CN109055374B

CN109055374B - shRNA for specifically inhibiting OCT1 gene expression and application thereof

Info

Publication number: CN109055374B
Application number: CN201810827539.2A
Authority: CN
Inventors: 屠志刚; 刘兰兰; 刘晗青; 卢子文; 沈艳婷
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2018-07-25
Filing date: 2018-07-25
Publication date: 2021-07-20
Anticipated expiration: 2038-07-25
Also published as: CN109055374A

Abstract

The invention belongs to the technical field of medical molecular biology, and particularly relates to shRNA for specifically inhibiting OCT1 gene expression and application thereof; the shRNA molecule is shOCT1-2 or shOCT1-5, and the shRNA is packaged into lentivirus through a lentivirus vector and then infects ovarian cancer cells, can be combined with mRNA of an OCT1 gene, and can efficiently interfere the translation of the mRNA after transcription of the gene, so that the migration and infiltration of the ovarian cancer cells in vitro and the transfer of the cells in a living mouse are inhibited; the shRNA is expected to be applied to the preparation of ovarian cancer targeted drugs with high efficiency, strong specificity and small side effect.

Description

shRNA for specifically inhibiting OCT1 gene expression and application thereof

Technical Field

The invention belongs to the technical field of medical molecular biology, and particularly relates to shRNA for specifically inhibiting OCT1 gene expression and application thereof.

Background

Ovarian cancer is a malignant tumor which occurs in female ovarian tissues, is one of the most common malignant tumors in female gynecological tumors, and has the incidence rate second to cervical cancer and uterine corpus cancer. Because the early onset symptoms of the ovary are not obvious and the current early diagnosis technology is not mature enough, most of patients diagnosed with ovarian cancer are in a middle and late stage state, the focus is separated from primary metastasis and invades other organs of the body including lymph nodes, lungs, livers, intestinal tracts, bladders and the like, so that the treatment and prognosis effects are extremely poor, and the 5-year survival rate is lower than 20%.

Different treatment schemes need to be applied to ovarian cancer due to different pathological typing, the standard treatment scheme comprises comprehensive treatment by combining surgical treatment with chemotherapy based on platinum and taxane, but the conventional treatment scheme has unsatisfactory effect, the postoperative recurrence rate is as high as 70%, and most of tumors after recurrence generate drug resistance to applied chemotherapeutic drugs, so that the search for a treatment method and means which are efficient, low in postoperative recurrence rate and not easy to cause tumor drug resistance becomes one of the hot spots and problems to be solved in the field of ovarian cancer treatment. A great deal of research shows that the main reason for causing high morbidity and mortality of ovarian cancer is the superstrong metastasis and invasion characteristics of the malignant tumor, so a method capable of effectively inhibiting metastasis and invasion of ovarian cancer cells is found, and the key points for reducing the postoperative recurrence rate of ovarian cancer patients and improving the survival rate of the patients are achieved.

RNA interference (RNAi) is a highly conserved gene defense mechanism in the evolution of species, a technique that relies on short sequences specific for double-stranded RNA to achieve post-transcriptional gene silencing. Currently, RNAi technologies commonly used in laboratories mainly include siRNA oligonucleotide vectors and shRNA lentiviral plasmid expression vectors. siRNA is a short double-stranded RNA, sized around 19-29 nt, with two free bases at the 3' end, which can activate interference of RNA, and specifically achieve mRNA degradation by binding to a complementary sequence of the target mRNA. However, the expression of siRNA in cells is transient and has a relatively short time to act. Relative to siRNA, shRNA can be stabilized by virus-mediated transfection, which can reduce off-target effects. The shRNA comprises two short inverted repeat sequences, and the middle of the shRNA is divided by a stem-loop structure to form a structure similar to a hairpin. The shRNA is firstly inserted into a lentiviral vector to form a recombinant lentiviral plasmid, the lentiviral plasmid and other helper plasmids form lentivirus by means of 293FT cells, and finally the cells are transfected by the lentivirus to play the silencing effect of the shRNA.

