CN107586781B - Liver cancer marker lncRNA ENST00000620463.1 and application thereof - Google Patents

Abstract

The invention discloses a liver cancer marker lncrnaenst00000620463.1 and application thereof. The invention provides a tumor marker, which is LncRNA, is named ENST00000620463.1 and is RNA shown in a sequence 1 of a sequence table. The DNA corresponding to ENST00000620463.1 is the DNA shown in sequence 2 of the sequence table. The invention discovers that ENST00000620463.1 in the liver cancer is remarkably up-regulated for the first time. Experiments prove that the shRNA interferes with silencing ENST00000620463.1, can effectively inhibit proliferation, migration and invasion of liver cancer cells, and provides a new way for personalized treatment of liver cancer.

Description

Liver cancer marker lncRNA ENST00000620463.1 and application thereof

Technical Field

The invention relates to the field of biological medicines, and particularly relates to a liver cancer marker lncRNA ENST00000620463.1 and application thereof.

Background

Primary hepatocellular carcinoma (HCC) (hereinafter, liver cancer) is a common malignant tumor in China, the mortality rate of the cancer is the second cancer, and about 35 ten thousand of the cancers occur each year. The incidence and mortality of liver cancer are always high, and most patients are in middle and late stages in clinical discovery due to high malignancy and fast disease progression, and the mortality and recurrence rate are high and the prognosis is poor. The conventional treatment means at present mainly comprise surgical excision, liver transplantation, local ablation treatment, chemical embolization through hepatic artery, radiotherapy, molecular targeted treatment, comprehensive treatment and the like, but the treatment effect is not ideal, and the treatment of liver cancer in middle and late stages is more unsatisfactory.

The development and progression of liver cancer is a complex biological process. Research shows that the occurrence and development of liver cancer are related to the up-regulation of transcription factors such as c-myc, c-jun, NF-kappa B, AP-2, beta-catenin and the like by HBV viral proteins (such as HBx and the like) in liver cells. In addition, a large number of studies indicate that the occurrence and development of human liver cancer are closely related to abnormal expression and signals of various growth factors such as insulin-like growth factor (IGF), Hepatocyte Growth Factor (HGF), transforming growth factor-alpha (TGF-alpha), Epidermal Growth Factor (EGF), transforming growth factor-beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), and the like. However, the molecular biological mechanism of liver cancer development, development and metastasis has not been clearly studied.

Long non-coding RNA (lncRNA) is a non-coding protein, and the non-coding RNA molecule with the transcript length of more than 200nt is originally regarded as a byproduct in the transcription process and has no biological function. There are roughly five types according to their positional relationship with mRNA: (1) a sense long non-coding RNA; (2) antisense long non-coding RNA; (3) bidirectional long-chain non-coding RNA; (4) intron-type long-chain non-coding RNA; (5) intergenic long non-coding RNA (i.e., large interactive coding RNA, llncRNA). The proportion of lncRNA is far greater than that of mRNA in the whole genome transcript. More and more researches show that lncRNA plays an important role in the life activities of gene transcription and translation, epigenetics, cell differentiation and the like, abnormal expression exists in various tumors, the lncRNA participates in the processes of occurrence, development, infiltration, metastasis and the like of the tumors, the occurrence of diseases can be comprehensively known and understood through researching the biological function of the lncRNA and the regulation mechanism of the lncRNA in the tumors, the lncRNA is expected to become a new potential tumor marker and a new medicine target, and a new research idea is provided for the diagnosis and prognosis of the tumors.

Disclosure of Invention

The invention aims to provide a marker for early diagnosis or targeted therapy of tumors, in particular for liver cancer.

The invention provides a tumor marker, which is LncRNA, is named ENST00000620463.1 and is RNA shown in a sequence 1 of a sequence table. The DNA corresponding to ENST00000620463.1 is the DNA shown in sequence 2 of the sequence table.

Also included within the scope of the present invention are polynucleotides derived from human and having at least 75% or more homology, at least 85% or more homology, at least 90% or more homology, at least 95% or more homology, at least 98% or more homology to said RNA or said DNA.

The invention also protects the application of a product (product A) for detecting the RNA or the DNA in the preparation of a kit; the kit is used for auxiliary diagnosis of tumors.

The invention also protects a kit comprising a product for detecting said RNA or said DNA (product a); the kit is used for auxiliary diagnosis of tumors.

