CN113151280B - Application of small-molecule non-coding RNA and host gene in diagnosis and treatment of gastric cancer - Google Patents
Application of small-molecule non-coding RNA and host gene in diagnosis and treatment of gastric cancer Download PDFInfo
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Abstract
The invention discloses a small-molecule non-coding RNA (microRNA-210) and a host gene (lncRNA MIR210 HG) thereof and application thereof. The microRNA-210 and lncRNA MIR210HG can be used as specific markers of gastric cancer. In addition, the invention carries out an in vitro transfer cell and an in vivo mouse tail vein injection tumor cell lung transfer model, which shows that microRNA-210 and lncRNA MIR210HG promote gastric cancer cell transfer, and the common transcription factor for inhibiting the microRNA-210 and lncRNA MIR210HG can inhibit gastric cancer cell transfer. Therefore, the microRNA-210 and lncRNA MIR210HG and the common transcription factor of the microRNA-210 and the lncRNA MIR210HG can be used for diagnosing gastric cancer, can also be used as a target point for preparing drugs for inhibiting metastasis of gastric cancer or resisting tumors, and can provide a new thought and a new way for treating gastric cancer.
Description
Technical Field
The invention relates to a small non-coding RNA (microRNA-210) and a host gene (lncRNA MIR210 HG) thereof, in particular to application of the small non-coding RNA (microRNA-210) and the host gene (lncRNA MIR210 HG) thereof in gastric cancer diagnosis and treatment, and belongs to the field of oncology.
Background
Gastric Cancer (Cancer) is the fourth most common Cancer worldwide, with the fourth most common incidence among men worldwide and the fifth most common among women. Although the incidence of gastric cancer is reduced in some countries, gastric cancer is second to the world in cancer-related deaths due to its high mortality. The Chinese is the country with high incidence of gastric cancer, and the latest statistics data show that the incidence rate of gastric cancer is up to 679.1 per 10 ten thousand people and the death rate is up to 498.0 per 10 ten thousand people in China. In recent years, thanks to popularization of gastrointestinal endoscopy and innovation of surgical techniques, early gastric cancer patients have significantly reduced five-year mortality; however, the five-year mortality rate of patients with advanced gastric cancer remains between 30 and 50%.
Because the gastric cancer is hidden, no obvious symptoms exist in early stage, and an effective screening means is lacked, 80% of gastric cancer patients are already in the development stage in diagnosis, and the opportunity of radical excision is missed; furthermore, the cause of death in patients with advanced gastric cancer is often associated with peritoneal, blood and lymph node metastasis. Therefore, early diagnosis and treatment and effective prevention and treatment of gastric cancer metastasis are effective ways for increasing survival rate of gastric cancer patients and reducing death rate of gastric cancer.
At present, gastric cancer is screened clinically mainly through tumor markers including carcinoembryonic antigen (CEA), glycoprotein antigens (CA 19-9, CA 125) and the like and gastrointestinal microscopy and imaging examination. However, tumor markers are not highly sensitive and specific. Although imaging methods such as ultrasound, CT, MRI and the like and gastroenteroscopy can be used as supplements to tumor markers, the detection rate of the methods depends on the tumor size and the experience of operators, so that misdiagnosis and missed diagnosis can occur. Therefore, the novel gastric cancer diagnosis marker which has high sensitivity, high specificity, low cost and easy detection is found to have important clinical significance.
Early gastric cancer patients were treated clinically with surgical resection as the primary treatment regimen, with adjuvant chemotherapy as the primary treatment regimen for patients who lost surgical opportunity and postoperative recurrence. Gastric cancer is less sensitive to radiotherapy and therefore less radiotherapy is used. The drugs currently used in gastric cancer chemotherapy mainly include fluorouracil (5-Fu), mitomycin (MMC), semustine (methylcyclohexylnitrourea), doxorubicin (doxorubicin), and the like. In recent years, targeted therapies have become a trend for cancer treatment. The targeted therapy refers to designing a therapeutic mode of corresponding drugs aiming at definite cancerogenic sites (such as target genes) on the cellular molecular level, and the drugs for targeted therapy enter the body to specifically bind the cancerogenic sites, so that tumor cells are specifically killed, and normal tissue cells around the tumor are not affected. At present, the targeting therapeutic agent trastuzumab is clinically used for treating Her2 positive gastric cancer patients, however, the research on targeting treatment of gastric cancer is still relatively few. Searching a new action target, researching and developing a gastric cancer specific targeting drug, and being beneficial to effective prevention and treatment of gastric cancer.