OCT1, also known as POU2F1, is the first identified transcription factor in the POU family. OCT1 is an important transcription regulatory factor, and plays an important role in the process of cancer occurrence and development. The research on the correlation of OCT1 and tumors shows that different types of cancers are accompanied by abnormal expression of OCT1 to different degrees, and OCT1 has been proved to have high expression phenomena in certain cancers and is closely related to low survival rate of patients. Cancer cell metastasis is a hallmark of tumor progression, and OCT1 can enhance the metastatic capacity of cancer by promoting migration and infiltration of tumor cells.

The abnormal metabolism of tumor cells is a necessary condition for maintaining the growth of the tumor cells, the OCT1 has the function of promoting the metabolic activity in the tumor cells, and the interference with the activity of the OCT1 protein makes the change of the tumor metabolism possibly applied to the treatment of cancers. However, no relevant report about the research on the expression condition and action of shRNA interference OCT1 gene in ovarian cancer tissues exists at present.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a shRNA that specifically inhibits OCT1 gene expression and its application in diagnosis, treatment and prognosis evaluation of ovarian cancer.

According to the invention, the specificity of the shRNA sequence of the silent OCT1 gene is judged by observing the influence of the silent OCT1 gene on migration and infiltration of ovarian cancer cells and the transfer capability in mice, and the inhibition effect of the shRNA capable of silencing the OCT1 gene on migration and infiltration of the ovarian cancer cells and the inhibition effect of the ovarian cancer cells on transfer in mice are verified.

The invention provides shRNA for specifically inhibiting OCT1 gene expression, wherein the shRNA is shOCT1-2 or shOCT1-5, and the sequences of shOCT1-2 and shOCT1-5 are shown as follows:

the shRNA can silence the expression of an OCT1 gene in ovarian cancer cells, so that the migration and infiltration capacity of the ovarian cancer cells is remarkably inhibited, and the metastasis of the ovarian cancer cells in mice is inhibited.

The shRNA sequence expressed by the silent OCT1 gene is used, and a gene medicament for inhibiting migration and infiltration of ovarian cancer cells can be developed and prepared based on the sequence.

The invention designs and screens 2 shRNAs based on the mRNA sequence of OCT1 gene, which can obviously reduce the expression of OCT1 gene in ovarian cancer cells. The method comprises the steps of constructing a lentivirus vector pLKO.1-TRC-shOCT1, transferring target plasmids and helper plasmids pLP1, pLP2 and pLPSVG into 293FT cells by lipofectmin2000, preparing and collecting viruses, infecting an ovarian cancer cell strain SKOV3 by using lentiviruses containing a target shOCT1 sequence, screening resistant cells for an experiment by using Puromycin, and establishing a control group (the lentiviruses without the target shOCT1 sequence). Detecting a target protein OCT1 by using a Western blotting technology to verify the silencing effect of shRNA on OCT1 gene expression in ovarian cancer cells; detecting the influence of shRNA on migration and infiltration capacity of SKOV3 cells by a Transwell chamber experiment; the effect of shRNA on the transfer of SKOV3 cells in mice was explored by in vitro animal experiments.

The invention has the beneficial effects that:

the shOCT1-2 and shOCT1-5 related in the invention have obvious silencing effect on the expression of OCT1 gene in SKOV3 cells; transwell chamber experiments prove that shOCT1-2 and shOCT1-5 can inhibit migration and infiltration of SKOV3 cells; in vitro animal experiments prove that shOCT1-2 and shOCT1-5 can obviously inhibit the transfer of SKOV3 cells in mice. The shRNA molecule provided by the invention has great potential value in the development and treatment of targeted gene drugs of future ovarian cancer.

Drawings

FIG. 1 shows the successful construction of recombinant plasmids verified by agarose gel electrophoresis, where M: DNA marker; 1 is an enzyme-digested no-load plasmid PLKO.1-TRC, which is marked as pLKO.1-control; 2-6 are plasmids prepared from a lentivirus vector pLKO.1-TRC-shRNA-OCT1 successfully constructed by enzyme digestion, and are respectively marked as pLKO.1-shOCT1-1, pLKO.1-shOCT1-2, pLKO.1-shOCT1-3, pLKO.1-shOCT1-4 and pLKO.1-shOCT 1-5.