Any of the above products (product A) may be a product for detecting the expression level of the DNA in a sample to be tested. The product can be specifically a primer, a probe, a chip, a nucleic acid membrane strip, a preparation or a kit for detecting the expression level of the DNA in a sample to be detected. The product can be specifically a specific primer pair, and consists of a primer F and a primer R;

the primer F is (e1) or (e 2):

(e1) a single-stranded DNA molecule shown in sequence 3 of the sequence table;

(e2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;

the primer R is (e3) or (e 4):

(e3) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

(e4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and has the same function as the sequence 4.

Any one of the above samples to be tested may be a human sample.

Any one of the above samples to be tested may be cells, tissues, organs, body fluids (blood, lymph fluid), digestive juice, expectoration, alveolar bronchial lavage fluid, urine or feces.

The system for auxiliary diagnosis of tumors is system A or system B.

The system A comprises a substance and a data processing device for detecting the expression level of the DNA; the data processing device is provided with a module 1 and a module 2, wherein the module 1 has the following functions (c1), and the module 2 has the following functions (c 2):

(c1) comparing the expression levels of said DNA in ex vivo samples taken from the subject and a healthy control, respectively;

(c2) determining whether the subject is a tumor patient according to the comparison result of (c1) as follows: (ii) if the expression level of said DNA in the ex vivo sample of said subject is up-regulated compared to said healthy control, then said subject is or is candidate for a tumor patient; otherwise, the subject is not, or is not a candidate for, a tumor patient.

The system B comprises a substance and data processing means for detecting the expression level of the DNA; the data processing device is provided with a module 3 and a module 4, wherein the module 3 has the following functions (d1), and the module 2 has the following functions (d 2):

(d1) respectively comparing the expression levels of the DNA in isolated samples taken from suspected tumor tissues of a tested person and other normal tissues except the suspected tumor tissues;

(d2) determining whether the subject is a tumor patient according to the comparison result of (d1) as follows: if the expression level of the DNA in the suspected tumor tissue of the subject is up-regulated compared with the normal tissue, the subject is or is candidate for being a tumor patient; otherwise, the subject is not, or is not a candidate for, a tumor patient.

Any one of the above isolated samples may be cells, tissues, organs, body fluids (blood, lymph fluid), digestive juices, expectorations, alveolar bronchial washes, urine or feces.

The specific primer pair also belongs to the protection scope of the invention.

The invention also protects the application of the RNA or the DNA as a target spot in the auxiliary diagnosis of tumor and/or the targeted therapy of tumor and/or the screening of tumor drugs.

The tumor medicament is a medicament for preventing and/or treating tumors.

The method for screening the tumor drug comprises the following steps: (1) treating a system expressing and/or containing said DNA with a candidate substance; setting a parallel control without candidate substance treatment; (2) detecting the expression level of said DNA in the system after step (1) is completed; compared with a parallel control, if the expression level of the DNA in a system treated by the candidate substance is obviously reduced (by more than 20 percent, more than 50 percent and more than 80 percent), the candidate substance can be used as a candidate tumor drug.

The system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

The invention also protects the application of the substance for inhibiting the expression of the RNA or the DNA in the preparation of a product (product B); the product has at least one of the following applications (f1) - (f 7):

(f1) inhibiting cancer cell proliferation;

(f2) promoting cancer cell apoptosis;

(f3) inhibiting cancer cell metastasis;

(f4) inhibiting cancer cell proliferation;

(f5) inhibiting tumor metastasis;

(f6) inhibiting tumor spread;

(f7) treating tumor.

The present invention also provides a product (product B) comprising, as an active ingredient, a substance which inhibits the expression of said RNA or said DNA; the product has the following use of at least one of (f1) - (f 7):

(f1) inhibiting cancer cell proliferation;

(f2) promoting cancer cell apoptosis;

(f3) inhibiting cancer cell metastasis;

(f4) inhibiting cancer cell proliferation;

(f5) inhibiting tumor metastasis;

(f6) inhibiting tumor spread;

(f7) treating tumor.

The "substance inhibiting the expression of the RNA or the DNA" may be an interfering molecule capable of inhibiting the expression or transcription of the DNA by using the DNA or the transcript thereof as a target sequence, and specifically may include shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

The "substance inhibiting the expression of said RNA or said DNA" may be a compound or a chemotherapeutic agent. The chemotherapeutic agent may include microtubule activators, alkylating agents, antineoplastic antimetabolites, platinum-based compounds, DNA-alkylating agents, antineoplastic antibiotic agents, antimetabolites, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cyclin inhibitors, myostatin inhibitors, metalloprotease inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, or molecularly modified viral, bacterial and exotoxin agents.

