cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, malignant tumor of small intestine, kidney cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, skin malignant melanoma, lymphoma.
  • diagnostic reagents include but are not limited to:
  • a primer / primer set that recognizes the MFR sequence or a fluorescently labeled primer / primer set that recognizes the MFR sequence;
  • Biomacromolecules that recognize the MFR sequence including but not limited to: antibodies or antibody functional fragments, fluorescently labeled antibodies or antibody functional fragments, RNA binding proteins or functional fragments thereof, fluorescently labeled RNA binding protein or its functional fragments.
  • the inhibitor targets the MFR sequence shown in SEQ ID NO.1.
  • inhibitors include but are not limited to:
  • siRNA siRNA, shRNA or functionally similar interfering small RNA that inhibits the MFR sequence
  • Oligonucleotide fragments that inhibit the MFR sequence, and the oligonucleotide fragments include but are not limited to: antisense oligonucleotide ASO, locked nucleic acid LNA, or functionally similar chemically modified oligonucleotide .
  • Biomacromolecules that inhibit the MFR sequence include but are not limited to: antibodies or antibody functional fragments, high substrate-specific enzymes or functional fragments thereof, and other protein molecules that inhibit MFR function .
  • tool molecules capable of knocking out or destroying MFR sequences include but are not limited to DNA homologous recombination plasmids, TALEN-TALEA targeted gene knockout plasmid system, Cre / Loxp plasmid system, tetracycline / interferon and other inducible Cre / Loxp plasmid system, FLP-frt plasmid system, CRISPR / Cas9 and other CRISPR gene editing plasmid systems, etc.
  • a tumor-associated long-chain non-coding RNA including the above MFR sequence sequence. Specifically, it may be GMAN shown in SEQ ID NO.2.
  • the beneficial effect of the present invention lies in the advantages compared with the prior art.
  • Figure 1.1 is a schematic diagram of lncRNA GMAN
  • Figure 2.1 is Northern blot detection of GMAN expression in human gastric cancer cell lines
  • Figure 2.2 is TCGA transcriptome data analysis of the expression of GMAN in human tissues and organs
  • Figure 3.1 shows the expression level of GMAN in gastric cancer tissues
  • Figure 3.2 is a cohort of 40 gastric cancer patients with 5-year survival information
  • Figure 3.3 shows the expression of GMAN in Cohort 3, Cohort 4, and TCGA databases of age and sex matched M0 gastric cancer tissues without metastasis and M1 gastric cancer tissues with distant metastasis; **, P ⁇ 0.01.
  • Figure 4.1 shows the Northern blot detection efficiency of GMAN knockdown
  • Figure 4.2 shows the effect of GMAN on cell proliferation by MTT and clone formation experiments
  • Figure 4.3 shows the effect of GMAN on cell cycle by cell flow cytometry
  • Figure 4.4 shows the effect of knocking down GMAN on the cell adhesion ability of gastric cancer cells; n.s., meaningless;
  • Figure 4.5 shows the effect of knocking down GMAN on the cell invasion ability of gastric cancer cells; ***, P ⁇ 0.001.
  • Figure 5.1 shows the effect of Northern blot detection of GMAN overexpression
  • Figure 5.2 shows the effect of GMAN on cell proliferation by MTT and clone formation experiments
  • Figure 5.3 shows the effect of GMAN on cell cycle by cell flow cytometry
  • Figure 5.4 shows the effect of overexpression of GMAN on the cell adhesion ability of gastric cancer cells; n.s., meaningless.
  • Figure 5.5 shows the effect of overexpression of GMAN on the cell invasion ability of gastric cancer cells; ***, P ⁇ 0.001.
  • Figure 6.1 shows the effect of knocking down GMAN on the metastatic ability of gastric cancer cells; ***, P ⁇ 0.001.
  • Figure 7.1 shows the use of CRISPR / Cas9 gene editing to construct a gastric cancer mutant strain ( ⁇ MFR) with deletion of the MFR segment of GMAN;
  • Figure 7.2 shows the effect of MFR-deficient cell lines on the proliferation of gastric cancer cells
  • Figure 7.3 shows the effect of MFR deletion on the cell cycle of gastric cancer cells
  • Figure 7.4 shows the effect of MFR deletion on the cell adhesion ability of gastric cancer cells; n.s., meaningless.
  • Figure 7.5 shows the effect of MFR deletion on the invasion ability of gastric cancer cells; ***, P ⁇ 0.001.
  • Figure 9.1 is a schematic diagram of CRISPR / Cas9 targeting the MFR segment of GMAN to treat lung metastasis of gastric cancer cells;
  • Figure 9.2 is the BLI real-time monitoring of lung metastasis of gastric cancer cells treated with CRISPR / Cas9 targeting the MFR segment of GMAN;
  • Figure 9.3 shows the cytotoxicity test related to the CRISPR / Cas9 delivery system
  • Figure 10.1 is the analysis of the expression of GMAN in colorectal cancer patients in the TCGA database.
  • A is the matching case of cancer and adjacent cancer
  • B is the expression of the overall cancer and adjacent cancer
  • C is the age and gender matching.
  • GMAN expression was analyzed in M0 colorectal cancer tissue with metastasis and M1 colorectal cancer tissue with distant metastasis. ***, P ⁇ 0.001.
  • Figure 10.2 is the analysis of the expression of GMAN in esophageal cancer patients in the TCGA database.
  • Panel A shows the cases of esophageal cancer and adjacent cancers.
  • Panel B shows the age-matched M0 esophageal cancer tissues with metastasis and distant GMAN expression was analyzed in metastatic M1 esophageal cancer tissues. *, P ⁇ 0.05.
  • Figure 10.3 is the analysis of the expression of GMAN in liver cancer patients in the TCGA database.
  • Panel A is the case of paired liver cancer and adjacent cancer.
  • Panel B is the overall expression of liver cancer and adjacent cancer. ***, P ⁇ 0.001.
  • the present invention proposes for the first time a new lncRNA GMAN, which significantly promotes the invasion and metastasis of gastric cancer cells, and has an important potential for treating gastric cancer metastasis.
  • the details are as follows:
  • 3'Race and 5'Race confirm the sequence of lncRNA and GMAN.
  • Northern blotting shows that GMAN expression can be detected in human gastric cancer tissues and cells.
  • GMAN has no significant effect on cell proliferation, cell cycle, clonal formation and cell adhesion of gastric cancer cells, but GMAN can significantly promote the invasion and metastasis of gastric cancer cells in vitro.
  • MFR main function
  • CRISPR / Cas9 technology targets the MFR segment of GMAN and constructs a gastric cancer cell line in which the MFR region of GMAN is deleted. The ability to invade and metastasize cells showing the absence of the MFR region was significantly inhibited.
  • GMAN especially the MFR segment of GMAN, can be used as an important target for the treatment of gastric cancer metastasis.
  • lncRNA and GMAN are relatively highly expressed in gastric cancer, especially in samples of M1 stage gastric cancer patients with distant metastasis, and the high expression of GMAN is significantly related to the poor prognosis of gastric cancer patients.
  • GMAN plays an important role in the occurrence and development of gastric cancer. GMAN can significantly promote the invasion and metastasis of gastric cancer cells. Further, the MFR section of GMAN is essential for GMAN to function.
  • the CRISPR / Cas9 treatment experiments show that GMAN, especially the MFR segment of GMAN, can effectively inhibit the metastasis of gastric cancer, and can be used as a new strategy for clinically controlling tumor metastasis.
  • Example 1 lncRNA GMAN is an 855nt lncRNA
  • RNA from human gastric cancer cell lines extract RNA from human gastric cancer cell lines and apply PolyA treatment and purification to the RNA
  • RNA isolation and extraction of total cellular RNA was carried out using TRIZOL reagent (purchased from Invitrogen, USA), and was carried out according to the standard operation of the reagent instructions. Take an appropriate amount of RNA and use DNase I kit (purchased from Invitrogen, USA) to digest the remaining genome in the RNA to remove genomic contamination.
  • RNA plus PolyA treatment using PolyA kit (purchased from NEB, USA). Take appropriate amount of RNA and mix with polyA enzyme, buffer, RNase inhibitor, and incubate at 37 °C. DNA gel electrophoresis was used to detect the PloyA tailing of RNA.
  • PCR tube in step 3.2 was incubated at 72 ° C for 3 minutes, 42 ° C for 2 minutes, cooled on ice, and centrifuged at 14000g for 10 seconds to make the solution at the bottom of the PCR tube.
  • step 3.4 The buffer mix in step 3.4 is added to step 4 (3'-RACE cDNA) and step 5 (5'-RACE cDNA) respectively, so that the final volume is 20ul. Mix gently and centrifuge to keep the solution at the bottom of the PCR tube. Incubate at 42 ° C for 90 minutes and 70 ° C for 10 minutes. Dilute 3'- and 5'-RACE-Ready cDNA with Tricine-EDTA Buffer and store at -20 ° C until use.
  • Reaction 1 5 cycles: 94 °C 30sec; 72 °C 3min *
  • Reaction 2 5 cycles: 94 °C 30sec; 70 °C 30sec; 72 °C 3min *
  • Reaction 3 20 cycles: 94 °C 30sec; 68 °C 30sec; 72 °C 3min *
  • Race DNA products were subjected to gel electrophoresis. Cut the gel to recover the purified DNA for use.
  • GMAN GMAN molecule and the specific sequence of GMAN exist in the human body. It was confirmed by 3'Race and 5'Race that GMAN exists in human gastric cancer cell lines, and GMAN is an 855nt lncRNA located on chromosome 1q22 (Table S1). GMAN is located in the gene cluster of EphrinA1, and most of GMAN is located in the intron of EphrinA1 ( Figure 1.1).
  • Example 2 GMAN expression in human gastric tissues and gastric cancer cell lines
  • a specific sequence of GMAN / GAPDH was obtained by PCR reaction.
  • the PCR reaction was carried out using the above primers to obtain the target gene (purchased from Japan Toyobo).
  • the target gene purchased from Japan Toyobo.
  • Take a PCR tube add 2ul of cDNA template, 1ul of KOD high-fidelity enzyme, 10xPCR buffer 5ul, dNTP 5ul, Forward primer 1ul, reverse primer 1ul, add ddH2O to 50ul, mix well, and perform PCR reaction according to the following reaction procedure:
  • the PCS107 vector contains SP6 and T7 promoters, we constructed a sequence of GMAN / GAPDH between the two promoters. Through single enzyme digestion, we can choose to transcribe the antisense chain of GMAN / GAPDH by SP6 transcriptase. Go out, or transcribe the GAMN / GAPDH sense chain by T7 transcriptase.
  • PCS107-GMAN and PCS107-GAPDH plasmids were digested with Xhol I and the linearized PCS107 vector was used as a template. Add the components required for transcription to the EP tube, mix well, and incubate in a 37 ° C water bath for 3 hours.
  • the specific components are as follows:
  • RNA After transcription, add 1ul DNase I, 37 °C, 15min to remove template DNA. Purify RNA with RNeasy Plus Mini Kit (purchased from QIAGEN, USA), measure the concentration and take a portion of the gel to make sure that the transcribed RNA has only a single target band. The purified RNA was aliquoted and stored at -80 ° C.
  • RNA was added to 1.2% denatured agarose gel well, 1 * MOPS buffer was used as electrophoresis buffer (purchased from Sigma, USA), 100V pre-run for 10min, 45V electrophoresis for 4-5h. The salt bridge was used to transfer the nitrocellulose membrane overnight. 1500V UV cross-links the membrane and stains with methylene blue.
  • the membrane at the position of the band of interest was cut out and incubated with pre-hybridization solution (purchased from Rohce, Switzerland) at 50 ° C for 1 h.
  • the hybridization solution containing Dig-labeled RNA probe was added and incubated at 50 ° C overnight (over 12h).
  • 0.2 * SSC solution wash the membrane at 50 °C for 15min, repeat twice.
  • Northern buffer purchased from Sigma, USA
  • Blocking buffer (purchased from Roche, Switzerland) is closed at room temperature for 30 minutes.
  • Anti-Dig-AP antibody (purchased from Roche, Switzerland) was incubated at room temperature for 30 min. Northern wash buffer 15min at room temperature, repeat twice. Detection buffer is incubated for 3 minutes. The color was developed and exposed with Dig-CDP star color developing solution (purchased from Roche, Switzerland).
  • GMAN is a 855nt long non-coding RNA located on chromosome 1q22.
  • the transcriptome data in the TCGA (The Cancer Genome Atlas) database to analyze the expression of GMAN.
  • GMAN is expressed in a variety of organizations ( Figure 2.2).
  • Example 3 GMAN is relatively highly expressed in gastric cancer tissues and is significantly associated with metastasis and poor prognosis of gastric cancer patients
  • the tissue was ground into a powder at a very low temperature using a mortar.
  • the isolation and extraction of the total RNA of the powdered tissue was carried out using TRIZOL reagent (purchased from Invitrogen, USA), and was carried out according to the standard operation of the reagent instructions.
  • the extracted RNA is subjected to reverse transcription PCR to synthesize cDNA products.
  • the PCR reaction system was prepared according to the instruction of Taqman probe Kit (purchased from Takara, Japan). Fluorescence quantitative PCR reaction was performed using BioFX Rad CFX-Touch Systerem fluorescence quantitative PCR instrument. All reactions were repeated three times. Obtain the ⁇ Ct value according to the fluorescence chart given by the instrument, so as to calculate the relative change of the corresponding expression level.
  • the primers are as follows:
  • Example 4 Knocking down the expression of GMAN can significantly inhibit the invasion of gastric cancer cells
  • siRNA design software design and synthesize two specific siRNAs targeting GMAN and silence GMAN expression (purchased from Gema, Shanghai, China). Plate the cells in the logarithmic growth phase. When the cell density reaches about 50%, dilute Lipo RNAi MAX (purchased from Invitrogen, USA) and siRNA with OPTI medium (purchased from Gibco, USA), and add the diluted siRNA to Lipo RNAi MAX In the tube, mix and let stand for 5min, then add to the cell culture fluid, shake well, and change the medium after 24.
  • dilute Lipo RNAi MAX purchased from Invitrogen, USA
  • siRNA with OPTI medium purchased from Gibco, USA
  • the transfected cells were resuspended in 1640 medium (purchased from Gibco, USA) of 10% FBS (purchased from Israel BI) and counted on a hemocytometer.
  • 1640 medium purchased from Gibco, USA
  • FBS purchased from Israel BI
  • 3000 cells 100 ul medium were added to each well in a 96-well plate, and five replicate wells per group were used. A total of 4 plates were needed to detect the proliferation of cells at different time points (0h, 24h, 48h, 72h).
  • add 5 mg / ml MTT purchasedd from Sigma
  • Add 150ul of DMSO purchasedd Since China National Medicine
  • the absorbance values of OD490 / OD570 at various time points were detected and analyzed on the M5 microplate reader.
  • Matrigel purchased from US BD coated cell plates, diluted 1:40 with pre-chilled FBS-free 1640 (purchased from Gibco), and coated with 50 ⁇ L per well; Fibronectin (purchased from BD) ) Coated cell plates, dilute fibronectin to 0.02 ⁇ g / ⁇ L with pre-chilled FBS-free 1640, and add 50 ⁇ L per well for coating.
  • the 96-well plates coated with matrigel and fibronectin were placed in a 37 ° C cell incubator for 24 hours.
  • Aspirate unsolidified matrigel and fibronectin add 100 ⁇ L of each well, 0.5% BSA (purchased from Shanghai, China) (PBS solution), and block at 37 ° C for 30 min. Discard BSA, wash twice with PBS gently, add 100 ⁇ L of each well to resuspend the cells in 1640 medium of 2x104 1% FBS, and incubate for 30 min in a 37 ° C incubator. Discard the supernatant, wash twice with PBS gently, add 100 ⁇ L per well, fix with 4% PFA for 30 min, discard PFA (China National Medicine), wash twice with PBS gently.
