| Objective: Ovarian cancer is a malignant tumor that seriously threatens the health of women.Because of the late appearance of clinical symptoms of ovarian cancer,it is easy to be widely spread in the pelvic and abdominal cavity.Therefore,although surgical treatment,chemotherapy,biological treatment and gene therapy are widely used in the treatment of ovarian cancer.The 5-year survival rate remains only 35-38%.Therefore,the research on the mechanism of ovarian cancer plays an important role in the early detection,early diagnosis and early treatment of ovarian cancer.Non-coding RNA refers to RNA molecules that are incapable of translating into proteins in the transcriptome,including small nuclear RNA(sn RNA)and small nucleolar RNAs of relatively small molecular weight(Sno RNA),micro RNA(micro RNA,mi RNA),piwi-interacting RNA(pi RNA),and long non-coding RNA(lnc RNA),circular RNA(circ RNA)with relatively large molecular weight.Lnc RNA,as a new type of gene regulatory factor,participates in regulating many processes of the body such as cell growth and development,apoptosis,proliferation,differentiation,cell autophagy,etc.,and is closely related to many diseases such as tumors.Because lnc RNA is a long-chain structure,there is an expression pattern of spatiotemporal specificity or tissue characteristics,so some lnc RNAs are more potential than mi RNAs to become specific targets for the treatment of some diseases.Lnc RNA TDRG1(human testicular development-related gene 1)is overexpressed in bone marrow mesenchymal stem cells,but its role in ovarian cancer has not been reported.This study intends to explore the role and mechanism of lnc RNA TDRG1 in the proliferation,apoptosis,migration,and invasion of ovarian cancer through histological,in vitro and in vivo cytology experiments,which may improve the network pathway of lnc RNA TDRG1,and explore whether lnc RNA TDRG1 can become a treatment for ovarian cancer.Methods: 1.Detect the expression level of lnc RNA TDRG1 in epithelial ovarian cancer tissue and normal ovarian tissue by q RT-PCR,and analyze the correlation between the expression level of lnc RNA TDRG1 and the clinical pathological type of ovarian cancer;2.Construct lnc RNA TDRG1 wild-type and mutant plasmids,establish lnc RNA TDRG1 over-expressing A2780 and OVCAR3 ovarian cancer cell lines,confirm transfection efficiency by q RT-PCR;3.MTT experiment and ki-67 protein detection was used to verify lnc RNA TDRG1 overexpression on cell proliferation in A2780 and OVCAR3 ovarian cancer cell line;4.The effect of lnc RNA TDRG1 overexpression on apoptosis was verified by Annexin V-PE / 7AAD staining in A2780 and OVCAR3 ovarian cancer cell lines;5.The effect of lnc RNA TDRG1 overexpression on cell migration ability was detected by cell scratch test in A2780 and OVCAR3 ovarian cancer cell lines;6.Transwell invasion experiment was used to validate the effect of lnc RNA TDRG1 overexpression on cell invasion in A2780 and OVCAR3 ovarian cancer cell lines;7.Construct lnc RNA TDRG1 si RNA,establish lnc RNA TDRG downregulated A2780 and OVCAR3 cell lines,and confirm transfection efficiency by q RT-PCR;8.Use MTT experiment and ki-67 protein test to verify the effect of lnc RNA TDRG1 downregulation on cell proliferation and viability in A2780 and OVCAR3 ovarian cancer cell lines;9.Annexin V-FITC / PI staining test the effect of lnc RNA TDRG1 downregulation on cell apoptosis in A2780 and OVCAR3 ovarian cancer cell lines;10.The effect of silencing lnc RNA TDRG1 on cell migration ability was detected by wound healing test in A2780 and OVCAR3 ovarian cancer cell lines;11.The effect of silencing lnc RNA TDRG1 on cell invasion was verified by Transwell invasion experiment in A2780 and OVCAR3 ovarian cancer cell lines;12.Biological Informatics website(micro RNA.org)was used to predict that lnc RNA TDRG1 has a possible mi RNA-93(mi R-93)binding site,and was verified through the double luciferase reporter gene;13.The function of lnc RNA TDRG1 overexpression / low expression on mi R-93 was checked by q