The "substance inhibiting the expression of the RNA or the DNA" may be specifically shRNA, which is shRNA1, shRNA2, or shRNA 3.

The shRNA1 consists of a sense strand 1 and an antisense strand 1; the sense chain 1 is shown as a sequence 5 in a sequence table; the antisense strand 1 is shown by a sequence 6 in a sequence table.

The shRNA2 consists of a sense strand 2 and an antisense strand 2; the sense strand 2 is shown as a sequence 7 in a sequence table; the antisense chain 2 is shown by a sequence 8 in a sequence table.

The shRNA3 consists of a sense strand 3 and an antisense strand 3; the sense strand 3 is shown as a sequence 9 in a sequence table; the antisense strand 3 is shown by a sequence 10 in a sequence table.

Any of the above products (product b) may be a pharmaceutical composition.

The pharmaceutical composition comprises any of the above "agents inhibiting the expression of said RNA or said DNA".

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, a physiological salt, an excipient, a filler, a coagulant and a blender, a surfactant, a dispersing agent or an antifoaming agent.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a virus, a microcapsule, a liposome, a nanoparticle, or a polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier can be a liposome, biocompatible polymer (including natural and synthetic polymers), lipoprotein, polypeptide, polysaccharide, lipopolysaccharide, artificial viral envelope, inorganic (including metal) particle, and bacterial or viral (e.g., baculovirus, adenovirus and retrovirus), phage, cosmid, or plasmid vector.

The pharmaceutical composition may also be used in combination with other drugs for the treatment of tumors, and the other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

The pharmaceutical compositions may also be administered separately with other therapeutic compounds, either as a separate composition or in a different dosage form than the primary active ingredient. Some of the doses of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

The shRNA1, the shRNA2 or the shRNA3 also belong to the protection scope of the invention.

Any of the above tumors may be a solid tumor or a hematological tumor. The tumor can be liver cancer, lung cancer, colon cancer, carcinoma of large intestine, breast cancer, ovarian cancer, cervical cancer, gastric cancer, renal cancer, pancreatic cancer, prostatic cancer, lymphoma, glioma or melanoma.

Any of the above cancer cells may be liver cancer cells. The liver cancer cell can be human liver cancer cell strain HepG2, Huh7 or SMMC 7721.

The inventor of the invention extensively and deeply researches, detects the expression level of lncRNA in liver cancer tissues and tissues beside the cancer by a high-throughput method and high-throughput sequencing, finds lncRNA fragments with obvious expression difference, and discusses the relation between the lncRNA fragments and the occurrence of the liver cancer, thereby finding better ways and methods for early detection and targeted therapy of the liver cancer. Through screening, the invention discovers that ENST00000620463.1 is remarkably upregulated in liver cancer for the first time. Experiments prove that the shRNA interferes with silencing ENST00000620463.1, can effectively inhibit proliferation, migration and invasion of liver cancer cells, and provides a new way for personalized treatment of liver cancer.

Drawings

FIG. 1 is a graph showing the expression of ENST00000620463.1 in a liver cancer patient by QPCR.

FIG. 2 is a ROC plot of ENST00000620463.1 in liver cancer patients.

FIG. 3 is a graph showing the detection of expression of ENST00000620463.1 in liver cancer cells by QPCR.

FIG. 4 is a graph showing the effect of transfected siRNA on the expression of ENST00000620463.1 in hepatoma cells.

FIG. 5 is a graph showing the effect of ENST00000620463.1 on cell proliferation measured by CCK 8.

FIG. 6 is a graph showing the effect of ENST00000620463.1 on colony formation by clones of cells.

FIG. 7 is a graph showing the effect of ENST00000620463.1 on apoptosis of hepatoma cells.

FIG. 8 is a graph showing the effect of Transwell chamber assay ENST00000620463.1 on migration and invasion of hepatoma cells; wherein, the graph A is the influence graph of ENST00000620463.1 on the migration of liver cancer cells, and the graph B is the influence graph of ENST00000620463.1 on the invasion of liver cancer cells.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data obtained were expressed as mean ± standard deviation, and the statistical analysis was performed by using SPSS 20.0 statistical software, and the difference between the two was considered to be statistically significant when P is less than 0.05 by using t-test.

