The Effect And Mechanism Of Timeless On Ovarian Cancer Cell Proliferation And Cisplatin Sensitivity

As a highly lethal and malignant tumor in gynecology,ovarian cancer has caused about 125000 deaths worldwide every year.Especially for advanced ovarian cancer,about 30%of patients have a survival rate of only 5 years after the initial diagnosis.The diagnosis is in advanced stage,70%of patients with advanced ovarian cancer is an important reason for the high mortality rate of ovarian cancer,and another reason is that many patients are generally resistant to chemotherapy drugs.Therefore,the screening and development of targeted therapeutic molecules and the use of targeting drugs to enhance the sensitivity of ovarian cancer to chemotherapy are urgent scientific and clinical issues to be solved.Through R&D and clinical research,the treatment efficacy of ovarian cancer will be improved,which will be of great value to improve the prognosis of patients.PART ONE:Functional research of TIMELESS in ovarian cancerObjective:To investigate the expression of TIMELESS in ovarian cancer and the effect of TIMELESS on proliferation,apoptosis,colony formation and cisplatin sensitivity of ovarian cancer cells.Method:The expression levels of TIMELESS mRNA and protein in normal ovarian tissues,ovarian cancer tissues and four ovarian cancer cell lines(A2780,HO-8910,OVCAR3,SKOV3)were detected by bioinformatics analysis,tissue microarray and Western Blot(WB).The expression of TIMELESS was knocked down in vitro by RNAi through co-transfection of chemically synthesized siRNAand lipofectamine3000.Real-time quantitative PCR(RT-qPCR)and Western Blot were used to detect the changes of TIMELESS at mRNA and protein levels,CCK-8 was used to detect the effects on the proliferation of A2780,HO-8910,OVCAR3 and SKOV3 cell lines,and the soft agar colony formation assay was used to determine the effects on the clone formation ability of the above cell lines.Annexin V/7-AAD staining was used to detect the apoptosis of ovarian cancer cells in each group,and Western Blot was used to detect apoptosis-related indicators,such as apoptosis protein(Cleaved Caspase-3)and senescence-related indicators,such as senescence marker P21.Finally,the combined administration of TIMELESS siRNA and cisplatin was used to detect its effect on cisplatin sensitivity of ovarian cancer cells by CCK-8.Results:1.Bioinformatics analysis based on in-depth database mining and RT-qPCR results showed that the expression of TIMELESS in ovarian cancer tissues was significantly higher than that in normal tissues(P<0.05),which suggested that TIMELESS might play an important role in the development and progression of ovarian cancer.2.The results of tissue microarray showed that the positive rate of TIMELESS was 53%(21/40)in all types of ovarian cancer.3.The expression of TIMELESS mRNA and protein in four ovarian cancer cell lines(A2780,HO-8910,OVCAR3,SKOV3)was significantly higher than that in normal ovarian epithelial cells by RT-PCR and Western Blot.4.In ovarian cancer cell lines SKOV3 and A2780,two siRNA targets siRNA-1(siTIM 1392)and siRNA-2(siTIM 3181)were successfully confirmed to significantly inhibit the expression of TIMELESS at both mRNA and protein levels(p<0.01).5.The results of CCK-8 proliferation assay and colony formation assay showed that TIMELESS knockdown could significantly inhibit the proliferation(p<0.01)and colony formation(p<0.05)of ovarian cancer cells.6.The results of Annexin V/7-AAD double staining showed that the apoptosis level of TIMELESS knockdown group was significantly higher than that of siRNA control group(p<0.05).7.Compared with the control group,the TIMELESS knockout group,Both apoptosis protein(Cleaved Caspase-3)and senescence protein(p21)were up-regulated(p<0.05);8.Under The condition of increasing the concentration of cisplatin,the proliferation of siRNA-NC,siTIM-1 and siTIM-2 was inhibited,and the IC50 values were significantly decreased.Therefore,the sensitivity of