It was found that 60% of human genomic DNA can be transcribed extensively, while less than 2% of the gene encodes a protein. However, these non-coding RNAs have important biological functions, are involved in regulating and controlling vital activities such as cell differentiation, proliferation, apoptosis, etc., and play an important role in various physiological and pathological processes such as embryonic development, organism metabolism, disease occurrence and development, etc. Currently, non-coding RNAs are known to be mainly microRNAs (microRNAs), long-chain non-coding RNAs (lncRNAs), cyclic RNAs (circRNAs), micronucleolar RNAs (snorRNAs), piwi-interacting RNAs (piRNAs), and the like. In recent years, researchers find that non-coding RNA such as microRNA can be used as a biomarker for diseases such as tumors and the like, and is suitable for screening high-risk groups; in addition, the non-coding RNA participates in the generation and development of tumors, plays a role of oncogenes or oncogene inhibitors, and can be used as potential targets for development of tumor drugs or for preparation of tumor drugs.
In the research process of the invention, the inventor determines that the expression level of small-molecule non-coding RNA (microRNA-210) and host gene (lncRNA MIR210 HG) thereof is increased in the gastric cancer nest based on the detection of gastric cancer paired cancer nest and paracancerous tissues, thereby indicating the potential of the two can be used as gastric cancer diagnosis biomarkers. Meanwhile, the inventor also indicates that microRNA-210 and lncRNA MIR210HG and common transcription factors of the microRNA-210 and the lncRNA MIR210HG can be used as targets for preparing medicines for inhibiting gastric cancer metastasis through in vitro metastasis cells and in vivo mouse tail intravenous injection tumor cell lung metastasis models.
Disclosure of Invention
Based on the above, the invention aims to overcome the defects and shortcomings of the prior art and provide a diagnosis marker which is simple and convenient to operate and can effectively diagnose gastric cancer.
In order to achieve the technical purpose, the invention provides a small non-coding RNA, wherein the small non-coding RNA is microRNA-210, and the nucleotide sequence of the small non-coding RNA is shown as SEQ ID NO. 1.
Further, the host gene of the small molecule non-coding RNA is MIR210HG, and the nucleotide sequence of MIR210HG is shown as SEQ ID NO. 2.
Further, the common transcription factor of the small non-coding RNA and the host gene thereof is c-Myc.
The application of the small-molecule non-coding RNA in gastric cancer diagnosis is provided.
The application of the small-molecule non-coding RNA in preparing drug targets for inhibiting gastric cancer metastasis and/or antitumor drug targets.
The application of the host gene of the small-molecule non-coding RNA in gastric cancer diagnosis.
The application of the host gene of the small molecular non-coding RNA in preparing a drug target for inhibiting gastric cancer metastasis and/or an anti-tumor drug target.
The small molecule non-coding RNA and the common promoter of host genes thereof are applied to the preparation of drug targets for inhibiting gastric cancer metastasis and/or the preparation of anti-tumor drug targets.
The invention also provides a kit for diagnosing gastric cancer, which comprises the small-molecule non-coding RNA microRNA-210 and a host gene MIR210HG thereof.
Compared with the prior art, the invention has the beneficial effects that: the microRNA-210 and lncRNAMIR210HG can be used as specific markers of gastric cancer. In addition, the microRNA-210, the lncRNA MIR210HG and the common transcription factors of the microRNA-210 and the lncRNA MIR210HG can also be used as targets for preparing medicines for inhibiting gastric cancer metastasis or resisting tumors.
For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.
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FIG. 1 shows the expression levels of microRNA-210 and MIR210HG in paired cancer nests and paracancerous tissues of a gastric cancer patient according to example 1 of the present invention.
FIG. 2 is a graph showing the results of in vitro transfer cells according to example 3 of the present invention. Wherein, (A) is the effect of knocking down microRNA-210 or/and MIR210HG on the migration capability of gastric cancer cells, and (B) is the effect of over-expressing microRNA-210 or/and MIR210HG on the migration capability of gastric cancer cells.