Example 1 screening of liver cancer markers

The cancer tissues and paired paracarcinoma tissues of 20 liver cancer patients were subjected to secondary sequencing analysis (patients all informed consent, all the above specimens were obtained with consent of the tissue ethics committee), and a liver cancer marker was found to be an LncRNA, named ENST00000620463.1, and shown in sequence 1 of the sequence listing. The DNA corresponding to ENST00000620463.1 is shown as sequence 2 in the sequence table. Compared with the tissues beside the cancer, the expression level of ENST00000620463.1 in the liver cancer tissues is obviously higher than that in the tissues beside the cancer.

Example 2 qPCR sequencing to verify differential expression of ENST00000620463.1

1. Cancer tissues and tissues adjacent to the cancer were collected from 102 patients with liver cancer, and all the specimens were obtained with informed consent from the tissue ethics committee.

2. Extracting the tissue RNA of each sample in the step 1, and performing reverse transcription to obtain cDNA.

3. qPCR was performed using each sample cDNA obtained in step 2 as a template to detect the expression of ENST00000620463.1 (GAPDH was used as an internal reference gene). The expression of ENST00000620463.1 was examined using primers F1 and R1, and the expression of GAPDH gene was examined using primers F2 and R2.

F1: 5'-GGTGTAAGCGGTGGTTA-3' (SEQ ID NO: 3 of the sequence Listing);

r1: 5'-CCAGTTCTGGGCTGGGATGT-3' (SEQ ID NO: 4 of the sequence Listing);

F2：5’-GGTGGTCTCCTCTGACTTCAACA-3’；

R2：5’-GTTGCTGTAGCCAAATTCGTTGT-3’。

the results are shown in FIG. 1. The results show that compared with the tissues beside the cancer, the expression level of ENST00000620463.1 is up-regulated in the liver cancer tissues, and the difference has statistical significance (P is less than 0.001).

Example 3 ROC Curve analysis of ENST00000620463.1 in cancer tissue and paracancer tissue of patients

The test subject working characteristics of ENST00000620463.1 were analyzed using the pROC package in the R language, a binomial exact confidence space was calculated, and ROC curves were plotted.

The results are shown in FIG. 2. The AUC value of ENST00000620463.1 is 0.85, and has better specificity and sensitivity, which shows that ENST00000620463.1 has higher accuracy when applied to the diagnosis of liver cancer.

Example 4 differential expression of ENST00000620463.1 in liver cancer cell lines

Total RNAs of human hepatoma cell lines HepG2 (Japanese JCRB cell bank, cat # JCRB1054), Huh7 (Japanese JCRB cell bank, cat # JCRB0403), SMMC7721 (Beijing synergic cell bank, resource # 3111C0001CCC000087) and normal liver cell line LO2 (Beijing synergic cell bank, resource # 3131C0001000200006) were extracted and reverse-transcribed into cDNAs, respectively. qPCR was performed using cDNA from each cell as a template to detect expression of ENST00000620463.1 (GAPDH was used as an internal reference gene). The expression of ENST00000620463.1 was examined using primers F1 and R1, and the expression of GAPDH gene was examined using primers F2 and R2.

F1：5’-GGTGTAAGCGGTGGTTA-3’；

R1：5’-CCAGTTCTGGGCTGGGATGT-3’；

F2：5’-GGTGGTCTCCTCTGACTTCAACA-3’；

R2：5’-GTTGCTGTAGCCAAATTCGTTGT-3’。

The results are shown in FIG. 3. The results show that compared with the normal liver cell line LO2, ENST00000620463.1 is up-regulated in liver cancer cells HepG2, Huh7 and SMMC7721, and the difference is statistically significant (P is less than 0.001).

Example 5 silencing of ENST00000620463.1

1. Four groups of shrnas were designed for ENST00000620463.1, as follows:

(1) negative control shRNA sequence (shranc):

sense strand: 5'-UUCUCCGAACGUGUCACGU-3', respectively;

antisense strand: 5'-ACGUGACACGUUCGGAGAA-3', respectively;

(2)shRNA1：

sense strand: 5'-AACUGGUCCUUCUCGAGGUAUAGCUU-3' (SEQ ID NO: 5 of the sequence Listing);

antisense strand: 5'-AAGCUAUACCUCGAGAAGGACCAGUU-3' (SEQ ID NO: 6 of the sequence Listing);

(3)shRNA2：

sense strand: 5'-ACAGGGCUCUCUCGAGGUAUAGCUU-3' (SEQ ID NO: 7 of the sequence Listing);

antisense strand: 5'-AAGCUAUACCUCGAGAGAGCCCUGU-3' (SEQ ID NO: 8 of the sequence Listing);

(4)shRNA3：

the sense strand is 5, -AAGACAAUCAUUCUCGAGGUAUAGC-3' (SEQ ID NO: 9 of the sequence listing);

the antisense strand is 5, -AAGCUAUACCUCGAGAGAGCCCUGU-3' (SEQ ID NO: 10 of the sequence Listing).