  • Transwell 8- ⁇ m Transwell (24-well, purchased from Corning, USA) was pre-laid with 10 times diluted matrix glue (purchased from BD, USA) and 0.5% gelatin (purchased from Shanghai, China) according to a volume ratio of 1: 1 Mix 50ul of glue and incubate in a 37 ° C cell incubator for 2h.
  • the transfected cells were resuspended in the manner of cell passage (140 FBS in 1640 medium to resuspend the cells) and the cell count was performed with a hemacytometer.
  • Add 200 ul of cell suspension (cell number: 5 ⁇ 10 4 ) to each well in the upper chamber of transwell, add 700 ul of 1040 FBS 1640 medium in the lower chamber, and place it in the incubator.
  • GMAN is closely related to the occurrence and development of gastric cancer. So, how does GMAN play a role in the process of gastric cancer, especially the metastasis of gastric cancer? This important issue has aroused our great interest. Because GMAN is relatively highly expressed in gastric cancer tissues, and GMAN is also abundantly expressed in most gastric cancer cell lines. We designed two siRNAs that specifically target GMAN and silence GMAN expression. Transfection of GMAN siRNA in gastric cancer cell line BGC823, knocked down the expression of GMAN, and detected the knockdown efficiency of GMAN by Northern blotting.
  • the full-length sequence of GMAN was obtained, the primers of GMAN were designed, and the DNA product of GMAN was obtained by PCR. After digestion, linking and other steps, the pcDNA3.1 overexpression plasmid containing the full length of GMAN was constructed. Plate the cells in the logarithmic growth phase until the cell density reaches about 80%. According to the transfection system in the table below, dilute Lipo3000 (purchased from Invitrogen, USA) and plasmids with OPTI medium, and add the diluted plasmids to Lipo3000 tubes. After mixing, let stand for 5 minutes, then add to the cell culture fluid, shake well, and change the medium after 24.
  • Example 6 Knockdown of GMAN can significantly inhibit the metastasis of gastric cancer cells
  • a target site sequence for GMAN was designed to synthesize the corresponding forward and reverse sequences (purchased from Shanghai Shengong, China). These sequence joints were designed and annealed to form Bam H I And EcoR I sticky end. After digestion, linking, identification, sequencing and other steps to construct GMAN shRNA lentiviral expression vector.
  • the target sequences are:
  • the cells in the logarithmic growth phase were plated, and when the cell density reached about 80%, they were replaced with fresh 1040 FBS 1640 medium. Add the appropriate amount of virus stock solution and add 5ug / mL Polybrene to promote the infection efficiency of the virus.
  • the efficiency of lentivirus infection can be demonstrated by observing the efficiency of the cells carrying GFP through a fluorescence microscope.
  • QRT-PCR was used to detect the knockdown effect of GMAN.
  • GMAN can significantly promote the invasion of gastric cancer cells.
  • the analysis of the correlation between the expression of GMAN and clinicopathology also shows that the high expression of GMAN is significantly correlated with the depth of tumor invasion (stage T), lymph node metastasis (stage N), and the progression of TNM stage in gastric cancer patients.
  • stage T the depth of tumor invasion
  • stage N lymph node metastasis
  • stage N the progression of TNM stage in gastric cancer patients.
  • GMAN is significantly overexpressed in metastatic M1 gastric cancer tissues.
  • Nude mice were injected into the tail vein of the control group and the stable cell line knocked down by GMAN to establish a lung metastasis model. Because the cells carry GFP, we used a mouse imager to detect the GFP signal in the lungs of nude mice to reflect the metastasis of gastric cancer cells, showing that the GFP signal of nude mice in the GMAN knockdown group was significantly weakened, suggesting that the GMAN knockdown group of gastric cancer Lung metastasis ability is inhibited. Both HE staining and visual observation of metastases in the lungs of nude mice showed that the number and size of metastases produced by gastric cancer cells in the GMAN knockdown group were significantly reduced (Figure 6.1). GMAN knockdown can significantly inhibit the invasion of gastric cancer cells in vitro and the ability of lung metastasis in vivo, which is consistent with the clinical and pathological correlation analysis of GMAN.
  • Example 7 GMAN mainly exerts the function of inhibiting the invasion of gastric cancer cells through the MFR segment
  • CRISPR / Cas9 gene editing targets protein-coding genes, which can cause frameshift mutations and other effects by changing a few bases of the coding gene, knocking out the expression of the encoded protein.
  • RNA knockout for long-chain non-coding RNA knockout, only changing a few bases may not change the function of lncRNA, requiring truncation of large fragments, changing the secondary structure of RNA or destroying it The main area of action is to knock out the expression of lncRNA.
  • GMAN-1-sense 5’-CACCGGAGTAGTATTAAGTGGCCC-3 ’
  • GMAN-1-antisense 5’-AAACGGGCCACTTAATACTACTCC-3 ’
  • GMAN-2-sense 5’-CACCGTTTCTTATTTAACCCCTGT-3 ’
  • GAMN-2-antisense 5’-AAACACAGGGGTTAAATAAGAAAC-3 ’
  • GMAN can significantly promote the invasion and metastasis of gastric cancer cells.
  • NCBI's Blast tool shows that the sequence of about 400 nt at the 5 'end of GMAN is non-specific, and many other genes or partial sequences of lncRNA are similar to the nucleotides of about 400 nt. of.
  • the sequence of less than 100 nt at the 3 'end of GMAN is the same as Exon3 and part of Exon4 of EphrinA1.
  • the sequence of about 300 nt in the middle section of GMAN (provisionally called MFR) is a GMAN-specific sequence.
  • MFR section of GMAN is a GMAN-specific sequence (Table S3), prompting whether GMAN is played through this specific sequence Features. Therefore, we used CRISPR / Cas9 gene editing technology to design two specific sgRNAs targeting GMAN. Through monoclonal screening, sequencing identification and off-target effect identification, we successfully established a gastric cancer mutant strain ( ⁇ MFR) that knocked out the MFR segment of GMAN ( Figure 7.1).
  • ⁇ MFR gastric cancer mutant strain
  • Example 8 MFR deletion can significantly inhibit the metastasis of gastric cancer cells
  • nude mice producing lung metastases from gastric cancer cells Wild-type gastric cancer cells and mutant gastric cancer cell lines with ⁇ MFR were injected through the tail vein to establish nude mice lung metastasis models. Observe the living conditions of nude mice in real time and record the survival time of two groups of nude mice, accurate to days. Using Kaplan-Meier survival curve analysis, the survival curves of nude mice with lung metastasis from wild-type gastric cancer cells and lung metastases from gastric cancer mutant strains of ⁇ MFR were drawn.
  • GMAN mainly promotes the invasion and metastasis of gastric cancer cells through the MFR segment.
  • CRISPR / Cas9 gene editing technology to target the MFR segment of GMAN, a GMAN-mutated gastric cancer cell line with a large deletion of the MFR segment in the main action region of GMAN was obtained.
  • GMAN knockdown can significantly inhibit the invasion and metastasis of gastric cancer cells. So, does the GMAN mutation have a similar function?
  • Example 9 The MFR segment of GMAN has the potential to treat gastric cancer metastasis
  • Gastric cancer cells with missing MFR segments of GMAN can significantly inhibit the metastasis of gastric cancer cells, and CRISPR / Cas9 is a multifunctional genome editing tool with broad therapeutic potential.
  • the MFR segment of GMAN plays a vital role in the function of GMAN.
  • the MFR segment of GMAN is located in the intron region of the genome and is a unique sequence of GMAN. Therefore, we reasonably assume that the MFR segment of GMAN can be regarded as important Intron target sites for anti-metastatic effects, using CRISPR / Cas9-mediated therapy to target the MFR segment of GMAN may be a promising anti-gastric cancer strategy.
  • the experimental group was injected with a liposome delivery buffer coated with GMAN-targeted CRISPR / Cas9 (CRISPR-GMAN) vector in the tail vein, and the control group was given a liposome delivery buffer without GMAN CRISPR / Cas9 vector, which was performed weekly Two injections.
  • BLI was used to monitor lung metastases and evaluate the therapeutic efficacy of CRISPR / Cas9 delivery.
  • SGC-Luc cells produced severe lung metastases in animals treated with control delivery buffer, but CRISPR / Cas9 targeted GMAN's MFR segment-treated drugs significantly blocked SGC-Luc cells from metastasizing into the lungs within one week after delivery.
  • the MFR segment targeting GMAN can effectively treat the metastasis of gastric cancer, and the MFR segment has potential value. Because the MFR segment of GMAN is a small intron sequence on the genome, and the MFR segment is a relatively specific nucleotide sequence in the human body. The sequence characteristics of the MFR segment of GMAN allow us to design drugs that target the MFR segment without affecting the expression of other genes or RNA and greatly reducing the possibility of drug off-target. These characteristics provide great convenience and possibility for us to reasonably use gene editing technology or synthesize stable nucleic acid small molecule drugs targeting MFR segments. Therefore, based on the effects of our treatment experiments and the sequence characteristics of the MFR segment of GMAN, the MFR segment of GMAN has important potential for the treatment of gastric cancer metastasis.
  • Example 10 GMAN is relatively highly expressed in other digestive system tumor tissues and is significantly associated with cancer patient metastasis
  • GMAN is highly expressed in various tumor tissues and is significantly associated with tumor metastasis, which is significantly higher in patients with metastatic tumors (M1) than in patients with non-metastatic tumors (M0).
  • M1 metastatic tumors
  • M0 non-metastatic tumors
  • the examples show the high expression of GMAN in colorectal cancer ( Figure 10.1), esophageal cancer ( Figure 10.2) and liver cancer (Figure 10.3).

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Abstract

Disclosed in the present invention are a tumor-related sequence, a long-chain non-coding RNA (lncRNA) and use thereof. lncRNA GMAN is relatively highly expressed in gastric cancer, and especially, is significantly highly expressed in a sample of a gastric cancer patient, which generates distant metastasis, in M1 stage. The high expression of GMAN is significantly related to the poor prognosis of the gastric cancer patient. Studies show that GMAN plays an important role in the development of gastric cancer, GMAN being capable of significantly promoting invasion and metastasis of gastric cancer cells. Further studies show that MFR segment of GMAN is critical to the function of the GMAN. Treatment experiments using CRISPR/Cas9 show that GMAN, particularly MFR segments of GMAN, can effectively inhibit metastasis of gastric cancer, and can serve as a new strategy for clinically controlling tumor metastasis.

Description

一种肿瘤相关序列、长链非编码RNA及其应用Tumor-related sequence, long-chain non-coding RNA and application thereof 技术领域Technical field
本发明涉及一种肿瘤相关序列、长链非编码RNA及其应用。The invention relates to a tumor-related sequence, long-chain non-coding RNA and its application.
背景技术Background technique
最近的转录组研究已经显示超过70％的人类基因组被转录为RNA，其中大多数是非编码转录物。目前已知的非编码转录物至少有20多种，一般分为两类，一类为非编码小RNA(sncRNA，Small non-coding RNA)，其转录长度少于200个核苷酸，另一类为长链非编码RNA(Long non-coding RNA，LncRNA)，被广泛的定义为转录长度超过200个核苷酸并且没有表观编码能力的RNA。根据LncRNA基因所处的位置可将其分为正义LncRNA、反义LncRNA、双向LncRNA、内含子内LncRNA以及基因间LncRNA等五大类。目前越来越多的证据显示，很多非编码RNA具有重要的生物学功能，在基因转录、转录后调控、RNA剪切、翻译、表观遗传调控、遗传印迹、X染色体失活以及疾病发生发展等过程中都发挥着重要作用。Recent transcriptome studies have shown that more than 70% of the human genome is transcribed into RNA, most of which are non-coding transcripts. At present, there are at least more than 20 types of non-coding transcripts, generally divided into two categories, one is non-coding small RNA (sncRNA, Small non-coding RNA), its transcript length is less than 200 nucleotides, another The class is long non-coding RNA (LncRNA), which is widely defined as RNA with a transcription length of more than 200 nucleotides and no apparent coding ability. According to the position of the LncRNA gene, it can be divided into five categories: sense LncRNA, antisense LncRNA, bidirectional LncRNA, intron LncRNA and intergenic LncRNA. More and more evidence shows that many non-coding RNAs have important biological functions, such as gene transcription, post-transcriptional regulation, RNA splicing, translation, epigenetic regulation, genetic imprinting, X chromosome inactivation, and disease development. They all play an important role in the process.
越来越多的研究发现，LncRNA的突变或异常与癌症的发生发展密切相关。对LncRNA与癌症关系的研究可以上溯至20世纪90年代，H19是第一个发现与癌症相关的LncRNA。研究表明LncRNA在多种肿瘤的发生、侵袭、转移过程中起重要作用，其中包括黑色素瘤、结肠癌、***癌、白血病、肝癌、乳腺癌等，然而，有关其具体作用机制报道较少。Rajnish等人发现LncRNA HOTAIR位于HOX家族基因的内含子中，其在乳腺癌中高表达，并与肿瘤转移高度相关；进一步研究显示HOTAIR过表达可以促进肿瘤的侵袭和转移能力。另外，还有一些LncRNA在抑制肿瘤发生过程中也扮演着重要角色，在乳腺癌中表达下调的LncRNA GAS5(growth arrest-specific5)，可通过抑制细胞生长及诱导凋亡，而发挥着肿瘤抑癌基因的功能。More and more studies have found that mutations or abnormalities of LncRNA are closely related to the occurrence and development of cancer. Research on the relationship between LncRNA and cancer can be traced back to the 1990s, H19 was the first to discover LncRNA related to cancer. Studies have shown that LncRNA plays an important role in the occurrence, invasion and metastasis of various tumors, including melanoma, colon cancer, prostate cancer, leukemia, liver cancer, breast cancer, etc. However, there are few reports on its specific mechanism of action. Rajnish et al. Found that LncRNA HOTAIR is located in the intron of the HOX family gene, which is highly expressed in breast cancer and highly correlated with tumor metastasis; further research shows that HOTAIR overexpression can promote tumor invasion and metastasis ability. In addition, some LncRNAs also play an important role in inhibiting tumorigenesis. LncRNAs that are down-regulated in breast cancer GAS5 (growth-arrest-specific5) can play a role in tumor suppression by inhibiting cell growth and inducing apoptosis Gene function.
LncRNA的肿瘤发生发展中的重要功能为肿瘤治疗开拓了一个新的视野，可能为有效的肿瘤治疗提供重要的潜在价值。在乳腺癌中，lncRNA MAYA在促进乳腺癌的骨转移中发挥重要的作用。在乳腺癌细胞中，ROR1磷酸化HER3，招募LLGL2-MAYA-NSUN6RNA蛋白复合物，促进Hippo/MST1的甲基化，导致MST1失活并使YAP靶向基因活化，进而促进乳腺癌的骨转移。MAYA的治疗实验显示，在产生乳腺癌细胞骨转移的小鼠中，注射MAYA的LNA能显著的 抑制乳腺癌细胞的骨转移。虽然关于lncRNA对于肿瘤治疗的研究还相对比较少，但是已有的这些研究都显示LncRNA不仅在肿瘤中发挥重要的调控作用而且具有有效***的极大潜能，表明lncRNA可作为肿瘤治疗的新型药物靶点。The important function of LncRNA in tumorigenesis and development has opened up a new field of vision for tumor therapy, and may provide important potential value for effective tumor therapy. In breast cancer, lncRNA MAYA plays an important role in promoting bone metastasis of breast cancer. In breast cancer cells, ROR1 phosphorylates HER3, recruits LLGL2-MAYA-NSUN6RNA protein complex, promotes the methylation of Hippo / MST1, leads to inactivation of MST1 and activates the YAP targeting gene, thereby promoting bone metastasis of breast cancer. MAYA's treatment experiments show that in mice that produce bone metastases from breast cancer cells, LNA injected with MAYA can significantly inhibit bone metastases from breast cancer cells. Although there are relatively few studies on lncRNA for tumor therapy, the existing studies have shown that LncRNA not only plays an important regulatory role in tumors but also has great potential for effective treatment of tumors, indicating that lncRNA can be used as a new drug for tumor therapy Target.