FIG. 3 is a graph showing the lung transfer results of gastric cancer cells by in vivo mouse tail intravenous injection in example 4 of the present invention. Wherein, (A) is a graph of the HE staining result of the lung tissue of the mouse, and (B) is a graph of the statistical result of the transfer nodule of the lung tissue of the mouse.
FIG. 4 is a graph showing the results of the identification of the transcription factor c-Myc shared by microRNA-210 and MIR210HG in example 5 of the present invention.
Wherein, (A) is the effect of knocking down the expression of the microRNA-210 and the MIR210HG by using siRNA, (B) is the effect of knocking down the expression of the microRNA-210 and the MIR210HG by using inhibitor, and (C) is the effect of over-expressing the expression of the C-Myc on the expression of the microRNA-210 and the MIR210HG.
FIG. 5 shows the effect of the c-Myc inhibitor of example 6 of the invention on lung metastasis of gastric cancer cells. Wherein, (A) is a graph of the HE staining result of the lung tissue of the mouse, and (B) is a graph of the statistical result of the transfer nodule of the lung tissue of the mouse.
Detailed Description
The following examples facilitate the understanding of the present invention, but do not limit the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The context and specific definitions of the term application used in the present invention will be set forth below to aid in the understanding of the present invention. These definitions are not intended to limit the scope of the present invention.
The term "gastric cancer" as used herein refers to a malignancy that originates from the epithelium of the gastric mucosa.
The term "microRNA" used in the present invention refers to a class of endogenous small-molecule non-coding RNA widely existing in eukaryotes, and the length of the RNA is 18-24 nucleotides (nt).
The term "lncRNA" as used herein refers to a class of endogenous non-coding RNAs that are widely found in eukaryotic organisms and are 200 nucleotides (nt) or more in length.
The term "host gene" as used herein refers to a coding or non-coding gene in which a class of intronic microRNAs are located.
The reagents used in the present invention, sequence related information will be listed below to aid in the understanding of the present invention.
The PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit used in the present invention was purchased from TAKARA (Japan), and the 2x SYBR Green qPCR Master Mix kit was purchased from Biotool (USA). The primer company purchases or designs by oneself, wherein U6, microRNA-210 primer purchase from Ruibo company (Guangzhou), the goods number is MQPS0000002-1, MQPS0000810-1 separately; the ACTB and MIR210HG primers were self-designed and had the DNA sequences:
ACTB F:5'-AAGATGACCCAGATCATGTTTGAG-3' (shown as SEQ ID NO. 3)
ACTB R:5'-GCAGCTCGTAGCTCTTCTCCAG-3' (shown as SEQ ID NO. 4)
MIR210HG F:5'-TTTGTGTGCTCCAGAGAAAG-3' (shown as SEQ ID NO. 5)
MIR210HG R:5'-ACAAGTTTGCCCTAGATCAT-3' (SEQ ID NO. 6).
The preferred foreign NC67 sequence is a double stranded RNA designed based on the mature nematode miR-67 sequence that is not homologous to the human genomic sequence, and its detection sequence is 5'-UCACAACCUCCUAGAAAGAGUAGA-3' (shown in SEQ ID NO. 7).
Example 1: detection of microRNA-210 and MIR210HG expression levels in clinical samples
The trial of the present invention was approved by the ethical committee of the south hospital, university of Zhongshan affiliated with the sixth hospital and the university of south medical science, and informed consent was obtained from the participants. The inventor respectively collects 31 pairs of cancer nests and 19 pairs of cancer-side tissues of gastric cancer patients in a sixth hospital affiliated to the university of Zhongshan and a south hospital of the university of south medical science, and the total is 50 pairs. Quick freezing with liquid nitrogen, and storing at-80deg.C.
1. RNA extraction
(1) Taking 50-100mg tissue, preferably adding 1ml Trizol lysate, shaking thoroughly, mixing well, and ice-bathing for 15min.
(2) 200 μl of pre-cooled chloroform was added, mixed by shaking, and centrifuged at 12000g for 15min at 4deg.C.
(3) Transferring the supernatant, adding equal volume of isopropanol, shaking and mixing uniformly, and centrifuging at 4 ℃ for 30min at 16000 g.
(4) The supernatant was carefully poured off and the pellet washed once with 1ml 70% ethanol and centrifuged at 16000g for 10min at 4 ℃.