2. Human hepatoma cell line Huh7 is expressed by 2 x 10⁵One cell/well was seeded into six well cell culture plates at 37 ℃ in 5% CO₂Culturing cells in an incubator for 24 h; the four sets of shRNAs (concentration 20 nM/well) from step 1 were transfected into Huh7 cells using Lipofectin 2000 (Invitrogen), 48h later the cells were harvested, total RNA extracted and reverse transcribed to cDNA. qPCR was performed using cDNA as a template to detect the expression of ENST00000620463.1 (GAPDH was used as a reference gene). The expression of ENST00000620463.1 was examined using primers F1 and R1, and the expression of GAPDH gene was examined using primers F2 and R2.

F1：5’-GGTGTAAGCGGTGGTTA-3’；

R1：5’-CCAGTTCTGGGCTGGGATGT-3’；

F2：5’-GGTGGTCTCCTCTGACTTCAACA-3’；

R2：5’-GTTGCTGTAGCCAAATTCGTTGT-3’。

The results are shown in FIG. 4. Results show that compared with a negative control shRNA group, the shRNA1 group, the shRNA2 group and the shRNA3 group can obviously reduce the expression of ENST00000620463.1, and the difference has statistical significance (P is less than 0.01, P is less than 0.05, and P is less than 0.05).

Example 6 CCK8 test for cell proliferation

1. The Huh7 cells in the logarithmic proliferation phase were seeded in 96-well plates at 2X 10 per well³Individual cells, 5% CO at 37 ℃₂The cells were cultured in an incubator for 24 h.

2. Taking the 96-well plate in the step 1, transfecting the shRNAC and shRNA1 designed in the example 5 into HepG2 cells (the transfection method refers to the specification of a body transfection reagent), and adding 10 mu l/well of CCK8 reagent after transfecting for 24h, 48h, 72h and 96h respectively; the absorbance of A450nm was measured 2h after the addition of CCK8 using a microplate reader.

The results are shown in FIG. 5. The results show that the cell growth rate of the transfected shRNA1 group is obviously lower than that of the control group, the difference has statistical significance (P is less than 0.01), and the results show that the expression of ENST00000620463.1 can promote the growth of the liver cancer cells.

Example 7 Soft agar colony formation experiment

1. Logarithmic phase of proliferation of Huh7 cells were seeded in 6-well plates at 1.5X 10 per well⁵Individual cells, 5% CO at 37 ℃₂Culturing in an incubator for 24 h.

2. The shranc and shRNA1 designed in example 5 were transfected into Huh7 cells (the transfection method is described in the specification of the transfection reagent) and collected 24h after transfection, and the cell concentration was adjusted to 5 × 10 using DMEM medium containing 20% fetal bovine serum³Each cell/ml, to obtain a cell suspension.

3. Two low melting point agarose solutions with concentrations of 1.2% (mass percent) and 0.7% (mass percent) were prepared, autoclaved, and maintained in a 40 ℃ water bath.

4. Mixing 1 volume part of the low melting point agar sugar solution (1.2 percent by mass) in the step 3 with 1 volume part of 2 XDMEM culture medium, and adding 2 Xgentamicin (100 mu g/mL) and 20 percent by volume of calf serum to obtain a mixed solution. 3ml of the mixed solution is poured into a dish with the diameter of 6cm, placed for 5min to be cooled and solidified, and placed in an incubator as bottom agar for later use.

5. Mixing 1 volume part of the low melting point agarose solution (0.7 percent by mass) obtained in the step 3 with 1 volume part of 2 XDMEM culture medium, adding 0.2ml of the cell suspension obtained in the step 1, fully mixing uniformly, injecting the mixture into a plate with the diameter of 6cm prepared in the step 3 to form a double agarose layer, and repeating 4 samples for each experimental group. After the upper agar solidified, the plate was incubated at 37 ℃ in a 5% CO2 incubator with 1.5ml of DMEM medium every 3 days for a total of 14 days.

6. The plate from step 4 was removed and stained with 1ml of 0.005% gentian violet for 90 min. The plate was placed under an inverted microscope for observation, 10 low power fields were randomly selected for each group of cells, and the number of colonies of cells formed was counted under the microscope.