目前，关于LncRNA在胃癌中的具体调控作用也有了一些报道。在胃癌中高表达的GClnc1作为WDR5和KAT2A的支架，影响组蛋白修饰，进而影响组蛋白调控的一系列基因，包括SOD2，影响胃癌细胞的生长和转移。LncRNA BC032469作为miR-1207-5P的海绵，调控hTERT表达，促进胃癌细胞的增殖。LncRNA HOXA11-AS作为染色体调控因子PRC2，LSD1，DNMT1的支架，促进胃癌细胞的增殖和侵袭等。但是，关于LncRNA作为胃癌治疗潜能的研究目前还尚未有报道。At present, there have been some reports on the specific regulatory role of LncRNA in gastric cancer. GClnc1, which is highly expressed in gastric cancer, serves as a scaffold for WDR5 and KAT2A, which affects histone modification, which in turn affects a series of genes regulated by histones, including SOD2, which affects the growth and metastasis of gastric cancer cells. LncRNA BC032469 acts as a sponge for miR-1207-5P, regulates hTERT expression, and promotes the proliferation of gastric cancer cells. LncRNA HOXA11-AS serves as a scaffold for the chromosomal regulatory factors PRC2, LSD1, DNMT1, and promotes the proliferation and invasion of gastric cancer cells. However, the research on the potential of LncRNA as a treatment for gastric cancer has not been reported yet.
而胃癌作为全球最常见的恶性肿瘤之一，是导致癌症死亡的重要因素之一。在全球范围内，胃癌是第五高发病率和第三高死亡率的恶性肿瘤，而中国是胃癌高发的国家。胃癌的发生与发展已严重危害到人类的健康。但是，胃癌器官转移起病隐匿，发现时多数患者都难以手术切除，因而预后极差，其5年生存率低于10％，是晚期胃癌患者死亡的主要原因之一。因此，我们迫切需要深入了解胃癌器官转移的发病机理，寻找调控胃癌细胞转移过程的关键因子。LncRNA在多种肿瘤中发挥重要的作用，提示LncRNA的功能可能为胃癌细胞转移的诊断预后和治疗提供新靶点和新思路。As one of the most common malignant tumors in the world, gastric cancer is one of the important factors leading to cancer death. Globally, gastric cancer is the malignant tumor with the fifth highest morbidity and third highest mortality, while China is a country with a high incidence of gastric cancer. The occurrence and development of gastric cancer have seriously endangered human health. However, the onset of gastric cancer metastasis is hidden, and most patients are difficult to remove when found, so the prognosis is extremely poor, and its 5-year survival rate is less than 10%, which is one of the main causes of death in patients with advanced gastric cancer. Therefore, we urgently need to deeply understand the pathogenesis of gastric cancer organ metastasis and look for key factors that regulate the metastasis process of gastric cancer cells. LncRNA plays an important role in a variety of tumors, suggesting that the function of LncRNA may provide new targets and new ideas for the diagnosis, prognosis and treatment of gastric cancer metastasis.
发明内容Summary of the invention
本发明的目的是针对现有技术的不足，提供一种肿瘤相关序列、长链非编码RNA及其应用。The purpose of the present invention is to provide a tumor-related sequence, long-chain non-coding RNA and its application in view of the deficiencies of the prior art.
本发明的目的是通过以下技术方案实现的：一种肿瘤相关序列，其序列为如SEQ ID NO.1所示的MFR序列。The object of the present invention is achieved by the following technical solution: a tumor-related sequence whose sequence is the MFR sequence shown in SEQ ID NO.1.
进一步地，所述肿瘤包括鼻腔及鼻窦恶性肿瘤，鼻咽癌，口腔癌，喉癌，涎腺肿瘤，颅内肿瘤，甲状腺癌，舌癌，肺癌，食管癌，贲门癌，乳腺癌，纵膈肿瘤，胃癌，大肠癌，直肠癌，肝癌，胰腺癌与壶腹周围癌，小肠恶性肿瘤，肾癌，***癌，膀胱癌，子***，卵巢癌，皮肤恶性黑色素瘤，淋巴瘤。Further, the tumors include malignant tumors of the nasal cavity and sinuses, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardia cancer, breast cancer, and mediastinum Tumor, stomach cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, malignant tumor of small intestine, kidney cancer, prostate cancer, bladder cancer, cervical cancer, ovarian cancer, skin malignant melanoma, lymphoma.
上述MFR序列在制备肿瘤诊断试剂中的应用，所述诊断试剂识别SEQ ID NO.1所示的MFR序列。The application of the above MFR sequence in the preparation of a tumor diagnostic reagent that recognizes the MFR sequence shown in SEQ ID NO. 1.
进一步地，诊断试剂包括但不限于：Further, diagnostic reagents include but are not limited to:
(1)识别所述MFR序列的引物/引物组，或荧光标记的识别所述MFR序列的 的引物/引物组；(1) A primer / primer set that recognizes the MFR sequence, or a fluorescently labeled primer / primer set that recognizes the MFR sequence;
(2)识别所述MFR序列的小分子化合物；(2) A small molecule compound that recognizes the MFR sequence;
(3)识别所述MFR序列的生物大分子，所述的生物大分子包括但不限于：抗体或抗体功能片段、荧光标记的抗体或抗体功能片段、RNA结合蛋白或其功能片段、荧光标记的RNA结合蛋白或其功能片段。(3) Biomacromolecules that recognize the MFR sequence, including but not limited to: antibodies or antibody functional fragments, fluorescently labeled antibodies or antibody functional fragments, RNA binding proteins or functional fragments thereof, fluorescently labeled RNA binding protein or its functional fragments.
进一步地，所述抑制剂靶向SEQ ID NO.1所示的MFR序列。Further, the inhibitor targets the MFR sequence shown in SEQ ID NO.1.
进一步地，抑制剂包括但不限于：Further, inhibitors include but are not limited to:
(1)抑制所述MFR序列的siRNA、shRNA或功能类似的干扰小RNA；(1) siRNA, shRNA or functionally similar interfering small RNA that inhibits the MFR sequence;
(2)抑制所述MFR序列的寡核苷酸片段，所述的寡核苷酸片段包括但不限于：反义寡核苷酸ASO、锁核酸LNA或功能类似的化学修饰的寡核苷酸。(2) Oligonucleotide fragments that inhibit the MFR sequence, and the oligonucleotide fragments include but are not limited to: antisense oligonucleotide ASO, locked nucleic acid LNA, or functionally similar chemically modified oligonucleotide .
(3)抑制所述MFR序列的小分子化合物；(3) Small molecule compounds that inhibit the MFR sequence;
(4)抑制所述MFR序列的生物大分子，所述的生物大分子包括但不限于：抗体或抗体功能片段、高底物专一性的酶或其功能片段、其他抑制MFR功能的蛋白分子。(4) Biomacromolecules that inhibit the MFR sequence, the biomacromolecules include but are not limited to: antibodies or antibody functional fragments, high substrate-specific enzymes or functional fragments thereof, and other protein molecules that inhibit MFR function .
(5)敲除或破坏所述MFR序列的工具分子。(5) Tool molecules that knock out or destroy the MFR sequence.
进一步地，能敲除或破坏MFR序列的工具分子，包括但不限于DNA同源重组质粒，TALEN-TALEA靶向基因敲除质粒***，Cre/Loxp质粒***，四环素/干扰素等诱导性Cre/Loxp质粒***，FLP-frt质粒***，CRISPR/Cas9等CRISPR基因编辑质粒***等。Further, tool molecules capable of knocking out or destroying MFR sequences include but are not limited to DNA homologous recombination plasmids, TALEN-TALEA targeted gene knockout plasmid system, Cre / Loxp plasmid system, tetracycline / interferon and other inducible Cre / Loxp plasmid system, FLP-frt plasmid system, CRISPR / Cas9 and other CRISPR gene editing plasmid systems, etc.
一种肿瘤相关长链非编码RNA，包含上述MFR序列序列。具体的，可以为SEQ ID NO.2所示的GMAN。A tumor-associated long-chain non-coding RNA, including the above MFR sequence sequence. Specifically, it may be GMAN shown in SEQ ID NO.2.
本发明的有益效果在于：相比于现有技术的优势。The beneficial effect of the present invention lies in the advantages compared with the prior art.
附图说明BRIEF DESCRIPTION
图1.1为lncRNA GMAN示意图；Figure 1.1 is a schematic diagram of lncRNA GMAN;
图2.1为Northern印迹检测人胃癌细胞系中GMAN的表达；Figure 2.1 is Northern blot detection of GMAN expression in human gastric cancer cell lines;
图2.2为TCGA转录组数据分析人的组织器官中GMAN的表达；Figure 2.2 is TCGA transcriptome data analysis of the expression of GMAN in human tissues and organs;
图3.1为GMAN在胃癌组织中的表达水平；Figure 3.1 shows the expression level of GMAN in gastric cancer tissues;
图3.2为40例具有5年生存信息的胃癌患者队列；Figure 3.2 is a cohort of 40 gastric cancer patients with 5-year survival information;
图3.3为Cohort 3，cohort 4和TCGA数据库中年龄，性别匹配的不具有转移的M0期胃癌组织与具有远端转移的M1期胃癌组织中分析GMAN的表达；**，P<0.01。Figure 3.3 shows the expression of GMAN in Cohort 3, Cohort 4, and TCGA databases of age and sex matched M0 gastric cancer tissues without metastasis and M1 gastric cancer tissues with distant metastasis; **, P <0.01.
图4.1为Northern印迹检测GMAN的敲低效率；Figure 4.1 shows the Northern blot detection efficiency of GMAN knockdown;
图4.2为MTT和克隆形成实验检测GMAN对细胞增殖的影响；Figure 4.2 shows the effect of GMAN on cell proliferation by MTT and clone formation experiments;
图4.3为细胞流式检测GMAN对细胞周期的影响；Figure 4.3 shows the effect of GMAN on cell cycle by cell flow cytometry;
图4.4为敲低GMAN对胃癌细胞的细胞粘附能力的影响；n.s.，没有意义；Figure 4.4 shows the effect of knocking down GMAN on the cell adhesion ability of gastric cancer cells; n.s., meaningless;
图4.5为敲低GMAN对胃癌细胞的细胞侵袭能力的影响；***，P<0.001。Figure 4.5 shows the effect of knocking down GMAN on the cell invasion ability of gastric cancer cells; ***, P <0.001.
图5.1为Northern印迹检测GMAN的过量表达效果；Figure 5.1 shows the effect of Northern blot detection of GMAN overexpression;
图5.2为MTT和克隆形成实验检测GMAN对细胞增殖的影响；Figure 5.2 shows the effect of GMAN on cell proliferation by MTT and clone formation experiments;
图5.3为细胞流式检测GMAN对细胞周期的影响；Figure 5.3 shows the effect of GMAN on cell cycle by cell flow cytometry;
图5.4为过量表达GMAN对胃癌细胞的细胞粘附能力的影响；n.s.，没有意义。Figure 5.4 shows the effect of overexpression of GMAN on the cell adhesion ability of gastric cancer cells; n.s., meaningless.
图5.5为过量表达GMAN对胃癌细胞的细胞侵袭能力的影响；***，P<0.001。Figure 5.5 shows the effect of overexpression of GMAN on the cell invasion ability of gastric cancer cells; ***, P <0.001.
图6.1为敲低GMAN对胃癌细胞的转移能力的影响；***，P<0.001。Figure 6.1 shows the effect of knocking down GMAN on the metastatic ability of gastric cancer cells; ***, P <0.001.
图7.1为利用CRISPR/Cas9基因编辑构建GMAN的MFR段缺失的胃癌细胞突变株(ΔMFR)；Figure 7.1 shows the use of CRISPR / Cas9 gene editing to construct a gastric cancer mutant strain (ΔMFR) with deletion of the MFR segment of GMAN;
图7.2为MFR缺失的细胞株对胃癌细胞的细胞增殖能力的影响；Figure 7.2 shows the effect of MFR-deficient cell lines on the proliferation of gastric cancer cells;
图7.3为MFR缺失对胃癌细胞的细胞周期的影响；Figure 7.3 shows the effect of MFR deletion on the cell cycle of gastric cancer cells;
图7.4为MFR缺失对胃癌细胞的细胞粘附能力的影响；n.s.，没有意义。Figure 7.4 shows the effect of MFR deletion on the cell adhesion ability of gastric cancer cells; n.s., meaningless.
图7.5为MFR缺失对胃癌细胞的侵袭能力的影响；***，P<0.001。Figure 7.5 shows the effect of MFR deletion on the invasion ability of gastric cancer cells; ***, P <0.001.
图8.1为MFR缺失对胃癌细胞的肺转移能力的影响；***，P<0.001(n＝6)。Figure 8.1 shows the effect of MFR deletion on the lung metastatic ability of gastric cancer cells; ***, P <0.001 (n = 6).
图8.2为小鼠肺部HE染色检测MFR缺失对胃癌细胞的肺转移能力的影响；***，P<0.001(n＝6)。Figure 8.2 shows the effect of HE staining on the lungs of mice to detect the effect of MFR deletion on the lung metastatic ability of gastric cancer cells; ***, P <0.001 (n = 6).
图8.3为接种ΔMFR的胃癌细胞对小鼠体重的影响；P<0.001(n＝6)。Figure 8.3 shows the effect of gastric cancer cells inoculated with ΔMFR on the body weight of mice; P <0.001 (n = 6).
图8.4为MFR缺失对肺转移裸鼠的生存的影响；使用对数秩检验计算P值，P<0.001(n＝8)，HR＝25.9(6.1,110.1)。Figure 8.4 shows the effect of MFR deletion on the survival of lung metastatic nude mice; P value was calculated using log-rank test, P <0.001 (n = 8), HR = 25.9 (6.1, 110.1).
图9.1为靶向GMAN的MFR区段的CRISPR/Cas9治疗胃癌细胞肺转移的示意图；Figure 9.1 is a schematic diagram of CRISPR / Cas9 targeting the MFR segment of GMAN to treat lung metastasis of gastric cancer cells;
图9.2为BLI实时监测靶向GMAN的MFR区段的CRISPR/Cas9治疗胃癌细胞肺转移的情况；Figure 9.2 is the BLI real-time monitoring of lung metastasis of gastric cancer cells treated with CRISPR / Cas9 targeting the MFR segment of GMAN;
图9.3为CRISPR/Cas9递送***相关的细胞毒性检测；Figure 9.3 shows the cytotoxicity test related to the CRISPR / Cas9 delivery system;
图10.1为TCGA数据库中分析GMAN在结直肠癌病人中的表达情况，A图为癌和癌旁配对的病例，B图为总体癌和癌旁的表达情况，C图为年龄，性别匹配的不具有转移的M0期结直肠癌组织与具有远端转移的M1期结直肠癌组织中分析GMAN的表达。***，P<0.001。Figure 10.1 is the analysis of the expression of GMAN in colorectal cancer patients in the TCGA database. A is the matching case of cancer and adjacent cancer, B is the expression of the overall cancer and adjacent cancer, and C is the age and gender matching. GMAN expression was analyzed in M0 colorectal cancer tissue with metastasis and M1 colorectal cancer tissue with distant metastasis. ***, P <0.001.
图10.2为TCGA数据库中分析GMAN在食管癌病人中的表达情况，A图 为食管癌和癌旁配对的病例，B图为年龄，性别匹配的不具有转移的M0期食管癌组织与具有远端转移的M1期食管癌组织中分析GMAN的表达。*，P<0.05。Figure 10.2 is the analysis of the expression of GMAN in esophageal cancer patients in the TCGA database. Panel A shows the cases of esophageal cancer and adjacent cancers. Panel B shows the age-matched M0 esophageal cancer tissues with metastasis and distant GMAN expression was analyzed in metastatic M1 esophageal cancer tissues. *, P <0.05.