FIG. 2 is a Western blot detection result of shRAN on the inhibition of protein expression level of OCT1 gene in SKOV3 cells, wherein Vector is a control group and is infected with pLKO.1-TRC no-load plasmid; and in other experimental groups, SKOV3 cells are respectively infected with lentiviral plasmids and are marked as shOCT1-1, shOCT1-2, shOCT1-3, shOCT1-4 and shOCT 1-5.

FIG. 3 shows the effect of successfully prepared plasmids shOCT1-1, shOCT1-2, shOCT1-5 on SKOV3 cell migration and infiltration; FIGS. 3A and 3C are the electron micrograph and the statistical result of using Transwell to detect the effect of shOCT1 on cell migration; fig. 3B and 3D are electron micrographs and statistical results of the effect of shOCT1 on SKOV3 cell infiltration; p < 0.01, P < 0.001, n = 3. Vector is a control group, shOCT1-1, shOCT1-2 and shOCT1-5 are experimental groups.

FIG. 4 shows that the successfully prepared plasmid shOCT1-2 and shOCT1-5 inhibit the metastasis of SKOV3 cells in vivo, wherein, a is the lung morphology of mice in a control group, a shOCT1-2 group and a shOCT1-5 group; FIG. B shows the statistical analysis results of lung nodule numbers of mice in the control group, shOCT1-2 group and shOCT1-5 group; and the graph C shows the lung weight statistical analysis results of mice in the control group, the shOCT1-2 group and the shOCT1-5 group.

Detailed Description

The invention will be explained and explained in more detail with reference to the figures and the embodiments.

Example 1: construction of lentivirus plasmid for silencing OCT1 gene expression

First, 5 shRNAs were designed based on the mRNA sequence of OCT1 gene (accession No. NM-002697 in GENABANK), and named as shRNA-OCT1 (5 shRNAs are shOCT1-1, shOCT1-2, shOCT1-3, shOCT1-4, and shOCT 1-5) as shown in Table 1. The 5 pairs of shRNA primers were annealed to form 5 double-stranded oligonucleotide short fragments, which were ligated to a double-digested lentiviral vector PLKO.1-TRC at EcoRI and Age I (Biotechnology engineering, Shanghai, Ltd.). The ligation product is transformed into DH5 alpha colibacillus competent cells (Novozan biotechnology limited) by a heat shock method, placed on LB solid culture medium containing ampicillin resistance for 16 h at 37 ℃, single colonies are selected and shaken in LB liquid culture medium containing ampicillin resistance for 16 h at 37 ℃, 220 rpm and 16 h, thallus extracting plasmids are collected, the plasmids are verified by enzyme digestion, and the positive plasmids obtained by enzyme digestion are sent to Huada gene for sequencing verification. Sequencing showed that the successfully constructed lentiviral plasmid was used in subsequent experiments.

TABLE 1 shRNA sequences

FIG. 1 shows the successful construction of recombinant plasmids verified by agarose gel electrophoresis, where M: DNA marker; 1 is an enzyme-digested no-load plasmid pLKO.1-TRC, which is marked as pLKO.1-control; 2-6 are plasmids prepared from a lentivirus vector pLKO.1-TRC-shOCT1 successfully constructed by enzyme digestion, and are respectively marked as pLKO.1-shOCT1-1, pLKO.1-shOCT1-2, pLKO.1-shOCT1-3, pLKO.1-shOCT1-4 and pLKO.1-shOCT 1-5. Plasmid transformation is carried out on the constructed pLKO.1-shOCT1 plasmid, monoclonal is selected for culture, then the plasmid is extracted, and double enzyme digestion verification is carried out on part of the plasmid. In the figure, pLKO.1-control and 5 pLKO.1-shOCT1 plasmids are subjected to double enzyme digestion respectively and then run on glue, and it can be seen that the product of pLKO.1-control (control group) enzyme digestion has two bands, wherein the lower band is located between 500 and 1000 bp, the size of the lower band is matched with 726 bp of a fragment between two sites of double-enzyme digestion EcoRI and BamHI, and the upper band is located between 7000 and 10000 bp, thus obtaining the linear vector after enzyme digestion. The 5 pLKO.1-shOCT1 plasmids all had only one band, the size was 7000-10000 bp and slightly larger than the linear vector band. This is because pLKO.1-shOCT1 plasmid construction destroys EcoRI restriction site, plasmid double restriction verification only cuts BamHI site to linearize plasmid, and the results prove that plasmid construction is successful.