The results are shown in FIG. 6. The results show that the colony formation number of single cell clones in the cell group transfected with shRNA1-ENST00000620463.1 is significantly reduced (P < 0.001) compared with the control group.

Example 8 Effect of ENST00000620463.1 on apoptosis of hepatoma cells

1. The log phase of Huh7 cells were seeded in 12-well plates at 1.0X 10 per well⁵Individual cells, 5% CO at 37 ℃₂Culturing in an incubator for 24 h.

2. Shranc and shRNA1 designed in example 5 were transfected into Huh7 cells (the transfection method was described in reference to the instructions of the transfection reagent) and harvested 24h after transfection, and Annexin/FITC staining was used to detect apoptosis.

The results are shown in FIG. 7. The result shows that the apoptosis rate of the cell group transfected with shRNA1-ENST00000620463.1 is increased (P is less than 0.001) compared with the control group, and the result shows that the expression of ENST00000620463.1 inhibits the apoptosis of the liver cancer cell.

Example 9 cell migration and invasion assay

1. The Matrigel was thawed in an ice bath under sterile conditions, diluted 20-fold with PBS and plated onto a polycarbonate membrane in a Transwell chamber at a volume of 50. mu.l/well. Standing at 37 deg.C for 4 hr, taking out after Matrigel gel polymerizes into gel, and sucking out supernatant liquid gently. 50 μ l of serum-free BSA-containing culture medium was added to each well to hydrate the basement membrane, and the membrane was left at 37 ℃ for 30 min.

2. Logarithmic growth phase of Huh7 cells were seeded in 24-well plates at 5X 10 cells per well⁴Individual cells, 5% CO at 37 ℃₂Culturing in an incubator for 24 h.

3. The shranc and shRNA1 designed in example 5 were transfected into Huh7 cells, respectively (the transfection method was described in reference to the instructions for the transfection reagent in vivo)) After 24h of transfection, the serum-free DMEM medium is replaced for culture for 12h, the cells are collected, and the cell concentration is adjusted to 5 multiplied by 10 by adopting the serum-free DMEM medium⁵Each cell/ml, to obtain a cell suspension.

4. 200. mu.l of the cell suspension prepared in step 3 (100. mu.l for migration experiments and 200. mu.l for invasion experiments) was taken and added to a Transwell chamber. 500. mu.l of 1640 medium containing FBS was added to the lower chamber of the 24-well plate. The cells were placed in a cell incubator for 24 h.

5. After completing step 4, the cell of the chamber is rinsed 2 times with PBS, and then placed in DAPI working solution for staining for 5-20min at room temperature. Rinsed 2 times with PBS, placed under a fluorescent microscope for observation and counted.

The results are shown in FIG. 8. The result shows that compared with a control group, the cell group cell migration and invasion capacity of the transfected shRNA1-ENST00000620463.1 is obviously reduced (P is less than 0.001), and the result shows that ENST00000620463.1 can promote the migration and invasion of the liver cancer cells.

Claims (2)

1. The application of the substance for inhibiting the expression of RNA shown in the sequence 1 of the sequence table or DNA shown in the sequence 2 of the sequence table in the preparation of products; the product has at least one of the following applications (f1) - (f 7):

(f1) inhibiting cancer cell proliferation;

(f2) promoting cancer cell apoptosis;

(f3) inhibiting cancer cell metastasis;

(f4) inhibiting cancer cell proliferation;

(f5) inhibiting tumor metastasis;

(f6) inhibiting tumor spread;

(f7) treating tumors;

the tumor is liver cancer; the cancer cell is a liver cancer cell.

2. Use according to claim 1, characterized in that: the substance for inhibiting the expression of the RNA shown in the sequence 1 of the sequence table or the DNA shown in the sequence 2 of the sequence table is shRNA1 or shRNA2 or shRNA 3;

the shRNA1 consists of a sense strand 1 and an antisense strand 1; the sense chain 1 is shown as a sequence 5 in a sequence table; the antisense strand 1 is shown by a sequence 6 in a sequence table;

the shRNA2 consists of a sense strand 2 and an antisense strand 2; the sense strand 2 is shown as a sequence 7 in a sequence table; the antisense strand 2 is shown by a sequence 8 in a sequence table;

the shRNA3 consists of a sense strand 3 and an antisense strand 3; the sense strand 3 is shown as a sequence 9 in a sequence table; the antisense strand 3 is shown by a sequence 10 in a sequence table.

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