图10.3为TCGA数据库中分析GMAN在肝癌病人中的表达情况，A图为肝癌和癌旁配对的病例，B图为总体肝癌和癌旁的表达情况，***，P<0.001。Figure 10.3 is the analysis of the expression of GMAN in liver cancer patients in the TCGA database. Panel A is the case of paired liver cancer and adjacent cancer. Panel B is the overall expression of liver cancer and adjacent cancer. ***, P <0.001.
具体实施方式detailed description
本发明首次提出一种新的lncRNA GMAN，显著的促进胃癌细胞的侵袭和转移，并且具有治疗胃癌转移的重要潜能。具体内容如下：The present invention proposes for the first time a new lncRNA GMAN, which significantly promotes the invasion and metastasis of gastric cancer cells, and has an important potential for treating gastric cancer metastasis. The details are as follows:
1)3’Race和5’Race确认lncRNA GMAN的序列，Northern印迹显示在人的胃癌组织及细胞中可检测到GMAN的表达。1) 3'Race and 5'Race confirm the sequence of lncRNA and GMAN. Northern blotting shows that GMAN expression can be detected in human gastric cancer tissues and cells.
2)发现了在胃癌组织中表达上调的lncRNA GMAN，GMAN的高表达与肿瘤的侵润深度，淋巴转移，远端转移和胃癌病人的不良预后显著相关。2) It was found that the up-regulated lncRNA GMAN in gastric cancer tissues, the high expression of GMAN is significantly correlated with the depth of tumor invasion, lymphatic metastasis, distant metastasis and poor prognosis of gastric cancer patients.
2)研究显示GMAN对胃癌细胞的细胞增殖，细胞周期，克隆形成和细胞粘附没有明显影响，但是GMAN能显著的促进胃癌细胞的体外侵袭和体内转移。2) Studies have shown that GMAN has no significant effect on cell proliferation, cell cycle, clonal formation and cell adhesion of gastric cancer cells, but GMAN can significantly promote the invasion and metastasis of gastric cancer cells in vitro.
3)证明了GMAN的MFR(main function region)区段是GMAN发挥促进肿瘤侵袭和转移的主要作用的区域。3) It is proved that the MFR (main function) region of GMAN is the region where GMAN plays a major role in promoting tumor invasion and metastasis.
4)CRISPR/Cas9技术靶向GMAN的MFR区段，构建GMAN的MFR区缺失的胃癌细胞系。显示MFR区域缺失的细胞侵袭和转移能力受到显著的抑制。4) CRISPR / Cas9 technology targets the MFR segment of GMAN and constructs a gastric cancer cell line in which the MFR region of GMAN is deleted. The ability to invade and metastasize cells showing the absence of the MFR region was significantly inhibited.
5)体内治疗实验证明了，GMAN尤其是GMAN的MFR区段可作为治疗胃癌转移的重要靶点。5) In vivo treatment experiments have proved that GMAN, especially the MFR segment of GMAN, can be used as an important target for the treatment of gastric cancer metastasis.
综上，lncRNA GMAN在胃癌中相对高表达，尤其是在产生远端转移的M1期胃癌病人的样本中显著高表达，而且GMAN的高表达与胃癌病人的不良预后显著相关。研究显示GMAN在胃癌的发生发展中发挥重要的作用，GMAN能显著促进胃癌细胞的侵袭和转移。进一步，GMAN的MFR区段对于GMAN发挥功能至关重要。通过CRISPR/Cas9的治疗实验表明，GMAN特别是GMAN的MFR区段能够有效的抑制胃癌的转移，可作为临床上控制肿瘤转移的一个新策略。In summary, lncRNA and GMAN are relatively highly expressed in gastric cancer, especially in samples of M1 stage gastric cancer patients with distant metastasis, and the high expression of GMAN is significantly related to the poor prognosis of gastric cancer patients. Studies have shown that GMAN plays an important role in the occurrence and development of gastric cancer. GMAN can significantly promote the invasion and metastasis of gastric cancer cells. Further, the MFR section of GMAN is essential for GMAN to function. The CRISPR / Cas9 treatment experiments show that GMAN, especially the MFR segment of GMAN, can effectively inhibit the metastasis of gastric cancer, and can be used as a new strategy for clinically controlling tumor metastasis.
下面结合实施例对本发明作进一步说明。The present invention will be further described below with reference to examples.
实施例1：lncRNA GMAN是一个855nt的lncRNAExample 1: lncRNA GMAN is an 855nt lncRNA
为了检测GMAN的5’和3’的末端终序列，我们根据SMARTer TMRACE cDNA Amplification Kit(购自美国Clontech)的使用说明进行实验。 In order to detect the 5 'and 3' terminal sequences of GMAN, we conducted experiments according to the instructions of SMARTer ™ RACE cDNA Amplification Kit (purchased from Clontech, USA).
1.按照Race说明书中设计引物的原则，设计Race的特异引物如下：1. According to the principle of primer design in Race specification, the specific primers for Race are designed as follows:
5'race for GMAN:5'-CAAGACTTCTATACCAT-3'5'race for GMAN: 5'-CAAGACTTCTATACCAT-3 '
3'race for GMAN:5'-CAGGCTGGTCTCGAACTCCT-3'3'race for GMAN: 5'-CAGGCTGGTCTCGAACTCCT-3 '
2.提取人胃癌细胞系的RNA并对RNA进行加PolyA处理和纯化2. Extract RNA from human gastric cancer cell lines and apply PolyA treatment and purification to the RNA
细胞总RNA的分离提取利用TRIZOL试剂(购自美国Invitrogen)，并按照试剂说明书的标准操作进行。取适量的RNA利用DNase I试剂盒(购自美国Invitrogen)消化RNA中残留的基因组，去除基因组污染。Isolation and extraction of total cellular RNA was carried out using TRIZOL reagent (purchased from Invitrogen, USA), and was carried out according to the standard operation of the reagent instructions. Take an appropriate amount of RNA and use DNase I kit (purchased from Invitrogen, USA) to digest the remaining genome in the RNA to remove genomic contamination.
RNA加PolyA处理，利用PolyA试剂盒(购自美国NEB)。取适量RNA与polyA酶，buffer，RNase inhibitor混匀，37℃孵育。DNA凝胶电泳检测RNA的PloyA加尾情况。RNA plus PolyA treatment, using PolyA kit (purchased from NEB, USA). Take appropriate amount of RNA and mix with polyA enzyme, buffer, RNase inhibitor, and incubate at 37 ℃. DNA gel electrophoresis was used to detect the PloyA tailing of RNA.
用RNeasy Plus Mini Kit(购自美国QIAGEN)纯化RNA，备用。RNA was purified using RNeasy Plus Mini Kit (purchased from QIAGEN, USA), and used for standby.
3.RACE-Ready first-strand cDNA合成3. RACE-Ready first-strand cDNA synthesis
3.1配制5’-and 3’-RACE-Ready cDNA合成的buffer mix3.1 Preparation of buffer for 5’-and 3’-RACE-Ready cDNA synthesis
4.0μl4.0μl 5X First-Strand Buffer5X First-Strand Buffer
0.5μl0.5μl DTT(100mM)DTT (100mM)
1.0μl1.0μl dNTPs(20mM)dNTPs (20mM)
5.5μl5.5μl Total VolumeTotal Volume
3.2取新的PCR管配制如下溶液3.2 Take a new PCR tube to prepare the following solution
5’-RACE-Ready cDNA的配制5’-RACE-Ready cDNA preparation
1ug1ug RNARNA
1μl1μl 5’-CDS Primer A5’-CDS Primer A
0-9μl0-9μl Sterile H2OSterile H2O
11μl11μl Total VolumeTotal Volume
3’-RACE-Ready cDNA的配制Preparation of 3’-RACE-Ready cDNA
1ug1ug RNARNA
1.0μl1.0μl 3’-CDS Primer A3’-CDS Primer A
0–10μl0–10μl Sterile H2OSterile H2O
12μl12μl Total VolumeTotal Volume
3.3步骤3.2中的PCR管在72℃孵育3分钟，42℃孵育2分钟，冰上冷却，14000g离心10秒使溶液都位于PCR管的底部。在5’-RACE-Ready cDNA的配制的PCR管中加入1μl SMARTer II A Oligonucleotide。3.3 The PCR tube in step 3.2 was incubated at 72 ° C for 3 minutes, 42 ° C for 2 minutes, cooled on ice, and centrifuged at 14000g for 10 seconds to make the solution at the bottom of the PCR tube. Add 1μl SMARTer II Oligonucleotide to the prepared PCR tube of 5’-RACE-Ready cDNA.
3.4步骤3.1中的buffer mix分别加入RNase inhibitor和转录酶，室温混匀。3.4 Add buffer and mix RNase and transcriptase in step 3.1, mix at room temperature.
5.5μl5.5μl Buffer Mix from Step 1Buffer, Mix from Step 1
0.5μl0.5μl RNase Inhibitor(40U/μl)RNaseInhibitor (40U / μl)
2.0μl2.0μl SMARTScribe Reverse Transcriptase(100U)SMARTScribeReverseTranscriptase (100U)
8.0μl8.0μl Total VolumeTotal Volume
3.5步骤3.4中的buffer mix分别加入到步骤4(3’-RACE cDNA)和步骤5(5’-RACE cDNA)中，使终体积为20ul。温和混匀，离心使溶液都位于PCR管的底部。42℃孵育90分钟，70℃孵育10分钟。用Tricine-EDTA Buffer稀释3’-和5’-RACE-Ready cDNA，-20℃保存备用。3.5 The buffer mix in step 3.4 is added to step 4 (3'-RACE cDNA) and step 5 (5'-RACE cDNA) respectively, so that the final volume is 20ul. Mix gently and centrifuge to keep the solution at the bottom of the PCR tube. Incubate at 42 ° C for 90 minutes and 70 ° C for 10 minutes. Dilute 3'- and 5'-RACE-Ready cDNA with Tricine-EDTA Buffer and store at -20 ° C until use.
4.cDNA末端快速扩增(Race)4. Rapid amplification of cDNA ends (Race)
4.1配制3’-和5’-RACE的PCR反应的mix4.1 Mixing 3’- and 5’-RACE PCR reactions
15.5μl15.5μl PCR-Grade H2OPCR-Grade H2O
25.0μl25.0μl 2X SeqAmp Buffer2XSeqAmpBuffer
1.0μl1.0μl SeqAmp DNA PolymeraseSeqAmp DNA Polymerase
41.5μl41.5μl Total VolumeTotal Volume
4.2配制PCR反应，如下4.2 Prepare PCR reaction as follows
Figure PCTCN2018125262-appb-000001
Figure PCTCN2018125262-appb-000001
4.3 PCR反应4.3 PCR reaction
反应一：5 cycles:94℃30sec；72℃3min*Reaction 1: 5 cycles: 94 ℃ 30sec; 72 ℃ 3min *
反应二：5 cycles:94℃30sec；70℃30sec；72℃3min*Reaction 2: 5 cycles: 94 ℃ 30sec; 70 ℃ 30sec; 72 ℃ 3min *
反应三：20 cycles:94℃30sec；68℃30sec；72℃3min*Reaction 3: 20 cycles: 94 ℃ 30sec; 68 ℃ 30sec; 72 ℃ 3min *
5.分析Race产物5. Analysis of Race products
5.1 Race产物凝胶电泳并纯化5.1 Race product gel electrophoresis and purification
Race的DNA产物进行凝胶电泳。切胶回收纯化的DNA备用。Race DNA products were subjected to gel electrophoresis. Cut the gel to recover the purified DNA for use.
5.2 Race产物与克隆载体融合5.2 Fusion of Race product and cloning vector
配制融合体系并混匀，如下：Prepare and mix the fusion system as follows:
1μl1μl Lineareized pRACE vector(provided with SMARTer RACE 5’/3’Lineareized pRACEvector (provided withSMARTerRACE5 ’/ 3’
 A Kit Components)Kit Components
7μl7μl Gel-purified RACE productGel-purified RACE product
2μl2μl In-Fusion HD Master MixIn-Fusion HD Master Mix
10μl10μl Total VolumeTotal Volume
50℃孵育15分钟后置于冰上。从10ul的融合体系中取出2.5ul加入到Stellar Competent Cells中，温和混匀。加入SOC培养基孵育一段时间，把溶液均匀的涂在带有Amp+抗性的LB平板上，37℃孵育过夜，挑取LB平板上的克隆进行测序与分析。Incubate at 50 ° C for 15 minutes and place on ice. Take 2.5ul from the 10ul fusion system and add it to Stellar Competent Cells, mix gently. Add SOC medium to incubate for a period of time, spread the solution evenly on LB plates with Amp + resistance, incubate at 37 ° C overnight, and pick the clones on LB plates for sequencing and analysis.
实验结果Experimental results
为了研究GMAN的功能，我们首先要确认在人体内是否存在GMAN这个分子以及GMAN的具体序列。通过3’Race和5’Race确认，在人的胃癌细胞系中存在GMAN，而且GMAN是位于染色体1q22位上的一个855nt的lncRNA(表S1)。GMAN位于EphrinA1的基因簇中，GMAN的大部分是位于EphrinA1的内含子中(图1.1)。In order to study the function of GMAN, we must first confirm whether the GMAN molecule and the specific sequence of GMAN exist in the human body. It was confirmed by 3'Race and 5'Race that GMAN exists in human gastric cancer cell lines, and GMAN is an 855nt lncRNA located on chromosome 1q22 (Table S1). GMAN is located in the gene cluster of EphrinA1, and most of GMAN is located in the intron of EphrinA1 (Figure 1.1).
实施例2：GMAN在人的胃组织和胃癌细胞系中表达Example 2: GMAN expression in human gastric tissues and gastric cancer cell lines
1.体外转录GMAN的特异性RNA探针(靶向MFR区域)1. In vitro transcription of GMAN specific RNA probe (targeting MFR region)
1.1通过NCBI的Blast工具，找到GMAN的一段特异序列，并设计引物，同时设计GAPDH的引物(购自上海生工)，如下：1.1 Use NCBI's Blast tool to find a specific sequence of GMAN, and design primers, as well as GAPDH primers (purchased from Shanghai Shengong), as follows:
Figure PCTCN2018125262-appb-000002
Figure PCTCN2018125262-appb-000002
利用PCR反应获得GMAN/GAPDH的一段特异序列，以人胃癌细胞系的cDNA为模板，用上述引物，进行PCR反应，获得目的基因(购自日本Toyobo)。取PCR管，加入cDNA模板2ul，KOD高保真酶1ul，10xPCR buffer 5ul，dNTP 5ul，Forward primer 1ul，reverse primer 1ul，加入ddH2O至50ul，混匀，按以下反应程序进行PCR反应：A specific sequence of GMAN / GAPDH was obtained by PCR reaction. Using the cDNA of human gastric cancer cell line as a template, the PCR reaction was carried out using the above primers to obtain the target gene (purchased from Japan Toyobo). Take a PCR tube, add 2ul of cDNA template, 1ul of KOD high-fidelity enzyme, 10xPCR buffer 5ul, dNTP 5ul, Forward primer 1ul, reverse primer 1ul, add ddH2O to 50ul, mix well, and perform PCR reaction according to the following reaction procedure:
95℃95 ℃ 2min2min
95℃95 ℃ 30s30s
Tm-5℃Tm-5 ℃ 30s30s
68℃68 ℃ 1min-2min(延伸1Kb/min)1min-2min (Extended 1Kb / min)
68℃68 ℃ 10min10min
循环数：30cycle，PCR扩增产物于4℃保存备用Number of cycles: 30cycle, PCR amplification products are stored at 4 ℃ for future use
1.2载体PCS107和PCR产物的双酶切。1.2 Double digestion of vector PCS107 and PCR products.
按照下表配制酶切反应体系(购自美国NEB)，37℃，酶切3hPrepare the enzyme digestion reaction system (purchased from NEB in the United States) according to the following table.