RT-PCR;14.The effect of overexpression / low expression of lnc RNA TDRG1 on the expression of Rho C and related proteins was detected by Western blot;15.The effect of lnc RNA TDRG1 overexpression on subcutaneous tumor formation experiments in nude mice was checked;16.SPSS was used to analyze the datas statistically,and samples between groups were tested by two-tailed t test.Results: 1.The q RT-PCR results showed that the expression level of lnc RNA TDRG1 in epithelial ovarian cancer tissues was significantly higher than that in normal ovarian tissues(p <0.05),and the expression level of lnc RNA TDRG1 in medium/low-differentiated ovarian cancer tissues was significantly higher than that of well-differentiated ovarian cancer tssues(p <0.05);2.The results of MTT experiment showed that compared with the the blank control group and negative control group,the proliferation rate of A2780 and OVCAR3 cells that overexpressed lnc RNA TDRG1 was accelerated(p <0.05),besides,the expression level of ki-67 protein increased after the lnc RNA TDRG1 overexpression;3.Cell apoptosis experiments showed that,compared with the control group,lnc RNA TDRG1 verexpression inhibited cell apoptosis(p <0.05);4.The results of wound healing experiments showed that: compared with the control group,lnc RNA TDRG1 overexpression promoted the migration of ovarian cancer cells(p <0.05);5.Transwell invasion experiments proved that compared with the control group,lnc RNA TDRG1 overexpression promoted the ovarian cancer cell invasion(p <0.05);6.The results of MTT experiments showed that compared with the control group,the activity of A2780 and OVCAR3 cells decreased after si RNA transfection silenced lnc RNA TDRG1 expression(p <0.05),besides,the protein expression of ki-67 was also decreased;7.Cell apoptosis experiment results showed that: compared with the control group,silencing of lnc RNA TDRG1 expression could promote ovarian cancer cell apoptosis(p <0.05);8.Wound healing test showed that compared with the control group,lnc RNA TDRG1 downregulation significantly inhibited ovarian cancer cell migration(p <0.05);9.Transwell invasion experiments demonstrated that compared with the control group,silenced lnc RNA TDRG1 expression significantly inhibited ovarian cancer cell invasion(p <0.05);10.Through biological informatics prediction,there exists binding sites between the 3'UTR region of lnc RNA TDRG1 and mi R-93,dual luciferase reporter gene experiments show that mi R-93 mimic can significantly downregulate lnc RNA TDRG1 3'UTR wild Type luciferase activity(p <0.01),but had no effect on lnc RNA TDRG1 3'UTR mutant type luciferase activity;11.Compared with the control group,q RT-PCR results showed that lnc RNA TDRG1 overexpression inhibited mi R-93 expression,while silencing lnc RNA TDRG1 could promote mi R-93 expression(p <0.0 5);12.Western blot results showed that Rho C,P70S6 K,Bcl-x L and MMP2 protein expression was higher in lnc RNA TDRG1 overexpression group than in the control group,promoted,while silencing lnc RNA TDRG1 reduced Rho C,P70S6 K,Bcl-x L and MMP2 protein expression.13.Subcutaneous tumor formation experiments in nude mice showed that the overexpression of lnc RNA TDRG1 promoted tumor formation in nude mice.Immunohistochemistry / q RT-PCR results further confirmed that overexpression of lnc RNA TDRG1 promoted Rho C protein expression and inhibited mi R-93 expression.Conclusion: 1.This study confirmed for the first time that the expression level of lnc RNA TDRG1 in epithelial ovarian cancer is significantly higher than that of normal ovarian tissue,and the expression is related to the degree of differentiation;2.lnc RNA TDRG1 promotes the proliferation,migration and Invasion ability,inhibit apoptosis,suggesting that it may function as an “oncogene”;3,lnc RNA TDRG1 competitively inhibits mi R-93,relieves the inhibition of target protein Rho C by mi R-93,regulates the expression of Rho C and related proteins,and promotes the development of ovarian cancer. |
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