Example 2: preparation of cell lentivirus and lentivirus transfected cell

Firstly, preparing a mixed solution A, adding the solution A of an experimental group into 0.75 mu g of pLP1+0.35 mu g of pLP2+0.49 mu g of pLPSVG +0.61 mu g of pLKO.1-TRC-shOCT1 and the solution A of a control group into 0.75 mu g of pLP1+0.35 mu g of pLP2+0.49 mu g of pLPSVG +0.61 mu g of PLKO.1-TRC, gently mixing uniformly, and incubating for 5min at room temperature.

Then, mixture B was prepared by adding 9. mu.L of liposome Lipofectmin2000 (available from Invitrogen, USA) to 0.5 mL of OPTI-MEM, gently mixing, and standing at room temperature for 5 min.

Mixing solution A and solution B of experimental group and control group, and incubating at room temperature for 20 min. 293FT cells (purchased from Thermo Fisher Scientific) were digested and counted under a microscope with cell density adjusted to one third of the area of the bottom of the dish. The 293FT cell suspension was gently mixed well with the AB mixture and transferred to a petri dish, gently shaken to allow the mixture to spread flat on the bottom. After the culture dish is placed in an incubator to be incubated for 12 hours, the cells are changed in liquid, and the culture is continued. After two days of culture, the cell supernatant was aspirated by a 5 mL sterile syringe and the virus was collected by filtration through a 0.45 μm microporous membrane.

SKOV3 cells (purchased from ATCC cell resource center, usa) were next transfected with the collected viruses: on the first day, plating a plate, counting cells, and adjusting the cell density to be one third of the bottom area of a culture dish of 6 cm; the next day, virus infection, 6 cm petri dish was removed, supernatant was discarded, virus and medium 1:1, adding polybrene (8 mg/mL) (Sigma Bio Inc, USA) to make the action concentration be 8 mug/mL, putting the mixture into an incubator, and incubating for 8 h; after incubation for 8 h, removing the supernatant, replacing the cell with a liquid, and continuously putting the cell into an incubator for two days; after two days of culture, the supernatant was discarded and purified by 1000 mg/mL puromycin (puromycin; PM) (Biosharp Biotech, China) at a ratio of 1: diluting 1000, and screening for 2-3 days; reseeding: the cells after two days of selection were digested and re-seeded back into the culture dish, at which time the viable cells were resistant and available for the experiment.

Example 3: western blotting detection of expression level of OCT1 gene in SKOV3 cells in control group and test group

Collecting SKOV3 cells surviving in experimental group and control group after Puromycin (PM) screening, respectively extracting total cell proteins, detecting the expression condition of target protein OCT1 in different groups of cells by Western blot, and calculating the silencing efficiency of shOCT1 according to the gray value.

The Western blotting experiment firstly determines the protein concentration, the membrane is rotated after running glue after the protein is denatured, the membrane is sealed by 5 percent skim milk for 1.5h, the membrane sealed by the milk is incubated by a primary antibody OCT1 and a secondary antibody which can be specifically combined with the primary antibody OCT1 for 1h respectively, the membrane is washed by 0.1 percent TBS-T solution for 3 times and 4 min each time, the membrane is placed in a dark box which is processed by dark treatment and is taken into a dark room for exposure, a film is fixed on the membrane during exposure, and strips of the protein are blotted on the film under the action of luminous liquid and developing liquid.

FIG. 2 is a representative Western blot detection result of the inhibition of shOCT1 on the protein expression level of OCT1 gene in SKOV3 cells, wherein Vector is a control group and is infected by pLKO.1-TRC empty Vector; other groups of experiments were prepared by infecting SKOV3 cells with plasmids prepared from lentiviral vectors pLKO.1-TRC-shOCT1, i.e., pLKO.1-shOCT1-1, pLKO.1-shOCT1-2, pLKO.1-shOCT1-3, pLKO.1-shOCT1-4 and shOCT1-5, respectively, as shown in FIG. 2 as shOCT1-1, shOCT1-2, shOCT1-3, shOCT1-4 and shOCT 1-5. The results show that shOCT1-1, shOCT1-2 and shOCT1-5 in the experimental group can obviously silence the expression of OCT1 gene in SKOV3 cells compared with the control group.