(5) The supernatant was discarded, and after ethanol had evaporated, 10. Mu.l of DEPC (diethyl pyrocarbonate) treated water was added for dissolution and stored at-80℃until use.
2. Real-time quantitative RT-qPCR detection and analysis
The invention adopts a PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit of TAKARA company to carry out reverse transcription, and the specific procedures are as follows: 1) 42 ℃ for 2min; 2) 15min at 37 ℃; 3) And 5s at 85 ℃. The obtained cDNA was stored at-20 ℃.
The invention adopts a 2x SYBR Green qPCR Master Mix kit of Biotool company, takes cDNA diluted 10 times as a template, and preferably adopts corresponding primers to carry out RT-qPCR detection. The kit comprises 5 pairs of primers, which are respectively: microRNA-210, MIR210HG, U6, ACTB and a primer for detecting the external reference NC 67. The PCR procedure was: 1) 95 ℃ for 10min; 2) 95 ℃ for 10 seconds and 60 ℃ for 1min, and keeping the temperature rising and falling speed of 1.6 ℃/s; 3) Repeating 2) in 39 cycles; 4) Melting curve analysis, 95 ℃ 20s,60 ℃ 20s, then heating to 95 ℃ at a speed of 0.11 ℃/s; 5) The procedure was ended at 40℃for 30 s.
The invention adopts the software of the LightCycler 480 to carry out RT-qPCR data analysis. Through corresponding reference calibration, the expression value 2 of microRNA-210 and MIR210HG is obtained -ΔCt (ΔCt＝Ct target -Ct reference ). The results show that microRNA-210 and MIR210HG are expressed in the cancer nest of gastric cancer patients more than in the paracancerous tissues (FIG. 1).
Example 2: preparation of diagnostic kit
The kit is used for gastric cancer detection and gastric cancer diagnosis, and consists of a sample RNA extraction system, a reverse transcription system, a real-time quantitative RT-qPCR system, a primer system and a result analysis system.
In the sample RNA extraction system of the kit, the inventor adopts a Trizol reagent extraction and isopropanol precipitation method to obtain RNA. Preferably, the blood sample is extracted with Trizol reagent with external reference NC67, and RNA is obtained by further combining phenol/chloroform extraction purification and glycogen assist precipitation. The inventors performed reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit from TAKARA corporation. The inventor adopts a primer of a Ruibo company or a self-designed primer as a primer system of the kit, and the primer comprises a microRNA-210, MIR210HG, U6, ACTB and NC67 (external reference). In the real-time quantitative RT-qPCR system, the inventor adopts a 2x SYBR Green qPCR Master Mix kit of Biotool company for detection. The inventor of the result analysis system adopts software of the LightCycler 480 to carry out RT-qPCR data analysis, and obtains the level 2 of microRNA-210 and MIR210HG in the sample to be detected through corresponding reference calibration -ΔCt (ΔCt＝Ct target -Ct reference ). Wherein U6 and ACTB are references to microRNA-210 and MIR210HG, respectively, when a tissue sample is tested, and NC67 is a common reference to microRNA-210 and MIR210HG when a blood sample is tested. The risk threshold values of microRNA-210 and MIR210HG are respectively 0.00333 and 0.04203, and are divided into two groups of high risk and low risk of gastric cancer.
Example 3: in vitro transfer laboratory experiments
In vitro metastasis cell experiments are experiments that evaluate the ability of tumor cells to migrate to invade by detecting tumor cells that migrate to the other side of the basement membrane of the metastasis cell. The invention researches the influence of microRNA-210 and MIR210HG on the migration capability of gastric cancer cells in vitro by combining the following in vitro transfer small room experiment with the missing sexual function experiment and the acquired sexual function experiment:
1. experiment of missing sexual function
(1) And respectively knocking down genes or microRNAs by adopting siRNA or microRNA antisense nucleotides. The siRNA is the siRNA of MIR210 HG: siMIR210HG-1 and siMIR210HG-2 are siRNA respectively targeting different sites of MIR210HG, and negative control is NC; the antisense nucleotide of the microRNA is antisense nucleotide Anti-miR-210 of the microRNA-210, and the negative control is Anti-NC. The final concentrations of the experiments were 50nM siRNA and 200nM antisense nucleotide, respectively. According to different combinations, the following groups are set:
negative control group: anti-NC+NC;
experiment group one: anti-miR-210+NC;
experimental group two: anti-NC+siMIR210HG-1;
experimental group three: anti-NC+siMIR210HG-2;
experimental group four: anti-miR-210+siMIR210HG-1;
experimental group five: anti-miR-210+siMIR210HG-2.
