The Study Of Regulatory Mechanism Of MiR-629 By Targeting TSPYL5 In Ovarian Cancer

The molecular mechanism of ovarian cancer is complex,studies have shown that mi RNA is its key regulator.This study aimed to explore the key role of mi R-629 in the development of ovarian cancer and to reveal its possible molecular mechanism.Testis-specific Y-like protein 5(TSPYL5)is a tumor suppressor gene in various cancers,but there is little for its role in OC.This study is divided into three parts.Part ?: The Role of mi R-629 and TSPYL5 in Ovarian CancerObjective: This study was to explore the different expressions of mi RNA and TSPYL5 in ovarian cancer tissues and the effect of mi RNA-629 on ovarian cancer cells.The levels of mi R-629 in cancer and adjacent tissues were detected by RT-PCR and the target mi RNA was screened.Methods: 1.RT-PCR and Western-blot was used to detect the levels of mi RNA and the expression of TSPYL5 in tumors and adjacent tissues.The results showed that mi RNA-629 and TSPYL5 were selected as target genes.At the same time,the pathological features and expressions of mi R-629 and TSPYL5 we examined in OC clinical tissues.2.OVCAR3 cells were transfected with mi R-629-inhibitor and mi RNA-629-mimics,respectively,and the function of mi R-629 was inhibited or enhanced as the experimental group,and the mock group served as the control.Flow Cytometry,Scratch test and Transwell was used for cell function experiments.Results: 1.The pathological result showed that,compared with the adjacent normal tissues,there were lots of large cell nuclei in OC tissues.Moreover,the expression of caspase-3 was significantly repressed in OC tissues.Mi R-629 was overexpressed in ovarian cancer tissues and the expression of TSPYL5 was down-regulated or absent(P<0.05).2.Flow Cytometry results show that mi R-629 overexpression significantly decreased the apoptosis rates of OVCAR3 cells,and there was a significant difference between the mimics group and the mock group.Cell cycle assays further confirmed the inhibitory effect of mi R-629 on apoptosis and proliferation.The scratch test demonstrated that overexpressing mi R-629 promoted cells migration effectively compared with mock group cells,where as the cell stransfected with mi R-629 inhibition had the widest cell scratch.At the same time,the transwell assay indicated that overexpression of mi R-629 significantly promoted the invasion of OVCAR3 cells,and there was a significant difference between the mimic group and the mock group.Conclusion: Mi R-629 promotes OC cell proliferation,migration and invasion in vitro.TSPYL5 inhibits both OC tissue and cell lines.Part ?: Mi R-629 Inhibits the Malignant Behavior of Ovarian Cancer by Targeting TSPYL5Objective: This study was to investigate the pivotal role of mi R-629 in the progression of OC and to reveal the possible molecular mechanism of its action.Methods: 1.The pc DNA3.1-TSPYL5 was transfected into OVCAR3 cells and empty vector transfected OVCAR3 as a control group.Transwell,Flow Cytometry and Scratch test was used to detect cell function.2.Mi RWALK predicts that TSPYL5 is the target of mi RNA-629.TSPYL5 3'UTR sequences were captured to construct psi-CHECK2 wild-type and mutant vectors and transfected with mi R-629.The dual-luciferase assay verified the relationship between mi R-629 and TSPYL5.3.The OVCAR3 cells were transfected with mi RNA-629 inhibitors and mi RNA-629 inhibitor+si TSPYL5 respectively as experimental group and Black group and negative control group were used as control group.After transfectio,the function of TSPYL5 was detected using Transwell,Scratch test and Flow Cytometry.Results:1.When OVCAR3 cells overexpress TSPYL5 in for cell function experiments,the results showed that overexpression of TSPYL5 could promote the apoptosis of OVCAR3 cells(P= 0.022);the result of Flow Cytometry showed that TSPYL5 inhibited the cell proliferation actively and shortened the S phase of cell cycle,and increased the ratio of G0/G1 obviously(P=0.022).TSPYL5 also repressed their migration and invasion.Compared with the NC,the rate of cell apoptosis was significantly increased(P<0.01),whereas the ability of migration and invasion were effectively reduced(P<0.01).2.The binding potential of TSPYL5 and mi R-629 in potential targets from mi RWALK is relatively high.The dual luciferase assay demonstrated that mi R-629 inhibited the luciferase activity of the wild-type TSPYL5 3'UTR but prevented this inhibition in the mutated 3'UTR.3.Cell function results showed that after the cells were transfected with the mi R-629 inhibitor,apoptosis was decreased,G0/G1 phase was increased,and cell migration rate was increased.However,co-transfection of TSPYL5-si RNA significantly blocked apoptosis,cell cycle,migration and invasion.OVCAR3 cells in the blank group had the most active migration and invasion.Conclusion: Mi R-629 suppressed the expression of TSPYL5 in OC cells by directly binding the 3'UTR of TSPYL5.On the contrary,the intervention of mi R-629 can inhibit the malignant behavior of ovarian cancer.TSPYL5 plays a role in tumor suppressor genes in ovarian cancer.Part ?:The study of mi R-629 inhibiting the growth of ovarian tumor in nude miceObjective: To study the effect of mi R-629 on ovarian tumor growth in vivo.Methods: Firstly,mi R-629 inhibitor lentivirus was used to infect OVCAR3 in order to obtain stable interfering tumor cells.Then it was injected into nude mice for subcutaneous tumorigenesis.The size of tumor was measured regularly.After 30 days,the tumors were removed.We used RT-PCR and Western blot to detect the expression levels of mi R-629 and TSPYL5 in the tumors.At the same time,HE staining was used to detect the pathological conditions of the tumor and immunohistochemistry was used to detect TSPYL5 expression.Results: The size of the tumors started to appear different on the 15 th day after the implantation.The differences continued to increase with continuing feeding.On the 30 th day,the tumors in the Inhibitor group were significantly smaller than those in the blank group and the control group.RT-PCR results showed that the expression of mi R-629 in the Inhibitor group was significantly lower than that in the blank group and the control group,whereas the expression of TSPYL5 was the opposite.Further Western blot results showed that TSPYL5 was highly expressed in inhibitors.At the same time,the results of immunohistochemistry showed that TSPYL5 was more highly expressed in the Inhibitor group than in the blank group and the control group;and the tissue showed necrosis.Conclusion: The study further verified in vivo that intervention of mi R-629 expression can increase the expression of TSPYL5 and inhibit the growth of ovarian tumors.