DNADNA 1ug1ug
10×Buffer10 × Buffer 5ul5ul
Bam H ⅠBam H 1ul1ul
Xhol ⅠXhol 1ul1ul
ddH2OddH2O 至50ulUp to 50ul
1.3酶切产物回收纯化、连接、转化1.3 Recovery, purification, ligation and transformation of digested products
1.4挑取单克隆菌落并培养、质粒小量提取与鉴定(购自美国Axygen)1.4 Pick and culture monoclonal colonies, small amount of plasmid extraction and identification (purchased from Axygen, USA)
1.5体外合成RNA探针1.5 RNA probes synthesized in vitro
由于PCS107载体上含有SP6和T7的启动子，我们把GMAN/GAPDH的一段序列构建在两个启动子之间，通过单酶切的方式，可以选择通过SP6转录酶把GMAN/GAPDH的antisense链转录出来，或者通过T7转录酶转录GAMN/GAPDH的sense链。Since the PCS107 vector contains SP6 and T7 promoters, we constructed a sequence of GMAN / GAPDH between the two promoters. Through single enzyme digestion, we can choose to transcribe the antisense chain of GMAN / GAPDH by SP6 transcriptase. Go out, or transcribe the GAMN / GAPDH sense chain by T7 transcriptase.
把构建的PCS107-GMAN和PCS107-GAPDH的质粒用Xhol I进行单酶切，把线性化的PCS107载体作为模板。把转录所需的组分加到EP管中，混匀，37℃水浴孵育3h，具体组分如下：The constructed PCS107-GMAN and PCS107-GAPDH plasmids were digested with Xhol I and the linearized PCS107 vector was used as a template. Add the components required for transcription to the EP tube, mix well, and incubate in a 37 ° C water bath for 3 hours. The specific components are as follows:
模板template 1ug1ug
SP6转录酶(购自瑞士Roche)SP6 transcriptase (purchased from Roche, Switzerland) 2ul2ul
10*转录buffer10 * transcription buffer 2ul2ul
RNA mix(dig标签，购自瑞士Roche)RNA Mix (dig tag, purchased from Roche, Switzerland) 2ul2ul
RNase inhibitor(购自美国Invitrogen)RNase inhibitor (purchased from Invitrogen, USA) 0.5ul0.5ul
DEPCDEPC 至20ulTo 20ul
转录后，加1ul DNase I，37℃，15min，去除模板DNA。用RNeasy Plus Mini Kit(购自美国QIAGEN)纯化RNA，测浓度并取一分部跑胶，确定转录的RNA只有单一的目的条带。把纯化的RNA分装并-80℃保存。After transcription, add 1ul DNase I, 37 ℃, 15min to remove template DNA. Purify RNA with RNeasy Plus Mini Kit (purchased from QIAGEN, USA), measure the concentration and take a portion of the gel to make sure that the transcribed RNA has only a single target band. The purified RNA was aliquoted and stored at -80 ° C.
2.人的多种组织和人胃癌细胞系中GMAN的表达情况2. Expression of GMAN in various human tissues and human gastric cancer cell lines
2.1组织或者细胞总RNA的分离提取利用TRIZOL试剂(购自美国Invitrogen)，并按照试剂说明书的标准操作进行，并检测RNA的浓度。RNA中加入变形剂，混匀，70℃变性15min。RNA加入1.2％的变性琼脂糖凝胶孔中，1*MOPS buffer作为电泳缓冲液(购自美国Sigma)，100V预跑10min，45V电泳4-5h。利用盐桥进行硝酸纤维素膜的转膜，过夜。1500V的UV进行膜的cross link，并用亚甲基蓝染色。剪出目的条带位置的膜，用预杂交液(购自瑞士Rohce)，50℃孵育1h。加入含有Dig标签的RNA探针的杂交液，50℃孵育过夜(12h以上)。2*SSC溶液，60℃洗膜15min，重复两次。1*SSC溶液，50℃洗膜15min，重复两次。0.2*SSC溶液，50℃洗膜15min，重复两次。Northern wash buffer(购自美国Sigma)常温洗5min。Blocking buffer(购自瑞士Roche)常温封闭30min。Anti-Dig-AP的抗体(购自瑞士Roche)常温孵育30min。Northern wash buffer常温洗15min，重复两次。Detection buffer孵育3min。用Dig-CDP star显色液(购自瑞士Roche)显色并曝光。2.1 Isolation and extraction of total RNA from tissues or cells Use TRIZOL reagent (purchased from Invitrogen, USA), and follow the standard operation of the reagent instructions, and detect the concentration of RNA. Add deformer to RNA, mix well, and denature at 70 ℃ for 15min. RNA was added to 1.2% denatured agarose gel well, 1 * MOPS buffer was used as electrophoresis buffer (purchased from Sigma, USA), 100V pre-run for 10min, 45V electrophoresis for 4-5h. The salt bridge was used to transfer the nitrocellulose membrane overnight. 1500V UV cross-links the membrane and stains with methylene blue. The membrane at the position of the band of interest was cut out and incubated with pre-hybridization solution (purchased from Rohce, Switzerland) at 50 ° C for 1 h. The hybridization solution containing Dig-labeled RNA probe was added and incubated at 50 ° C overnight (over 12h). 2 * SSC solution, wash the membrane at 60 ℃ for 15min, repeat twice. 1 * SSC solution, wash the membrane at 50 ℃ for 15min, and repeat twice. 0.2 * SSC solution, wash the membrane at 50 ℃ for 15min, repeat twice. Northern buffer (purchased from Sigma, USA) was washed at room temperature for 5 minutes. Blocking buffer (purchased from Roche, Switzerland) is closed at room temperature for 30 minutes. Anti-Dig-AP antibody (purchased from Roche, Switzerland) was incubated at room temperature for 30 min. Northern wash buffer 15min at room temperature, repeat twice. Detection buffer is incubated for 3 minutes. The color was developed and exposed with Dig-CDP star color developing solution (purchased from Roche, Switzerland).
实验结果Experimental results
GMAN是一个位于染色体1q22位的855nt的长链非编码RNA。我们进一步检测GAMN在人的组织以及人胃癌细胞系中的表达情况。Northern印迹显示，无论从RNA Marker的位置还是从GAPDH的相对位置都显示，GMAN的特异性RNA探针在鉴定的855nt位置能显示出一条比较特异的条带(图2.1)，验证了Race的结果。同时，我们利用TCGA(The Cancer Genome Atlas)数据库中转录组的数据分析GMAN表达情况，GMAN在多种组织中均有表达(图2.2)。GMAN is a 855nt long non-coding RNA located on chromosome 1q22. We further examined the expression of GAMN in human tissues and human gastric cancer cell lines. Northern blotting shows that whether it is from the position of the RNA marker or the relative position of GAPDH, the specific RNA probe of GMAN can show a more specific band at the identified 855nt position (Figure 2.1), verifying the results of Race . At the same time, we used the transcriptome data in the TCGA (The Cancer Genome Atlas) database to analyze the expression of GMAN. GMAN is expressed in a variety of organizations (Figure 2.2).
实施例3：GMAN在胃癌组织中相对高表达并与胃癌病人的转移和不良预后显著相关Example 3: GMAN is relatively highly expressed in gastric cancer tissues and is significantly associated with metastasis and poor prognosis of gastric cancer patients
1.胃癌组织及其配对的正常组织的RNA反转录为cDNA1. Reverse transcription of RNA from gastric cancer tissues and their matched normal tissues to cDNA
组织在极低温度下利用研钵研磨成粉末状，粉末状的组织的总RNA的分离提取利用TRIZOL试剂(购自美国Invitrogen)，并按照试剂说明书的标准操作进行。提取的RNA进行反转录PCR合成cDNA产物。The tissue was ground into a powder at a very low temperature using a mortar. The isolation and extraction of the total RNA of the powdered tissue was carried out using TRIZOL reagent (purchased from Invitrogen, USA), and was carried out according to the standard operation of the reagent instructions. The extracted RNA is subjected to reverse transcription PCR to synthesize cDNA products.
2.实时荧光定量PCR(QRT-PCR)检测GMAN在胃癌组织及其配对的正常组织中的表达情况2. Real-time fluorescence quantitative PCR (QRT-PCR) to detect the expression of GMAN in gastric cancer tissues and their matched normal tissues
根据Taqman probe Kit的说明书(购自日本Takara)并配制PCR反应体系。使用Bio Rad公司CFX-Touch Systerem荧光定量PCR仪进行荧光定量PCR反应。所有的反应都重复三次。根据仪器给出的荧光图得到ΔCt值，，从而计算相 应表达水平的相对变化。引物如下：The PCR reaction system was prepared according to the instruction of Taqman probe Kit (purchased from Takara, Japan). Fluorescence quantitative PCR reaction was performed using BioFX Rad CFX-Touch Systerem fluorescence quantitative PCR instrument. All reactions were repeated three times. Obtain the ΔCt value according to the fluorescence chart given by the instrument, so as to calculate the relative change of the corresponding expression level. The primers are as follows:
Primer NamesPrimer Names Sequences(5'-3')Sequences (5'-3 ')
GMAN ForwardGMANForward CGGAGGAATGAAGGATGAAACGGAGGAATGAAGGATGAAA
GMAN ReverseGMAN Reverse CCTGCTTTCTCAGCTCCCTACCTGCTTTCTCAGCTCCCTA
GMAN probeGMAN probe TGAACATTGGGCAGGAAGTTAGCAAAAACTGAACATTGGGCAGGAAGTTAGCAAAAAC
实验结果Experimental results
通过实时荧光定量PCR反应(qRT-PCR)对111对胃癌组织与其配对的正常组织进行GMAN表达的分析，GMAN在76例(68.5％)胃癌肿瘤组织中相对上调表达(图3.1)。同时，我们也检测了GMAN的表达与111例胃癌患者的临床病理的相关性。结果显示，GMAN的相对高表达与严重的肿瘤侵袭深度(T阶段)，***转移(N阶段)，TNM阶段的进展具有明显相关性(表S2)。此外，40例具有5年生存信息的胃癌患者进行Kaplan-Meier生存曲线分析，引人注目的是，GMAN的高表达与胃癌病人的不良预后显著相关(图3.2)。By real-time fluorescence quantitative PCR reaction (qRT-PCR), 111 pairs of gastric cancer tissues and their matched normal tissues were analyzed for GMAN expression. GMAN was relatively up-regulated in 76 (68.5%) gastric cancer tumor tissues (Figure 3.1). At the same time, we also tested the correlation between the expression of GMAN and the clinicopathology of 111 gastric cancer patients. The results showed that the relatively high expression of GMAN was significantly correlated with the severity of tumor invasion (T stage), lymph node metastasis (N stage), and the progression of TNM stage (Table S2). In addition, Kaplan-Meier survival curve analysis was performed on 40 gastric cancer patients with 5-year survival information. It is striking that the high expression of GMAN is significantly associated with the poor prognosis of gastric cancer patients (Figure 3.2).
进一步分析GMAN的转移的胃癌样本中的表达情况。通过分析cohort 3(n＝11)，cohort 4(n＝13)，TCGA数据库(n＝25)的转移的胃癌病人样本M1与年龄，性别匹配的未发生转移的胃癌病人样本M0中的表达情况，结果显示，GMAN在具有转移的M1样本中显著高表达(图3.3)。这些结果提示，GMAN可能在胃癌的转移过程中发挥重要的作用。Further analysis of the expression of GMAN in metastatic gastric cancer samples. By analyzing the expression of cohort 3 (n = 11), cohort 4 (n = 13), TCGA database (n = 25) metastatic gastric cancer patient sample M1 and age, gender matching non-metastatic gastric cancer patient sample M0 The results showed that GMAN was significantly overexpressed in the M1 samples with metastasis (Figure 3.3). These results suggest that GMAN may play an important role in the metastasis of gastric cancer.
实施例4：敲低GMAN的表达能显著的抑制胃癌细胞的侵袭Example 4: Knocking down the expression of GMAN can significantly inhibit the invasion of gastric cancer cells
1.细胞转染siRNA1. Cell transfection with siRNA
通过siRNA设计软件，设计并合成两条特异靶向GMAN，沉默GMAN表达的siRNA(购自中国上海吉玛)。取对数生长期的细胞铺平板，待细胞密度达到50％左右，分别用OPTI培养基(购自美国Gibco)稀释Lipo RNAi MAX(购自美国Invitrogen)和siRNA，将稀释的siRNA加入Lipo RNAi MAX管中，混匀后静置5min，然后加至细胞培养液中，摇匀，24后更换培养基。Through siRNA design software, design and synthesize two specific siRNAs targeting GMAN and silence GMAN expression (purchased from Gema, Shanghai, China). Plate the cells in the logarithmic growth phase. When the cell density reaches about 50%, dilute Lipo RNAi MAX (purchased from Invitrogen, USA) and siRNA with OPTI medium (purchased from Gibco, USA), and add the diluted siRNA to Lipo RNAi MAX In the tube, mix and let stand for 5min, then add to the cell culture fluid, shake well, and change the medium after 24.
2.细胞增殖(MTT)与克隆形成实验2. Cell proliferation (MTT) and clone formation experiments
转染后的细胞用10％FBS(购自以色列BI)的1640培养基(购自美国Gibco)进行重悬，用血球计数板计数。对于MTT实验，在96孔板中每孔加入3000个细胞(100ul培养基)，每组五个复孔，共需铺4块板用于检测细胞在不同时间点的增殖情况(0h，24h，48h，72h)。在每个时间点，每孔加5mg/ml MTT(购自美国Sigma)在37℃培养箱孵育4h，用枪头小心吸取孔中溶液(注意不要碰到孔的底部)，加入150ul DMSO(购自中国国药)，混匀。在M5酶标仪上检测 各时间点的OD490/OD570的吸收光值并分析。The transfected cells were resuspended in 1640 medium (purchased from Gibco, USA) of 10% FBS (purchased from Israel BI) and counted on a hemocytometer. For the MTT experiment, 3000 cells (100 ul medium) were added to each well in a 96-well plate, and five replicate wells per group were used. A total of 4 plates were needed to detect the proliferation of cells at different time points (0h, 24h, 48h, 72h). At each time point, add 5 mg / ml MTT (purchased from Sigma) to each well and incubate in a 37 ° C incubator for 4 hours. Use the pipette tip to carefully absorb the solution in the well (be careful not to touch the bottom of the well). Add 150ul of DMSO (purchased Since China National Medicine), mix well. The absorbance values of OD490 / OD570 at various time points were detected and analyzed on the M5 microplate reader.
克隆形成实验，在6孔板中每孔加入500个细胞(2mL培养基)，在37℃培养箱培养2周，弃去培养基，PBS温和洗两遍，4％PFA固定10min，0.1％结晶紫染色15min，PBS洗4-5遍，显微镜下进行观察并拍照分析。In the colony formation experiment, 500 cells (2 mL medium) were added to each well in a 6-well plate, and cultured in a 37 ° C incubator for 2 weeks. The medium was discarded, washed twice with PBS, fixed with 4% PFA for 10 min, and 0.1% crystal Violet staining for 15 min, washing with PBS 4-5 times, observation under a microscope and analysis by taking pictures.