(2) The RNA of the different combinations was diluted to 100. Mu.l OPTI-MEMI, and after mixing, 1. Mu. lLipofectamine RNAiMAX was added to each tube, gently mixed, and left at room temperature for 15min.
(3) Mu.l of the transfection system described above was added to each well of a 24-well plate, followed by 500. Mu.l of the cell suspension.
(4) Cells were transfected for 48 hours, counted by pancreatin digestion, and washed once with serum-free DMEM medium.
(5) 600. Mu.l of DMEM medium containing 10% serum was added to the lower chamber of the transfer chamber, and 100. Mu.l of serum-free DMEM medium was added to the upper chamber to resuspend 6X 10 4 The cells are digested.
(6) After 20 hours incubation at 37℃the chamber was fixed with methanol for 15 minutes and stained with 0.1% crystal violet for 15 minutes. Subsequently, the upper cell layer of the cell was scraped off with a cotton swab, observed with a microscope, photographed, and the number of migrating cells was counted.
The results show that knocking down microRNA-210 or MIR210HG can inhibit gastric cancer cell migration, and the inhibition effect of both the knockdown is further enhanced (figure 2A).
2. Experiment of acquired sexual function
(1) Obtaining gastric cancer cell-stable strains by slow virus infection, comprising:
negative control stable strain one: AGS-GFP;
negative control stable strain two: AGS-RFP;
microRNA-210 overexpression stable strain: AGS-miR-210;
MIR210HG over-expression stable strain: AGS-MIR210HG;
microRNA-210 and MIR210HG simultaneously overexpress the stable strain: AGS-miR-210& MIR210HG.
(2) Pancreatin cells were counted and the cells were washed once with serum-free DMEM medium.
(3) 600. Mu.l of DMEM medium containing 10% serum was added to the lower chamber of the transfer chamber, and 100. Mu.l of serum-free DMEM medium was added to the upper chamber to resuspend 6X 10 4 And (3) cells.
(4) After 13 hours incubation at 37℃the chamber was fixed with methanol for 15 minutes and stained with 0.1% crystal violet for 15 minutes. Subsequently, the upper cell layer of the cell was scraped off with a cotton swab, observed with a microscope, photographed, and the number of migrating cells was counted.
The results show that microRNA-210 and MIR210HG over-expressed stable strain cells have enhanced migration ability compared with the control group, and the stable strain cells co-over-expressed with the microRNA-210 and MIR210HG have further enhanced migration ability (figure 2B).
Example 4: in vivo mouse tail intravenous injection tumor cell lung metastasis model
The mouse tail intravenous tumor cell lung metastasis model is characterized in that tumor cells are injected into the tail vein of a mouse, after a certain period of time, the lung tissues of the mouse are collected for paraffin embedding and slicing, then tumor nodules are counted under a microscope through HE staining, and thus the lung metastasis capacity of the tumor cells is reflected. NOD-SCID immunodeficient mice were selected as the study material in this example. The method comprises the following specific steps:
1. taking 5 gastric cancer stable strains (AGS-GFP, AGS-RFP, AGS-miR-210, AGS-MIR210HG and AGS-miR210& MIR210 HG) in logarithmic growth phase, digesting the cell-forming suspension by trypsin, and centrifuging for 3 minutes at 500 g.
2.1 XPBS cells were washed and counted to prepare 5×10 cells 6 cell suspensions of cells/ml, 2X 10 per NOD-SCID immunodeficient mice per group 6 And (3) gastric cancer cells.
3. After 10 weeks of tail vein injection, mice were euthanized and their lung tissue was collected for paraffin-embedded sections, followed by HE staining and microscopic counting of tumor nodules.
The results show that the lung transfer capacity of microRNA-210 and MIR210HG over-expressed stable strain cells in mice is enhanced compared with that of a control group, and the lung transfer capacity of the stable strain cells which over-express both cells in mice is further enhanced (figure 3).