Example 4: transwell chamber experiments examined the effect of shpad 6 on migration infiltration of SKOV3 cells.

Transwell migration: the surviving SKOV3 cells of the experimental and control groups after Puromycin (PM) screening in example 2 were digested and adjusted to a cell density of 20X 10 with serum-free medium⁴Cell suspension per mL; removing the Transwell chambers in the 24-well plate by using sterile forceps, adding 500 mu L of complete culture medium containing 10% serum to the bottom of the 24-well plate, then slightly returning the chambers, ensuring that no air bubbles exist at the bottoms of the chambers in contact with the culture medium, adding 300 mu L of cell suspension into each chamber, and returning the chambers to the incubator for incubation for 10 hours; after the incubator is incubated for 10 h, stopping the Transwell small-hole experiment; taking a clean 24-well plate, and adding 1 mL of PBS and 500 mu L of 0.1% crystal violet solution into each well; taking out the Transwell 24-hole plate from the incubator, moving the chamber out by using a pair of tweezers, slightly wiping off cells in the upper chamber by using an absorbent cotton swab, air-drying a membrane at the bottom of the chamber in an air port for dyeing, and placing the chamber into the hole for dyeing for 15-20 min. If the staining is too deep, washing with PBS; dropping a drop of clear water or PBS solution on a clean glass slide, placing the bottom membrane of the Transwell chamber on the drop, observing the bottom membrane by a microscope to clearly see the cells attached to the bottom membrane of the chamber, randomly selecting 5 to 10 visual fields for photographing, and counting.

Transwell infiltration: the first day, excellular Matrix (ECM) matrigel (Sigma, usa) was transferred from-20 ℃ to 4 ℃ overnight for thawing; the next day, placing the Transwell (8 μm) small hole, 24-hole plate, EP tube, and gun head in refrigerator at 4 deg.C for precooling, diluting matrigel with serum-free culture medium at a ratio of 1:12, adding 45 μ L ECM diluent into each small chamber, quickly spreading until no bubble is formed, and adding 5% CO₂Culturing in a cell culture box at 37 ℃; the following procedure was identical to the Transwell migration procedure except that the incubator incubation time was 24 h.

FIG. 3 shows the results of the effects of shOCT1-1, shOCT1-2 and shOCT1-5 on SKOV3 cell migration and infiltration. Fig. 3A and 3C are graphs showing the effect and statistical result of shOCT1 on cell migration detected by Transwell, and fig. 3B and 3D are graphs showing the effect and statistical result of shOCT1 on SKOV3 cell infiltration; p < 0.01, P < 0.001, n = 3. Vector is a control group, shOCT1-1, shOCT1-2 and shOCT1-5 are experimental groups. The experimental results show that shOCT1 can indeed inhibit the migration and infiltration capacities of SKOV3 cell strains (FIGS. 3A and 3B), wherein the migration capacities of shOCT-1, shOCT-2 and shOCT-5 are respectively 63 +/-4.3%, 21 +/-5.6% and 31 +/-4.3% of the control group (FIG. 3C), and the infiltration capacities are respectively 100 +/-8.6%, 59 +/-5.1% and 58 +/-1.9% of the control group (FIG. 3D). The results show that: the shOCT1-2 and shOCT1-5 silent OCT1 gene expression has obvious inhibiting effect on migration and infiltration of ovarian cancer cell SKOV 3.

Example 5: SKOV3 cells of an experimental group and a control group are injected into the mice through tail veins, and the effect of shOCT1 on the transfer of the SKOV3 cells in the mice is observed.

Based on a successfully constructed stable ovarian cancer cell strain SKOV3 lung metastasis model (research on action mechanism of PARD6A in ovarian cancer, Raney Linling), SKOV3 cells of an experimental group and a control group are respectively injected into a mouse body in a tail vein mode, the growth state of the mouse is regularly observed, the mice of the control group and the experimental group are killed at 60 days, lung organs of the mouse are photographed, the volume of the lung organs is measured, and the number of lung nodules is counted.