3.细胞周期实验3. Cell cycle experiment
1)转染48h后的细胞，弃去培养基，PBS洗两遍，用胰酶(购自美国Thermo Fisher)进行消化。PBS重悬细胞并转移至1.5mL EP管中，800g，4℃离心5min。弃去上清，保留细胞沉淀，用250μL PBS重悬细胞。将250μL细胞重悬液逐滴加入到750μL的95％乙醇(购自中国国药)，4℃固定2h。将固定后的细胞进行离心，800g，4℃离心5min。弃去上清，500μL PBS重悬细胞，800g，4℃离心5min。弃去上清，500μL PBS重悬细胞，将细胞悬液通过细胞筛过滤，在过滤后的细胞悬液中加入PI染料(购自美国Solarbio)和RNase A(购自美国Thermo Fisher)，37℃水浴孵育30min。用流式细胞仪(美国Beckman)检测各处理组细胞的细胞周期分布。1) After 48 hours of transfection, cells were discarded, washed twice with PBS, and digested with trypsin (purchased from Thermo Fisher, USA). Resuspend the cells in PBS and transfer to 1.5mL EP tube, 800g, centrifuge at 4 ℃ for 5min. Discard the supernatant, retain the cell pellet, and resuspend the cells in 250 μL of PBS. 250 μL of cell resuspension was added dropwise to 750 μL of 95% ethanol (purchased from Sinopharm), and fixed at 4 ° C for 2 h. The fixed cells were centrifuged at 800g and centrifuged at 4 ° C for 5min. Discard the supernatant, resuspend the cells in 500 μL PBS, 800g, and centrifuge at 4 ° C for 5 min. Discard the supernatant, resuspend the cells in 500 μL PBS, filter the cell suspension through a cell sieve, add PI dye (purchased from Solarbio, USA) and RNase A (purchased from Thermo Fisher, USA) to the filtered cell suspension, 37 ° C Incubate for 30 minutes in a water bath. The cell cycle distribution of cells in each treatment group was detected by flow cytometry (Beckman, USA).
4.细胞粘附实验4. Cell adhesion experiment
取96-well板进行预处理。Matrigel(购自美国BD)包被的细胞板，用预冷的无FBS的1640(购自美国Gibco)对matrigel进行1:40的稀释，每孔加入50μL进行包被；Fibronectin(购自美国BD)包被的细胞板，用预冷的无FBS的1640稀释fibronectin至0.02μg/μL，每孔加入50μL进行包被。将matrigel和fibronectin包被的96-well板放入37℃细胞培养箱中孵育24h。吸去未凝固的matrigel和fibronectin，每孔加入100μL，0.5％BSA(购自中国上海生工)(PBS溶)，37℃封闭30min。弃去BSA，PBS温和洗两遍，每孔加入100μL，2x104的1％FBS的1640培养基重悬的细胞，37℃培养箱培养30min。弃去上清，PBS温和洗两遍，每孔加100μL，4％PFA固定30min，弃去PFA(中国国药)，PBS温和洗两遍。每孔加100μL亚甲基蓝染色30min，弃去亚甲基蓝染色液，用PBS温和洗4-5次。配制裂解液(无水乙醇与0.1M HCl体积比1:1混合)，每孔加入150μL裂解液，混匀。并在无细胞的孔中加入150μL裂解液作为空白对照。在M5酶标仪上检测OD620的吸收光值并分析。Take a 96-well plate for pretreatment. Matrigel (purchased from US BD) coated cell plates, diluted 1:40 with pre-chilled FBS-free 1640 (purchased from Gibco), and coated with 50 μL per well; Fibronectin (purchased from BD) ) Coated cell plates, dilute fibronectin to 0.02 μg / μL with pre-chilled FBS-free 1640, and add 50 μL per well for coating. The 96-well plates coated with matrigel and fibronectin were placed in a 37 ° C cell incubator for 24 hours. Aspirate unsolidified matrigel and fibronectin, add 100 μL of each well, 0.5% BSA (purchased from Shanghai, China) (PBS solution), and block at 37 ° C for 30 min. Discard BSA, wash twice with PBS gently, add 100 μL of each well to resuspend the cells in 1640 medium of 2x104 1% FBS, and incubate for 30 min in a 37 ° C incubator. Discard the supernatant, wash twice with PBS gently, add 100 μL per well, fix with 4% PFA for 30 min, discard PFA (China National Medicine), wash twice with PBS gently. Add 100 μL of methylene blue to each well for 30 min, discard the methylene blue staining solution, and wash gently with PBS 4-5 times. Prepare lysis solution (mixture of absolute ethanol and 0.1M HCl volume ratio of 1: 1), add 150μL of lysis solution to each well and mix well. Add 150 μL of lysate to the cell-free wells as a blank control. The absorbance value of OD620 was detected and analyzed on the M5 microplate reader.
5.细胞侵袭实验5. Cell invasion experiment
在8μm的Transwell(24-well，购自美国Corning)中预先铺上10倍稀释的基质胶(购自美国BD)和0.5％的明胶(购自中国上海生工)按照体积比1:1的混合胶50ul，37℃细胞培养箱孵育2h。转染后的细胞按照细胞传代的方式进行重悬(1％FBS的1640培养基重悬细胞)并用血球计数板进行细胞计数。在 transwell的上层小室中每孔中加入200ul细胞悬液(细胞数目：5×10 4)，在下室中加入700ul的10％FBS的1640培养基，放入培养箱中。待细胞迁移适当时间后，取出transwelll并用4％PFA固定10min。0.1％的结晶紫染色15min。PBS清洗掉多余的结晶紫，用棉签小心的擦去transwell小室上层膜的细胞，在显微镜下观察侵袭到transwell下层膜上的细胞并进行拍照和统计分析。 8-μm Transwell (24-well, purchased from Corning, USA) was pre-laid with 10 times diluted matrix glue (purchased from BD, USA) and 0.5% gelatin (purchased from Shanghai, China) according to a volume ratio of 1: 1 Mix 50ul of glue and incubate in a 37 ° C cell incubator for 2h. The transfected cells were resuspended in the manner of cell passage (140 FBS in 1640 medium to resuspend the cells) and the cell count was performed with a hemacytometer. Add 200 ul of cell suspension (cell number: 5 × 10 4 ) to each well in the upper chamber of transwell, add 700 ul of 1040 FBS 1640 medium in the lower chamber, and place it in the incubator. After the cells migrated for an appropriate time, remove transwelll and fix with 4% PFA for 10 min. Stain with 0.1% crystal violet for 15 min. PBS washes away excess crystal violet, and carefully wiped the cells of the upper membrane of the transwell chamber with a cotton swab. The cells invading the lower membrane of the transwell were observed under a microscope and photographed and statistically analyzed.
实验结果Experimental results
临床病理分析提示，GMAN的表达与胃癌的发生发展密切相关。那么，GMAN在胃癌的进程中，特别是胃癌的转移过程中是如何发挥作用的？这个重要的问题引起了我们极大的兴趣。由于GMAN在胃癌组织中相对高表达，而且GMAN在大部分胃癌细胞系中也表达丰富。我们设计了两个特异靶向GMAN，沉默GMAN表达的siRNA。在胃癌细胞系BGC823中转染GMAN的siRNA，敲低GMAN的表达，并通过Northern印迹检测GMAN的敲低效率，结果显示与对照组的siRNA相比较，特异性靶向GMAN的siRNA下调GMAN的水平至对照组的30％左右(图4.1)。在胃癌细胞系BGC823上检测敲低GMAN对胃癌细胞系的影响。Clinical pathological analysis suggests that the expression of GMAN is closely related to the occurrence and development of gastric cancer. So, how does GMAN play a role in the process of gastric cancer, especially the metastasis of gastric cancer? This important issue has aroused our great interest. Because GMAN is relatively highly expressed in gastric cancer tissues, and GMAN is also abundantly expressed in most gastric cancer cell lines. We designed two siRNAs that specifically target GMAN and silence GMAN expression. Transfection of GMAN siRNA in gastric cancer cell line BGC823, knocked down the expression of GMAN, and detected the knockdown efficiency of GMAN by Northern blotting. The results showed that compared with the siRNA of the control group, the siRNA specifically targeting GMAN downregulated the level of GMAN To about 30% of the control group (Figure 4.1). The effect of knocking down GMAN on gastric cancer cell line was detected on gastric cancer cell line BGC823.
由于肿瘤细胞的两个重要特征是能够无限增殖和容易转移。我们通过MTT实验和克隆形成实验检测GMAN对胃癌细胞的细胞增殖能力的影响，结果显示，GMAN的敲低对胃癌细胞的细胞增殖和克隆形成能力没有明显影响(图4.2)。进一步的细胞周期实验也显示，GMAN的下调几乎不影响胃癌细胞的细胞周期(图4.3)。通过细胞侵袭实验和细胞粘附实验检测GMAN对肿瘤转移方面的影响。细胞粘附实验显示，GMAN的敲低对胃癌细胞与细胞外基质的粘附能力没有明显影响(图4.4)。但是，GMAN的敲低能够显著的抑制胃癌细胞的细胞侵袭能力(图4.5)。提示，GMAN可能通过影响胃癌细胞的侵袭能力影响胃癌的发生与发展。Because the two important characteristics of tumor cells are the ability to proliferate indefinitely and easily metastasize. We examined the effect of GMAN on the cell proliferation ability of gastric cancer cells through MTT experiments and clone formation experiments. The results showed that GMAN knockdown had no significant effect on the cell proliferation and clone formation ability of gastric cancer cells (Figure 4.2). Further cell cycle experiments also showed that the down-regulation of GMAN hardly affected the cell cycle of gastric cancer cells (Figure 4.3). The effects of GMAN on tumor metastasis were detected by cell invasion experiments and cell adhesion experiments. Cell adhesion experiments showed that GMAN knockdown had no significant effect on the ability of gastric cancer cells to adhere to the extracellular matrix (Figure 4.4). However, GMAN knockdown can significantly inhibit the cell invasion ability of gastric cancer cells (Figure 4.5). It is suggested that GMAN may affect the occurrence and development of gastric cancer by affecting the invasion ability of gastric cancer cells.
实施例5：过量表达GMAN能显著的促进胃癌细胞的侵袭Example 5: Overexpression of GMAN can significantly promote the invasion of gastric cancer cells
根据Race的结果获得GMAN的全长序列，设计GMAN的引物，通过PCR的方法获得GMAN的DNA产物，经过酶切，链接等步骤构建含有GMAN全长的pcDNA3.1过表达质粒。取对数生长期的细胞铺平板，待细胞密度达到80％左右，按照下表转染体系，分别用OPTI培养基稀释Lipo3000(购自美国Invitrogen)和质粒，将稀释的质粒加入Lipo3000管中，混匀后静置5min，然后加至细胞培养液中，摇匀，24后更换培养基。According to the results of Race, the full-length sequence of GMAN was obtained, the primers of GMAN were designed, and the DNA product of GMAN was obtained by PCR. After digestion, linking and other steps, the pcDNA3.1 overexpression plasmid containing the full length of GMAN was constructed. Plate the cells in the logarithmic growth phase until the cell density reaches about 80%. According to the transfection system in the table below, dilute Lipo3000 (purchased from Invitrogen, USA) and plasmids with OPTI medium, and add the diluted plasmids to Lipo3000 tubes. After mixing, let stand for 5 minutes, then add to the cell culture fluid, shake well, and change the medium after 24.
实验结果Experimental results
在GMAN含量相对丰富的BGC823细胞中敲低GMAN的表达能够显著的 抑制胃癌细胞的侵袭能力。那么，过量表达GMAN对胃癌细胞又会有如何的影响？我们挑选了一株GMAN表达相对较少的细胞HGC27，检测在人胃癌细胞系HGC27细胞中过量表达GMAN对胃癌细胞的生物学功能的影响。Northern印迹检测GMAN的过表达效率，与作为对照的空载质粒相比，GMAN过量表达的效果是其表达的3-5倍(图5.1)。研究结果显示，在HGC27细胞中过量表达GMAN，对胃癌细胞的细胞增殖，克隆形成，细胞周期和细胞粘附(图5.2-5.4)没有明显影响，但是能显著的促进胃癌细胞的侵袭(图5.5)。这些数据与GMAN敲低的数据结果是一致的，显示GMAN调控胃癌细胞的侵袭。Knocking down the expression of GMAN in BGC823 cells with relatively rich GMAN content can significantly inhibit the invasion ability of gastric cancer cells. So, how does overexpression of GMAN affect gastric cancer cells? We selected a cell HGC27 with relatively low GMAN expression, and examined the effect of overexpression of GMAN on the biological function of gastric cancer cells in human gastric cancer cell line HGC27. Northern blot was used to detect the overexpression efficiency of GMAN. Compared with the empty plasmid as a control, GMAN overexpression effect was 3-5 times that of its expression (Figure 5.1). The results show that overexpression of GMAN in HGC27 cells has no significant effect on cell proliferation, clonal formation, cell cycle and cell adhesion (Figure 5.2-5.4) of gastric cancer cells, but it can significantly promote the invasion of gastric cancer cells (Figure 5.5 ). These data are consistent with the results of GMAN knockdown data, showing that GMAN regulates the invasion of gastric cancer cells.
实施例6：敲低GMAN能显著的抑制胃癌细胞的转移Example 6: Knockdown of GMAN can significantly inhibit the metastasis of gastric cancer cells
1.慢病毒载体构建1. Lentiviral vector construction
根据shRNA设计原则，以GMAN RNA为靶序列，设计了一条针对GMAN的靶位点序列，合成相对应的正反向序列(购自中国上海生工)，这些序列接头设计退火后形成Bam H Ⅰ和EcoR Ⅰ的粘性末端。经过酶切，链接，鉴定，测序等步骤构建GMAN的shRNA慢病毒表达载体。靶点序列分别为：According to the shRNA design principles, using GMAN RNA as the target sequence, a target site sequence for GMAN was designed to synthesize the corresponding forward and reverse sequences (purchased from Shanghai Shengong, China). These sequence joints were designed and annealed to form Bam H I And EcoR I sticky end. After digestion, linking, identification, sequencing and other steps to construct GMAN shRNA lentiviral expression vector. The target sequences are:
GMAN sense：GMANsense:
5’-GATCCGGCTTATCTTGCAGCCAAATTCAAGAGATTTGGCTGCAAGATAAGCCTTTTTTG-3’5’-GATCCGGCTTATCTTGCAGCCAAATTCAAGAGATTTGGCTGCAAGATAAGCCTTTTTTG-3 ’
GMAN antisense：GMAN antisense:
5’-AATTCAAAAAAGGCTTATCTTGCAGCCAAATCTCTTGAATTTGGCTGCAAGATAAGCCG-3'5’-AATTCAAAAAAGGCTTATCTTGCAGCCAAATCTCTTGAATTTGGCTGCAAGATAAGCCG-3 '
2.慢病毒包装2. Lentivirus packaging
将HEK293T细胞铺入T25瓶中，待细胞密度达到80-90％时，进行转染。取15μL Lippo3000混入250ul OPTI中(具体转染方法详见质粒转染步骤)。收集48h，72h的细胞培液，过滤分装后，浓缩，测滴度，-80℃冰箱中保存。Spread HEK293T cells into T25 flasks and transfect when the cell density reaches 80-90%. Mix 15μL Lippo3000 into 250ul OPTI (see the plasmid transfection step for the specific transfection method). Collect 48h and 72h cell culture fluid, filter and aliquot, concentrate, measure titer, and store in -80 ℃ refrigerator.
3.筛选GMAN敲低的稳定细胞系3. Screening of stable cell lines knocked down by GMAN
取对数生长期的细胞铺平板，待细胞密度达到80％左右，换成新鲜的10％FBS的1640培养基。加入适量的病毒原液，并加入5ug/mL的Polybrene促进病毒的感染效率，后续可通过荧光显微镜观察细胞带有GFP的效率说明慢病毒的感染效率。同时，通过向细胞施加puromycin药物，筛选慢病毒表达的细胞，QRT-PCR检测GMAN的敲低效果。The cells in the logarithmic growth phase were plated, and when the cell density reached about 80%, they were replaced with fresh 1040 FBS 1640 medium. Add the appropriate amount of virus stock solution and add 5ug / mL Polybrene to promote the infection efficiency of the virus. The efficiency of lentivirus infection can be demonstrated by observing the efficiency of the cells carrying GFP through a fluorescence microscope. At the same time, by applying puromycin drugs to the cells to screen the cells expressing the lentivirus, QRT-PCR was used to detect the knockdown effect of GMAN.