Example 5: identification of a shared transcription factor for microRNA-210 and MIR210HG
Abnormal gene expression is mainly regulated by epigenetic regulation and transcriptional regulation. In this example, the inventors predicted microRNA-210 and MIR210HG upstream promoters and transcription factors that could bind to the promoter regions by informatics. The predicted result shows that the transcription factor c-Myc can be combined with a common promoter of the microRNA-210 and the MIR210HG, so that the transcription of the microRNA-210 and the MIR210HG is regulated. Further, the following three experiments prove that the c-Myc regulates the transcription of microRNA-210 and MIR210 HG:
1. the expression of microRNA-210 and MIR210HG can be inhibited by knocking down c-Myc through siRNA
(1) Knocking down c-Myc with siRNA: the sic-Myc-1 and the sic-Myc-2 are siRNA respectively targeting different sites of c-Myc, and the negative control is NC. The final concentration of the experiment was 50nM siRNA and the following groups were set:
negative control group: NC;
experiment group one: sic-Myc-1;
experimental group two: sic-Myc-2.
(2) The RNA was diluted in 100. Mu.l of OPTI-MEMI, and after mixing, 1. Mu. l Lipofectamine RNAiMAX was added to each tube, and the mixture was gently mixed and left at room temperature for 15min.
(3) Mu.l of the transfection system described above was added to each well of a 24-well plate, followed by 500. Mu.l of gastric cancer cell suspension.
(4) After 48 hours of cell transfection, 500. Mu.l Trizol lyses the cells and RNA was extracted.
(5) Reverse transcription was performed using the PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit from TAKARA.
(6) Further adopting a 2x SYBR Green qPCR Master Mix kit of Biotool company to perform RT-qPCR detection, performing RT-qPCR data analysis by software of a LightCycler 480, and obtaining the level 2 of microRNA-210 and MIR210HG in a sample to be detected by corresponding reference calibration -ΔCt (ΔCt＝Ct target -Ct reference )。
The results indicate (FIG. 4A) that knockdown of c-Myc can inhibit transcription of microRNA-210 and MIR210HG.
2. The expression of microRNA-210 and MIR210HG can be inhibited by adopting an inhibitor to knock down c-Myc
(1) Planting 1×10 in 24-well plate one day in advance 7 And (3) gastric cancer cells.
(2) The c-Myc inhibitor 10058-F4 was used to knock down c-Myc and the following groups were set:
negative control group: DMSO;
experiment group one: 50. Mu.M inhibitor 10058-F4;
experimental group two: 100 μM inhibitor 10058-F4;
experimental group three: 200. Mu.M inhibitor 10058-F4.
(3) After 48 hours of cell treatment, 500. Mu.l Trizol lyses cells and RNA was extracted.
(4) Reverse transcription was performed using the PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit from TAKARA.
(5) Further adopting a 2x SYBR Green qPCR Master Mix kit of Biotool company to perform RT-qPCR detection, performing RT-qPCR data analysis by software of a LightCycler 480, and obtaining the level 2 of microRNA-210 and MIR210HG in a sample to be detected by corresponding reference calibration -ΔCt (ΔCt＝Ct target -Ct reference )。
The results indicate (FIG. 4B) that treatment with the c-Myc inhibitor 10058-F4 can inhibit transcription of microRNA-210 and MIR210HG.
3. Overexpression of c-Myc promotes expression of microRNA-210 and MIR210HG
(1) Planting 1×10 in 24-well plate one day in advance 7 And (3) gastric cancer cells.
(2) Plasmid transfection with Lipofectamine 3000, set the following groups:
negative control group: 500ng pCDH;
experimental group: 500ng of pCDH-c-Myc.
(3) The plasmid was diluted in 25. Mu.l of OPTI-MEMI, and after mixing, 1. Mu. l p3000 was added to each tube, and gently mixed to form tube A. Simultaneously, another 25. Mu.l OPTI-MEMI B tube containing 1. Mu.l Lipofectamine 3000 was prepared and gently mixed. Mixing the tube A and tube B gently, and standing at room temperature for 5-10min.
(4) 50 μl of the transfection system described above was added to a 24-well plate seeded with gastric cancer cells.
(5) After 48 hours of cell transfection, 500. Mu.l Trizol lyses the cells and RNA was extracted.
(6) Reverse transcription was performed using the PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit from TAKARA.