FIG. 4 shows that shOCT1-2 and shOCT1-5 inhibit the metastasis of SKOV3 cells in vivo. When the lungs of the mice were taken out, the surface of the lungs of the control mice was uneven, and formed translucent irregular or round nodules, and the morphology of the lungs was significantly different from that of normal mice, and the morphology of the lungs of the shOCT1-2 and shOCT1-5 mice was more normal (FIG. 4A). Statistical analysis of the lung nodule counts in mice was performed and it was observed that although lung nodules were also present in the lungs of some mice in the shOC1-2 and shOCT1-5 groups, the lung nodule counts were significantly reduced (fig. 4B) and statistically significant compared to the control group. These results show that the shOC1-2 and shOCT1-5 can significantly inhibit the ability of ovarian cancer cell strain SKOV3 to migrate and infiltrate into the lung in mice. Statistical analysis of lung weights of mice in each group revealed that although the shOCT1 test group had a slightly lower wet lung weight than the control group, it was not statistically significant, possibly because the individual differences were large and the increased lung nodules were not enough in weight to make the lung weights significantly different (fig. 4C). The results show that: by using the shOC1-2 and shOCT1-5 to silence the expression of the OCT1 gene in SKOV3 cells, the transfer of the SKOV3 cells in a mouse living body can be obviously reduced.

Sequence listing

<110> university of Jiangsu

shRNA for specifically inhibiting OCT1 gene expression and application

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ccgggaccag cagctcacct attaactcga gttaataggt gagctgctgg tcttttt 57

<210> 2

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aattaaaaag accagcagct cacctattaa ctcgagttaa taggtgagct gctggtc 57

<210> 3

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccgggcaact gggaacctgg tatttctcga gaaataccag gttcccagtt gcttttt 57

<210> 4

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aattaaaaag caactgggaa cctggtattt ctcgagaaat accaggttcc cagttgc 57

<210> 5

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccggtttcac tctgcagtgt gattgctcga gcaatcacac tgcagagtga aattttt 57

<210> 6

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aattaaaaat ttcactctgc agtgtgattg ctcgagcaat cacactgcag agtgaaa 57

<210> 7

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccgggaggac agataactgg gcttactcga gtaagcccag ttatctgtcc tcttttt 57

<210> 8

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aattaaaaag aggacagata actgggctta ctcgagtaag cccagttatc tgtcctc 57

<210> 9

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccggccagtc aacaccaaag cgaatctcga gattcgcttt ggtgttgact ggttttt 57

<210> 10

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aattaaaaac cagtcaacac caaagcgaat ctcgagattc gctttggtgt tgactgg 57

Claims

1. shRNA for specifically inhibiting OCT1 gene expression is shOCT1-2 or shOCT1-5, wherein a sense strand template sequence of shOCT1-2 is shown as SEQ ID No.3, an antisense strand template sequence is shown as SEQ ID No.4, a sense strand template sequence of shOCT1-5 is shown as SEQ ID No.9, and an antisense strand template sequence is shown as SEQ ID No. 10; the accession number of the OCT1 gene in GENABANK is NM-002697.

2. An shRNA lentiviral expression vector pLKO.1-TRC-shRNA-OCT1 for specifically inhibiting OCT1 gene expression, wherein the pLKO.1-TRC-shRNA-OCT1 vector comprises the shRNA of claim 1.

3. The use of the shRNA for specifically inhibiting OCT1 gene expression in claim 1 for non-diagnostic therapeutic purposes in specifically inhibiting OCT1 gene expression in ovarian cancer cells.

4. The use according to claim 3, wherein the use is for inhibiting migration or/and infiltration of ovarian cancer cells.

5. The use according to claim 3 for obtaining information on the inhibition of metastasis of ovarian cancer cells for non-diagnostic therapeutic purposes.

6. The application of shRNA (short hairpin ribonucleic acid) for specifically inhibiting OCT1 gene in claim 1 to preparing a medicament for inhibiting ovarian cancer cell metastasis, wherein the accession number of the OCT1 gene in GENABANK is NM-002697.

7. A drug for inhibiting metastasis of ovarian cancer cells, which comprises the shRNA of claim 1, which specifically inhibits the OCT1 gene, wherein the OCT1 gene has accession No. NM _002697 in GENABANK.