4.裸鼠肺转移实验4. Nude mouse lung metastasis experiment
10 6的带有GFP标记的慢病毒稳定转染的胃癌细胞尾静脉注射到裸鼠的体内，每周观察小鼠的状态及体重变化，5-6周后，解剖观察肺转移情况。将肺组 织固定、石蜡包埋、切片，后续进行苏木精伊红染色，或者ephrin A1抗体染色。 Tagged with GFP lentivirus 106 stably transfected gastric cancer cells injected intravenously to nude mice in vivo, condition and body weight change in mice were observed weekly, 5-6 weeks, dissected lung metastasis was observed. The lung tissue was fixed, embedded in paraffin, sectioned, and then stained with hematoxylin-eosin or ephrin A1 antibody.
实验结果Experimental results
GMAN能显著的促进胃癌细胞的侵袭。GMAN的表达与临床病理相关性的分析也显示，GMAN的高表达与胃癌病人的肿瘤侵袭深度(T阶段)，***转移(N阶段)，TNM阶段的进展具有明显相关性。而且与年龄，性别匹配的M0期胃癌组织相比较，GMAN在具有转移的M1期胃癌组织中显著高表达。这些数据都提示，GMAN可能在胃癌的转移中发挥重要的作用。我们构建GMAN敲低的稳定细胞系做裸鼠的肺转移实验。把对照组和GMAN敲低的稳定细胞系尾静脉注射裸鼠，建立肺转移模型。由于细胞带有GFP，我们利用小鼠成像仪检测裸鼠肺部的GFP信号来体现胃癌细胞的转移情况，显示GMAN敲低组的裸鼠GFP信号明显减弱，提示GMAN敲低组的胃癌细胞的肺转移能力受到抑制。无论是HE染色还是肉眼观察裸鼠肺部的转移灶，都显示GMAN敲低组的胃癌细胞产生的转移灶数目和转移灶大小都显著减弱(图6.1)。GMAN敲低能显著的抑制胃癌细胞的体外侵袭和体内肺转移能力，这与GMAN的临床病理相关性分析一致。GMAN can significantly promote the invasion of gastric cancer cells. The analysis of the correlation between the expression of GMAN and clinicopathology also shows that the high expression of GMAN is significantly correlated with the depth of tumor invasion (stage T), lymph node metastasis (stage N), and the progression of TNM stage in gastric cancer patients. Moreover, compared with age and sex-matched M0 gastric cancer tissues, GMAN is significantly overexpressed in metastatic M1 gastric cancer tissues. These data suggest that GMAN may play an important role in the metastasis of gastric cancer. We constructed a stable cell line with GMAN knockdown for lung metastasis experiments in nude mice. Nude mice were injected into the tail vein of the control group and the stable cell line knocked down by GMAN to establish a lung metastasis model. Because the cells carry GFP, we used a mouse imager to detect the GFP signal in the lungs of nude mice to reflect the metastasis of gastric cancer cells, showing that the GFP signal of nude mice in the GMAN knockdown group was significantly weakened, suggesting that the GMAN knockdown group of gastric cancer Lung metastasis ability is inhibited. Both HE staining and visual observation of metastases in the lungs of nude mice showed that the number and size of metastases produced by gastric cancer cells in the GMAN knockdown group were significantly reduced (Figure 6.1). GMAN knockdown can significantly inhibit the invasion of gastric cancer cells in vitro and the ability of lung metastasis in vivo, which is consistent with the clinical and pathological correlation analysis of GMAN.
实施例7：GMAN主要通过MFR区段发挥抑制胃癌细胞侵袭的功能Example 7: GMAN mainly exerts the function of inhibiting the invasion of gastric cancer cells through the MFR segment
1.靶向GMAN的CRISPR/Cas9基因编辑1. CRISPR / Cas9 gene editing targeting GMAN
CRISPR/Cas9基因编辑靶向蛋白编码基因，可通过改变编码基因的几个碱基引起移码突变等效应，敲除编码的蛋白的表达。与用于敲除蛋白表达的方法不同，对于长链非编码RNA的敲除，仅仅改变几个碱基很可能不能改变lncRNA的功能，需大片段的截短，改变RNA的二级结构或破坏主要作用区域，才能敲除lncRNA的表达。CRISPR / Cas9 gene editing targets protein-coding genes, which can cause frameshift mutations and other effects by changing a few bases of the coding gene, knocking out the expression of the encoded protein. Unlike the method used for knockout protein expression, for long-chain non-coding RNA knockout, only changing a few bases may not change the function of lncRNA, requiring truncation of large fragments, changing the secondary structure of RNA or destroying it The main area of action is to knock out the expression of lncRNA.
设计并合成两个特异性靶向GMAN的sgRNA，构建CRISPR/Cas9-GMAN sgRNA的载体。序列如下：Design and synthesize two sgRNAs that specifically target GMAN, and construct a CRISPR / Cas9-GMAN sgRNA vector. The sequence is as follows:
GMAN sgRNA 1GMAN sgRNA1
GMAN-1-sense：5’-CACCGGAGTAGTATTAAGTGGCCC-3’GMAN-1-sense: 5’-CACCGGAGTAGTATTAAGTGGCCC-3 ’
GMAN-1-antisense：5’-AAACGGGCCACTTAATACTACTCC-3’GMAN-1-antisense: 5’-AAACGGGCCACTTAATACTACTCC-3 ’
GMAN sgRNA 2 GMAN sgRNA 2
GMAN-2-sense：5’-CACCGTTTCTTATTTAACCCCTGT-3’GMAN-2-sense: 5’-CACCGTTTCTTATTTAACCCCTGT-3 ’
GAMN-2-antisense：5’-AAACACAGGGGTTAAATAAGAAAC-3’GAMN-2-antisense: 5’-AAACACAGGGGTTAAATAAGAAAC-3 ’
按照质粒转染的方法，把两个带有GMAN sgRNA的CRISPR/Cas9载体共转染人胃癌细胞系，通过单克隆筛选，并且利用基因组PCR及测序、cDNA的PCR及测序等方法验证鉴定出敲除GMAN的MFR区段的突变细胞株。另一方 面，为避免CRISPR/Cas9产生的脱靶效益，我们除了设计序列特异的sgRNA，同时根据sgRNA预测的潜在脱靶位点，进行PCR及测序验证，保证没有出现脱靶。According to the method of plasmid transfection, two CRISPR / Cas9 vectors with GMAN sgRNA were co-transfected into human gastric cancer cell lines, through monoclonal screening, and using genomic PCR and sequencing, cDNA PCR and sequencing to verify and identify knocks Mutant cell lines except for the MFR segment of GMAN. On the other hand, in order to avoid the off-target benefits generated by CRISPR / Cas9, in addition to designing sequence-specific sgRNA, we also performed PCR and sequencing verification based on the potential off-target sites predicted by sgRNA to ensure that no off-target occurred.
实验结果Experimental results
以上的研究显示GMAN能显著的促进胃癌细胞的侵袭和转移。我们想进一步探索GMAN主要发挥作用的区域。在NCBI的Blast工具中分析GMAN的全长序列的特征显示，GMAN的5’端约400nt的序列是非特异的，许多其他的基因或者lncRNA的部分序列是与这段约400nt的核苷酸是类似的。GMAN的3’端不到100nt的序列是与EphrinA1的Exon3和部分Exon4是相同的。但是GMAN的中间段(暂称为MFR)的约300nt的序列是GMAN特异的序列，可以说GMAN的MFR区段是GMAN特有的序列(表S3)，提示GMAN是不是通过这段特异的序列发挥功能。于是，我们利用CRISPR/Cas9基因编辑技术设计两个靶向GMAN的特异的sgRNA，通过单克隆筛选，测序鉴定以及脱靶效应鉴定，成功建立敲除GMAN的MFR区段的胃癌细胞突变株(ΔMFR)(图7.1)。检测ΔMFR的胃癌细胞突变株的细胞生物学功能发现，与正常的野生型胃癌细胞相比较，GMAN的MFR区段缺失的胃癌突变株的细胞增殖，克隆形成，细胞周期和细胞粘附(图7.2-7.4)几乎不受影响。但是GMAN的MFR区段缺失的胃癌细胞突变株的细胞侵袭能力明显受到抑制(图7.5)。这些数据显示GMAN的MFR区段缺失的细胞生物学功能与GMAN敲低引起的细胞生物学功能的变化是高度一致的，显示GMAN的MFR区段是GMAN主要发挥作用的区域。同时，也说明我们利用CRISPR/Cas9技术建立的细胞株是成功敲除GMAN的突变株。The above research shows that GMAN can significantly promote the invasion and metastasis of gastric cancer cells. We want to further explore the areas where GMAN plays a major role. Analysis of the characteristics of the full-length sequence of GMAN in NCBI's Blast tool shows that the sequence of about 400 nt at the 5 'end of GMAN is non-specific, and many other genes or partial sequences of lncRNA are similar to the nucleotides of about 400 nt. of. The sequence of less than 100 nt at the 3 'end of GMAN is the same as Exon3 and part of Exon4 of EphrinA1. However, the sequence of about 300 nt in the middle section of GMAN (provisionally called MFR) is a GMAN-specific sequence. It can be said that the MFR section of GMAN is a GMAN-specific sequence (Table S3), prompting whether GMAN is played through this specific sequence Features. Therefore, we used CRISPR / Cas9 gene editing technology to design two specific sgRNAs targeting GMAN. Through monoclonal screening, sequencing identification and off-target effect identification, we successfully established a gastric cancer mutant strain (ΔMFR) that knocked out the MFR segment of GMAN (Figure 7.1). Detection of the cell biological function of the gastric cancer mutant strain of ΔMFR revealed that, compared with normal wild-type gastric cancer cells, the gastric cancer mutant strain with the MFR segment of GMAN deleted had cell proliferation, clone formation, cell cycle and cell adhesion (Figure 7.2 -7.4) hardly affected. However, the cell invasion ability of gastric cancer mutant strains with deletion of the MFR segment of GMAN was significantly inhibited (Figure 7.5). These data show that the cell biological function of the deletion of the MFR segment of GMAN is highly consistent with the change of the cell biological function caused by GMAN knockdown, showing that the MFR segment of GMAN is the region where GMAN mainly plays a role. At the same time, it also shows that the cell line we established using CRISPR / Cas9 technology is a mutant strain that successfully knocked out GMAN.
实施例8：MFR缺失能显著的抑制胃癌细胞的转移Example 8: MFR deletion can significantly inhibit the metastasis of gastric cancer cells
产生胃癌细胞肺转移的裸鼠的预后分析：把野生型的胃癌细胞和ΔMFR的胃癌细胞突变株通过尾静脉注射建立裸鼠的肺转移模型。实时观测裸鼠的生活状态并记录两组裸鼠的存活时间，精确到天数。利用Kaplan-Meier生存曲线分析，绘制野生型胃癌细胞产生肺转移的裸鼠和ΔMFR的胃癌突变株产生肺转移的裸鼠的生存曲线。Prognostic analysis of nude mice producing lung metastases from gastric cancer cells: Wild-type gastric cancer cells and mutant gastric cancer cell lines with ΔMFR were injected through the tail vein to establish nude mice lung metastasis models. Observe the living conditions of nude mice in real time and record the survival time of two groups of nude mice, accurate to days. Using Kaplan-Meier survival curve analysis, the survival curves of nude mice with lung metastasis from wild-type gastric cancer cells and lung metastases from gastric cancer mutant strains of ΔMFR were drawn.
实验结果Experimental results
研究显示GMAN主要通过MFR区段发挥促进胃癌细胞侵袭和转移的功能。利用CRISPR/Cas9基因编辑技术靶向GMAN的MFR区段，获得GMAN的主要作用区域MFR区段的大片段缺失的GMAN突变的胃癌细胞株。由于GMAN的敲低能显著的抑制胃癌细胞的侵袭和转移。那么，GMAN突变是不是会有类似的功能呢？我们把构建的ΔMFR的胃癌细胞突变株通过尾静脉注射建立裸鼠 的肺转移模型，研究MFR缺失对胃癌细胞转移的影响。裸鼠接种胃癌细胞5周后，评估裸鼠的肺转移情况。通过裸鼠解剖取肺观察显示，对照组野生型的胃癌细胞发展严重肺转移，肺的体积显著增大，肺的重量也明显增重，肺部含有大量的肿瘤细胞，形成实质的瘤块，几乎观察不到完整的肺泡结构；而ΔMFR组裸鼠的肺部几乎与正常的小鼠的肺部一样，呈现白色并具有相对完整的肺部结构(图8.1)。进一步的肺部HE染色也显示，对照组的肺部产生许多大的转移灶，但是ΔMFR组的肺部只观察到少许的微转移灶(图8.2)。同时，接种胃癌细胞的裸鼠，我们每周观察裸鼠的生长状态，并测量裸鼠的体重显示，对ΔMFR组的小鼠相比，对照组的裸鼠由于产生严重的肺转移，进而危害小鼠的健康状态，生活状态每况愈下，使小鼠在转移的后期体重明显下降(图8.3)。研究显示，UC-Mut胃癌细胞能显著降低胃癌的肺转移能力。另外，我们也做一组独立平行实验，检测GMAN对肺转移的裸鼠的生存的影响。把野生型胃癌细胞和ΔMFR的胃癌细胞尾静脉注射裸鼠后，实时观察裸鼠的生存状况，并记录每只老鼠的生存寿命。结果显示，对照组裸鼠在接种后35天开始出现死亡，到43天对照组的8只裸鼠全部死亡。但是我们观察到，ΔMFR组的裸鼠在接种后的60天内没有出现死亡。提示，与用对照组的小鼠相比，注射ΔMFR细胞的小鼠显示出优异的生活质量和更长的总存活率(图8.4)。这些分析表明，MFR缺失能够显著抑制胃癌细胞的肺转移。Studies have shown that GMAN mainly promotes the invasion and metastasis of gastric cancer cells through the MFR segment. Using CRISPR / Cas9 gene editing technology to target the MFR segment of GMAN, a GMAN-mutated gastric cancer cell line with a large deletion of the MFR segment in the main action region of GMAN was obtained. Because GMAN knockdown can significantly inhibit the invasion and metastasis of gastric cancer cells. So, does the GMAN mutation have a similar function? We constructed a gastric cancer cell mutant strain of ΔMFR through tail vein injection to establish a lung metastasis model in nude mice, and studied the effect of MFR deletion on gastric cancer cell metastasis. Five weeks after the nude mice were inoculated with gastric cancer cells, the lung metastasis of the nude mice was evaluated. Observation of the lungs by dissection of nude mice showed that wild-type gastric cancer cells in the control group developed severe lung metastases, the volume of the lungs increased significantly, and the weight of the lungs also increased significantly. The lungs contained a large number of tumor cells, forming substantial tumor masses. Almost no complete alveolar structure was observed; the lungs of nude mice in the ΔMFR group were almost the same as those of normal mice, appearing white and having relatively complete lung structures (Figure 8.1). Further HE staining of the lungs also showed that the lungs of the control group produced many large metastases, but only a few micrometastases were observed in the lungs of the ΔMFR group (Figure 8.2). At the same time, in nude mice inoculated with gastric cancer cells, we observe the growth status of nude mice every week and measure the body weight of nude mice. Compared with the mice in the ΔMFR group, the nude mice in the control group have serious lung metastasis, which is harmful. The health and living conditions of the mice deteriorated gradually, causing the mice to lose weight significantly in the later stages of transfer (Figure 8.3). Studies have shown that UC-Mut gastric cancer cells can significantly reduce the lung metastatic ability of gastric cancer. In addition, we also conducted a set of independent parallel experiments to examine the effect of GMAN on the survival of nude mice with lung metastasis. After injecting wild-type gastric cancer cells and ΔMFR gastric cancer tail veins into nude mice, the survival status of nude mice was observed in real time, and the survival life of each mouse was recorded. The results showed that the nude mice of the control group began to die 35 days after the inoculation, and all 43 nude mice of the control group died by 43 days. However, we observed that the nude mice in the ΔMFR group did not die within 60 days after vaccination. It is suggested that the mice injected with ΔMFR cells showed superior quality of life and a longer overall survival rate compared to the mice with the control group (Figure 8.4). These analyses indicate that MFR deletion can significantly inhibit lung metastasis of gastric cancer cells.