(7) Further adopting a 2x SYBR Green qPCR Master Mix kit of Biotool company to perform RT-qPCR detection, performing RT-qPCR data analysis by software of a LightCycler 480, and obtaining the level 2 of microRNA-210 and MIR210HG in a sample to be detected by corresponding reference calibration -ΔCt (ΔCt＝Ct target -Ct reference )。
The results indicate (FIG. 4C) that overexpression of C-Myc can promote transcription of microRNA-210 and MIR210HG.
Example 6: inhibition of the common transcription factor of microRNA-210 and MIR210HG reduces in vivo mouse lung metastasis
1. AGS gastric cancer cell lines in the logarithmic growth phase were taken, the cell suspension was digested with trypsin, and centrifuged at 500g for 3 minutes.
2.1 XPBS cells were washed and counted to prepare 5×10 cells 6 cell suspensions of cells/ml, each NOD-SCID immunodeficient mouse tail intravenous injection2×10 6 And (3) gastric cancer cells.
3. After 1 week of tail vein injection, mice were randomly divided into three groups: (1) PBS group (2) corn oil solvent group (3)c-Myc inhibitor group (10058-F4, selleck, # S7153). Wherein the PBS group and the corn oil solvent group were intraperitoneally injected with 100. Mu.l of PBS or corn oil solvent per 25g of mice, and the c-Myc inhibitor group was intraperitoneally injected in an amount of 20mg inhibitor (10058-F4)/kg of mice.
4. The intraperitoneal administration was performed according to the body weight of mice at the same time every week for a total of 8 times.
5. Two weeks after dosing, mice were euthanized and lung tissue was collected for paraffin-embedded sections, followed by HE staining and microscopic counting of tumor nodules.
The results indicate (FIG. 5) that treatment with the c-Myc inhibitor 10058-F4 can inhibit lung metastasis of gastric cancer cells in mice.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Sequence listing
<110> and university of south China
<120> application of small-molecule non-coding RNA and host gene in diagnosis and treatment of gastric cancer
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> RNA
<213> Homo sapiens (Homo sapiens)
<400> 1
CUGUGCGUGU GACAGCGGCU GA 22
<210> 2
<211> 1991
<212> DNA
<213> Homo sapiens (Homo sapiens)
<400> 2
GAGGGTGCCA GCGGCCGCAG CTGAAGTTGG GCCGAGAGCC GGCGACGGCC CCGCGCCGGG 60
GTCGCAGGCC TGCAGGAGTT GAGGGCTGCA CCTGCTCGCT GGAGAGGGAG AGGCAGATTT 120
AGTGGACGCC TGGCATGGAC TCGGACTGGC CTTTGGAAGC TCCCTGCCCT GACGGGGTTG 180
CCTGTCACCA CTGCGAAGTG AGGCTTGGCA GGACCTGCAC CTGAGAAAGG CTGTGTGTGG 240
TCTTGGGGTC CACACCTGCA GAGCTAACTT ACTGCCAGAC GGCGACTTAC TGTGGGCCAC 300
CCTCAGTGAA CCGGGGTGTC CTCAGCTGGC CCTACAGAGC ACTTCTGTGC TGGGGATGAG 360
TAGGAACTCT GGGCGAGGAG GGTCCCAGCG CCGCCCCTCG ATACAGCCTG GCTCTGCCCT 420
CTGCCCGTAC TTACACCAGG TGGGATCCCT GCCCTGCATT GCCTGGGGAT TGGCTGGGCT 480
TGGGCCCGCC CTGCTGTGGA ACTGGATGTT TTCAGGGAGC CCAGCCTTTC CTCATGTCAA 540
CACAGTTCAC AATATAGTTT TCAAAGTACA GTTTAAAACT CAAAAGTAAA CTTTTCAGCA 600
ACTCAAAGGT TTGCTGAGTG ATCTGAAGCA CTCTGGCCAC TTTTTGGGGC CATGGGATTT 660
GGTTCACCTG AAACAGCCAG TGAGAGGCCG GGTGTGGTGG CTCACACCCG TAATCCCAAC 720