实施例9：GMAN的MFR区段具有治疗胃癌转移的潜能Example 9: The MFR segment of GMAN has the potential to treat gastric cancer metastasis
体内治疗实验：对于靶向GMAN的CRISPR/Cas9治疗测定，将10 6个荧光素酶标记的SGC7901细胞(S胃癌-Luc)尾静脉注射到SCID小鼠(10只小鼠)中。接种后一天，将动物随机分配到两组，进行4周治疗。实验组尾静脉注射包裹有靶向GMAN的CRISPR/Cas9(CRISPR-GMAN)载体(每只小鼠2.5μg)的脂质体递送缓冲液(成分：用于体内的脂质体与10％葡萄糖溶液按照质量比为1:14混匀)，对照组为不含GMAN的CRISPR/Cas9载体的脂质体递送缓冲液，每周注射两次。每周通过BLI使用Xenogen IVIS 200成像***监测SCID老鼠活体中胃癌细胞的luciferase信号，观察胃癌细胞的肺转移情况，并进行统计分析。 Vivo therapy experiments: For the targeting GMAN CRISPR / Cas9 therapeutic assay, 106 of luciferase-labeled cells SGC7901 (S gastric -Luc) tail vein injection into SCID mice (10 mice). One day after the inoculation, the animals were randomly assigned to two groups for 4 weeks of treatment. The experimental group was injected with a liposome delivery buffer wrapped with GMAN-targeted CRISPR / Cas9 (CRISPR-GMAN) vector (2.5 μg per mouse) via tail vein (ingredients: liposomes used in vivo and 10% glucose solution (Mix according to the mass ratio of 1:14), the control group is the liposome delivery buffer without GMAN CRISPR / Cas9 vector, injected twice a week. The Xenogen IVIS 200 imaging system was used to monitor the luciferase signal of gastric cancer cells in vivo in SCID mice every week through BLI, observe the lung metastasis of gastric cancer cells, and perform statistical analysis.
同时，为了排除CRISPR-GMAN递送***相关的细胞毒性抑制SGC-Luc胃癌肺转移的可能性，我们使用体内剂量以及高于体内剂量5倍的药物处理胃癌细胞系，评估CRISPR-GMAN治疗对SGC-Luc细胞活力的影响。At the same time, in order to rule out the possibility of cytotoxicity related to CRISPR-GMAN delivery system inhibiting lung metastasis of SGC-Luc gastric cancer, we used in vivo doses and drugs that were 5 times higher than the in vivo dose to treat gastric cancer cell lines, and evaluated CRISPR-GMAN treatment for SGC- Effect of Luc cell viability.
实验结果Experimental results
GMAN的MFR区段缺失的胃癌细胞能显著的抑制胃癌细胞的转移，而且 CRISPR/Cas9是具有广泛治疗潜力的多功能基因组编辑工具。GMAN的MFR区段对GMAN的功能发挥起到至关重要的作用，GMAN的MFR区段位于基因组的内含子区域，是GMAN的特有序列，所以我们合理的设想GMAN的MFR区段可作为重要的抗转移效应的内含子靶位点，利用CRISPR/Cas9介导的治疗方法靶向GMAN的MFR区段可能是一种有前景的抗胃癌策略。Gastric cancer cells with missing MFR segments of GMAN can significantly inhibit the metastasis of gastric cancer cells, and CRISPR / Cas9 is a multifunctional genome editing tool with broad therapeutic potential. The MFR segment of GMAN plays a vital role in the function of GMAN. The MFR segment of GMAN is located in the intron region of the genome and is a unique sequence of GMAN. Therefore, we reasonably assume that the MFR segment of GMAN can be regarded as important Intron target sites for anti-metastatic effects, using CRISPR / Cas9-mediated therapy to target the MFR segment of GMAN may be a promising anti-gastric cancer strategy.
为了检测GMAN的MFR区段的抗肿瘤治疗的效应，我们合理的设计了靶向GMAN的MFR区段的体内抗肿瘤转移的治疗实验并绘制了治疗实验的示意图(图9.1)。我们将荧光素酶标记的SGC7901胃癌细胞(S胃癌-Luc)尾静脉内注射到SCID小鼠中。接种后一天，将动物随机分配到两组，进行4周的治疗。实验组尾静脉注射包裹有靶向GMAN的CRISPR/Cas9(CRISPR-GMAN)载体的脂质体递送缓冲液，对照组为不含GMAN的CRISPR/Cas9载体的脂质体递送缓冲液，每周进行两次注射。利用BLI以监测肺转移并评估CRISPR/Cas9递送的治疗功效。SGC-Luc细胞在对照递送缓冲液处理的动物中产生严重的肺转移，但CRISPR/Cas9靶向GMAN的MFR区段处理的药物递送后一周内显著阻断SGC-Luc细胞转移到肺中。CRISPR/Cas9靶向GMAN的MFR区段处理的药物连续的，每周两次的治疗增强了对SGC-Luc肺转移性生长的抑制作用。最终的组织学分析证实，与对照递送治疗的动物相比，在CRISPR/Cas9靶向GMAN的MFR区段递送治疗的动物的肺转移情况受到显著的抑制，转移性肺损伤显著减少(图9.2)。In order to detect the effect of anti-tumor therapy of the MFR segment of GMAN, we rationally designed an in vivo anti-tumor metastasis treatment experiment targeting the MFR segment of GMAN and drawn a schematic diagram of the treatment experiment (Figure 9.1). We injected luciferase-labeled SGC7901 gastric cancer cells (S gastric cancer-Luc) into SCID mice via tail vein. One day after the inoculation, the animals were randomly assigned to two groups and treated for 4 weeks. The experimental group was injected with a liposome delivery buffer coated with GMAN-targeted CRISPR / Cas9 (CRISPR-GMAN) vector in the tail vein, and the control group was given a liposome delivery buffer without GMAN CRISPR / Cas9 vector, which was performed weekly Two injections. BLI was used to monitor lung metastases and evaluate the therapeutic efficacy of CRISPR / Cas9 delivery. SGC-Luc cells produced severe lung metastases in animals treated with control delivery buffer, but CRISPR / Cas9 targeted GMAN's MFR segment-treated drugs significantly blocked SGC-Luc cells from metastasizing into the lungs within one week after delivery. The treatment of CRISPR / Cas9 targeting GMAN's MFR segment continuous, twice-weekly treatment enhances the inhibitory effect on SGC-Luc lung metastatic growth. The final histological analysis confirmed that compared with the control delivery animals, the lung metastasis of animals delivered in the CRISPR / Cas9 MFR segment targeting GMAN was significantly inhibited and metastatic lung injury was significantly reduced (Figure 9.2) .
同时，为了排除CRISPR/Cas9递送***相关的细胞毒性抑制SGC-Luc胃癌肺转移的可能性，我们使用体内剂量以及高于体内剂量5倍的药物评价了CRISPR/Cas9治疗对SGC-Luc细胞活力的影响。靶向GMAN的MFR区段的CRISPR/Cas9递送缓冲液和对照递送缓冲液处理都不影响体外培养细胞的细胞活力。这表明靶向GMAN的MFR区段的CRISPR/Cas9处理对SGC-Luc肺转移的体内抑制作用不是由于非特异性细胞毒性导致的。At the same time, in order to rule out the possibility of cytotoxicity related to CRISPR / Cas9 delivery system inhibiting lung metastasis of SGC-Luc gastric cancer, we evaluated the effect of CRISPR / Cas9 treatment on the viability of SGC-Luc cells using in vivo doses and drugs 5 times higher than in vivo doses influences. Treatment of CRISPR / Cas9 delivery buffer and control delivery buffer targeting the MFR segment of GMAN did not affect the cell viability of cultured cells in vitro. This indicates that the CRISPR / Cas9 treatment targeting the MFR segment of GMAN on SGC-Luc lung metastasis in vivo is not due to non-specific cytotoxicity.
体内治疗实验的数据显示，靶向GMAN的MFR区段能够有效的治疗胃癌的转移，MFR区段具有潜在的价值。由于GMAN的MFR区段是基因组上的一小段内含子序列，而且MFR区段是一段在人体内相对特异的核苷酸序列。GMAN的MFR区段的序列特征使我们设计靶向MFR区段的药物既不会影响到其他基因或者RNA的表达又极大的降低了出现药物脱靶的可能。这些特征为我们合理的利用基因编辑技术或者合成靶向MFR区段的稳定核酸小分子药物提供了极大的便利和可能。因此，基于我们的治疗实验的效果以及GMAN的MFR区段的序列特征显示，GMAN的MFR区段具有重要的治疗胃癌转移的潜能。Data from in vivo treatment experiments show that the MFR segment targeting GMAN can effectively treat the metastasis of gastric cancer, and the MFR segment has potential value. Because the MFR segment of GMAN is a small intron sequence on the genome, and the MFR segment is a relatively specific nucleotide sequence in the human body. The sequence characteristics of the MFR segment of GMAN allow us to design drugs that target the MFR segment without affecting the expression of other genes or RNA and greatly reducing the possibility of drug off-target. These characteristics provide great convenience and possibility for us to reasonably use gene editing technology or synthesize stable nucleic acid small molecule drugs targeting MFR segments. Therefore, based on the effects of our treatment experiments and the sequence characteristics of the MFR segment of GMAN, the MFR segment of GMAN has important potential for the treatment of gastric cancer metastasis.
实施例10：GMAN在其他消化***肿瘤组织中相对高表达并与癌症病人的转移显著相关Example 10: GMAN is relatively highly expressed in other digestive system tumor tissues and is significantly associated with cancer patient metastasis
分析TCGA公共癌症数据库，发现GMAN在多种肿瘤组织中高表达，并且与肿瘤转移显著相关，其在转移性肿瘤患者(M1)中显著高于未转移的肿瘤患者(M0)。实施例显示的是GMAN在结直肠癌(图10.1)、食管癌(图10.2)和肝癌(图10.3)中的高表达情况。Analysis of the TCGA public cancer database revealed that GMAN is highly expressed in various tumor tissues and is significantly associated with tumor metastasis, which is significantly higher in patients with metastatic tumors (M1) than in patients with non-metastatic tumors (M0). The examples show the high expression of GMAN in colorectal cancer (Figure 10.1), esophageal cancer (Figure 10.2) and liver cancer (Figure 10.3).

Claims (9)

  1. 一种肿瘤相关序列，其特征在于，其序列为如SEQ ID NO.1所示的MFR序列。A tumor-related sequence, characterized in that its sequence is the MFR sequence shown in SEQ ID NO.1.
  2. 根据权利要求1所述的序列，其特征在于，所述肿瘤包括鼻腔及鼻窦恶性肿瘤，鼻咽癌，口腔癌，喉癌，涎腺肿瘤，颅内肿瘤，甲状腺癌，舌癌，肺癌，食管癌，贲门癌，乳腺癌，纵膈肿瘤，胃癌，大肠癌，直肠癌，肝癌，胰腺癌与壶腹周围癌，小肠恶性肿瘤，肾癌，***癌，膀胱癌，子***，卵巢癌，皮肤恶性黑色素瘤，淋巴瘤。The sequence according to claim 1, wherein the tumors include malignant tumors of nasal cavity and sinuses, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophagus Cancer, Cardiac Cancer, Breast Cancer, Mediastinal Tumor, Gastric Cancer, Colorectal Cancer, Rectal Cancer, Liver Cancer, Pancreatic Cancer and Periampullary Cancer, Small Intestine Malignant Tumor, Kidney Cancer, Prostate Cancer, Bladder Cancer, Cervical Cancer, Ovarian Cancer, Skin malignant melanoma, lymphoma.
  3. 如权利要求1所述的序列在制备肿瘤诊断、预后试剂中的应用，所述诊断试剂识别SEQ ID NO.1所示的MFR序列。The use of the sequence according to claim 1 in the preparation of tumor diagnosis and prognosis reagents, the diagnostic reagents recognize the MFR sequence shown in SEQ ID NO.1.
  4. 根据权利要求3所述的应用，其特征在于，诊断、预后试剂包括但不限于：The application according to claim 3, wherein the diagnostic and prognostic reagents include but are not limited to:
    (1)识别所述MFR序列的引物/引物组，或荧光标记的识别所述MFR序列的的引物/引物组；(1) A primer / primer set identifying the MFR sequence, or a fluorescently labeled primer / primer set identifying the MFR sequence;
    (2)识别所述MFR序列的小分子化合物；(2) A small molecule compound that recognizes the MFR sequence;
    (3)识别所述MFR序列的生物大分子，所述的生物大分子包括但不限于：抗体或抗体功能片段、荧光标记的抗体或抗体功能片段、RNA结合蛋白或其功能片段、荧光标记的RNA结合蛋白或其功能片段。(3) Biomacromolecules that recognize the MFR sequence, including but not limited to: antibodies or antibody functional fragments, fluorescently labeled antibodies or antibody functional fragments, RNA binding proteins or functional fragments thereof, fluorescently labeled RNA binding protein or its functional fragments.
  5. 如权利要求1所述的序列在制备肿瘤抑制剂中的应用，所述抑制剂靶向SEQ ID NO.1所示的MFR序列。The use of the sequence according to claim 1 in the preparation of a tumor suppressor targeting the MFR sequence shown in SEQ ID NO.1.
  6. 根据权利要求5所述的应用，其特征在于，抑制剂包括但不限于：The use according to claim 5, wherein the inhibitors include but are not limited to:
    (1)抑制所述MFR序列的siRNA、shRNA或功能类似的干扰小RNA；(1) siRNA, shRNA or functionally similar interfering small RNA that inhibits the MFR sequence;
    (2)抑制所述MFR序列的寡核苷酸片段，所述的寡核苷酸片段包括但不限于：反义寡核苷酸ASO、锁核酸LNA或功能类似的化学修饰的寡核苷酸；(2) Oligonucleotide fragments that inhibit the MFR sequence, and the oligonucleotide fragments include but are not limited to: antisense oligonucleotide ASO, locked nucleic acid LNA, or functionally similar chemically modified oligonucleotide ;
    (3)抑制所述MFR序列的小分子化合物；(3) Small molecule compounds that inhibit the MFR sequence;
    (4)抑制所述MFR序列的生物大分子，所述的生物大分子包括但不限于：抗体或抗体功能片段、高底物专一性的酶或其功能片段、其他抑制MFR功能的蛋白分子；(4) Biomacromolecules that inhibit the MFR sequence, the biomacromolecules include but are not limited to: antibodies or antibody functional fragments, high substrate-specific enzymes or functional fragments thereof, and other protein molecules that inhibit MFR function ;
    (5)敲除或破坏所述MFR序列的工具分子。(5) Tool molecules that knock out or destroy the MFR sequence.
  7. 根据权利要求6所述的应用，其特征在于，能敲除或破坏MFR序列的工具分子，包括但不限于DNA同源重组质粒，TALEN-TALEA靶向基因敲除质粒***，Cre/Loxp质粒***，四环素/干扰素等诱导性Cre/Loxp质粒***，FLP-frt质粒***，CRISPR/Cas9等CRISPR基因编辑质粒***等。The application according to claim 6, characterized in that the tool molecules capable of knocking out or destroying MFR sequences include but are not limited to DNA homologous recombination plasmids, TALEN-TALEA targeted gene knockout plasmid system, Cre / Loxp plasmid system , Inducible Cre / Loxp plasmid system such as tetracycline / interferon, FLP-frt plasmid system, CRISPR / Cas9 and other CRISPR gene editing plasmid system, etc.
  8. 一种肿瘤相关长链非编码RNA，其特征在于，包含权利要求1所述的序列。A tumor-related long-chain non-coding RNA, characterized by comprising the sequence of claim 1.
  9. 根据权利要求8所述的长链非编码RNA，其特征在于，为SEQ ID NO.2所示的GMAN。The long-chain non-coding RNA according to claim 8, which is GMAN shown in SEQ ID NO.2.
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