ACTTCAGGAG GCAGACGCGG GTGATCGCTC ACTTGAGATC AGGAGTTCAA GACCAGCCTG 780
GGCAACATGG TGAAACCTCG TCTCTACTAA AAATACAAAA ATTAGCTAGG CATGGTGGTG 840
GGCACCTGTA ATCCCAGCTA CTTGGAAGGC TGAGGCAAGA GAATCGCTTG AACCTGGGAG 900
GTGGAGGTTG CAGCGAGACG AGATTACGCC GCTGCACTCC AGCCTGGGTG ACGAGAGACT 960
CTGCCTCAAA AAAATAAAAA AATGAAACAG CCAGTGAGGA GGAAGGCTCC CCGCCTTCCC 1020
CCCGCCGGAA CATAGCCATA GCTGCTGCTG GGACACCCTC TTGGTGGGGA AGAAGGCTGG 1080
TTAGCTTCAT CAGAGCCAGC AGCAGCAGAC CAGGGACGGG CACCTAGGCA GTGGCCTCAG 1140
AGTGAACAGG AGTTCCTCAG AAACACACAC AGGGACGGCG TGGCGCATGC TCTGCCAGCT 1200
CCATGCCTCC TTCCCATTGT GGGGCTGGGG TACGTAGGGC AGAGCTCATG ACCTCCGGGA 1260
GGACATGGGG GTGGGCTCTG GATGGCACCT GGCATTGCCC CCTGCTGGCC TATGTGACGG 1320
TGTGGAGGGC TGGTCACAGA GGTACGACCA TCCCTCCAGA ATGTGGGTCG GGGCTGTGGA 1380
TGGAGGAGTA GGCCCCTCAT ATCCCAGGCC TGCTGCCCAG GCACAACCCA CTTGGCCTAT 1440
GCATTCCAGG CTCCATCCCA TGTGACTCTG GGCTTAGCCC CTTCTGGGGC CACAGGTCAG 1500
GCAGGTCCAG GCCCCAAGGA CCTCCCAGTG ACAGGCGACT GTGAGCTGGG CAGACAGGAG 1560
TGAAGTCAGG TGGGGGTTCT GGCTTGCTGA CACCAGCGTT TGGAGCCTCC TGCTGCTGCC 1620
TGGCTTCCCT GCATTCCCTG TTCCCTGCCT CAGGCAAGAA ATAACCAAGC CGAGTTGCCT 1680
CTGCACAGCA GTGAGCTCCT GGTGGCCCTG GCTTCTGGGG AGCCCTGTGG ATGGCTTCCT 1740
TGCCCAAGTC CAGGCCTTCT TGTTCCCTTT GTGTGCTCCA GAGAAAGGGG GCAGCACCAG 1800
ATCCAGATCC AGGGCCAACC AACAGAAAGC TGAGTCCATC CCAAACTCGC CCATTCTCAG 1860
AGCACAAAGA CCCCATGATC TAGGGCAAAC TTGTCCAACT GTTGGCCCAT GGAACAGCTT 1920
TGAATGCAGC CCAACACAAA TCTATAAATT TTCTTAAACA TCAAAAAAAA AAAAAAAAAA 1980
AAAAAAAAAA A 1991
<210> 3
<211> 24
<212> DNA
<213> artificial sequence
<400> 3
AAGATGACCC AGATCATGTT TGAG 24
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<400> 4
GCAGCTCGTA GCTCTTCTCC AG 22
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<400> 5
TTTGTGTGCT CCAGAGAAAG 20
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
ACAAGTTTGC CCTAGATCAT 20
<210> 7
<211> 24
<212> RNA
<213> artificial sequence
<400> 7
UCACAACCUC CUAGAAAGAG UAGA 24
Claims (1)
1. The application of a reagent for detecting the expression level of a molecular marker in preparing an auxiliary gastric cancer diagnosis reagent, wherein the molecular marker is a combination of small-molecule non-coding RNA microRNA-210 and long-chain non-coding RNA MIR210HG, the nucleotide sequence of the microRNA-210 is shown as SEQ ID NO.1, and the nucleotide sequence of the MIR210HG is shown as SEQ ID NO. 2.
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| US20110166200A1 (en) * | 2008-04-24 | 2011-07-07 | Zhan Zhang | Methods of using mir210 as a biomarker for hypoxia and as a therapeutic agent for treating cancer |
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| KR102194215B1 (en) * | 2019-07-02 | 2020-12-22 | 사회복지법인 삼성생명공익재단 | Biomarkers for Diagnosing Gastric Cancer And Uses Thereof |
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| Title |
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| NCBI Reference Sequence: NR_029623.1;Li X et al.;《Genbank》;20201219;ORIGIN * |
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