ovarian cancer cells to cisplatin can be significantly increased by siRNA knockdown of TIMELESS.Conclusion:TIMELESS is highly expressed in ovarian cancer tissues and cells,suggesting that TIMELESS plays an important role in the development of ovarian cancer.In ovarian cancer cells,silencing TIMELESS by RNAi technology revealed that TIMELESS knockdown could significantly inhibit proliferation and colony formation,promote apoptosis and senescence,and enhance the chemosensitivity of ovarian cancer cells to cisplatin.PART TWO:Influence of TIMELESS silencing on cisplatin sensitivity of ovarian cancer cellsObjective:To investigate the role of TIMELESS in the DNA damage response of ovarian cancer cells,and explore the possible mechanism of its effect on cisplatin sensitivity of ovarian cancer cells,so as to provide new clues for clinical chemotherapy.Method:Western Blot was used to detect the expression of γ-H2AX in A2780 and SKOV3 cells after TIMELESS knockdown,and comet assay was used to detect the level of DNA damage.The siRNA targeting the 3 ’-UTR region of TIMELESS was designed and co-transfected with TIMELESS over-expression plasmid,and the role of TIMELESS in DNA damage repair was verified again by rescue experiment.After Treatment with TIMELESS siRNA and cisplatin(1 μg/ml),Western Blot was used to detect DNA damage and repair-related proteins such as γ-H2AX.Rad51 and DNA damage repair pathway proteins p-ATR and p-CHK1;Western Blot was used to detect the changes of apoptosis-senescence pathway related protein p53-p21.Result:1.TIMELESS knockdown resulted in the up-regulation of γ-H2AX,the lengthening of comet tail and the increase of gray scale,suggesting that a higher level of DNA damage occurred in TIMELESS knockdown A2780 cells.2.Exogenous TIMELESS expression reverses DNA damage caused by siRNA targeting the 3 ’-UTR region of TIMELESS;3.Knock-down of TIMELESS can down-regulate the expression of homologous recombinant protein Rad51.The DNA damage repair function of ovarian cancer cells was impaired.4.Although the ATR-CHK1 pathway was activated in the TIMELESS knockdown group after treatment with TIMELESS siRNA and cisplatin,it was significantly stronger than that in the control group,so the level of γ-H2AX in the group was also higher than that in the control group,resulting in sensitive response to DNA damage.5.The expression of p21 protein in A2780 and SKOV3 cells increased,which was independent of p53 pathway.The expression of p21 protein in TIMELESS knockout group was always higher than that in control group.Conclusion:TIMELESS plays an important role in the response of ovarian cancer cells to DNA damage.When DNA damage was induced by cisplatin,the activation of ATR-CHK1 pathway was inhibited and the repair function was impaired in TIMELESS knockdown group,which promoted the sensitivity of ovarian cancer cells to cisplatin.These studies may reveal the mechanism of TIMELESS in ovarian cancer and provide a new target for the treatment of ovarian cancer,and provide innovative strategies to improve patient outcomes.PART THREE:The effect of TIMELESS on cisplatin sensitivity of ovarian cancer cells in vivoObjective:To explore the effect of TIMELESS knockdown on cisplatin sensitivity of ovarian cancer cells in vivo,and to provide new evidence for chemotherapeutic clinical application.Methods:TIMELESS shRNA lentiviral vector was constructed and lentivirus are packaged.The stable cell line of TIMELESS knock-down was obtained by lentiviral infection and puromycin screening.The knockdown efficiency of mRNA expression was verified by real-time PCR.The knockdown efficiency at the protein level was verified by Western blot.A2780 stable cell line of TIMELESS knock-down was used to construct an animal model of transplanted tumor in nude mice.After tumor formation,cisplatin was injected subcutaneously at a dose of 3 mg/kg every 3 days,and the growth curve was recorded.One week after drug withdrawal,the nude mice were sacrificed,the tumor tissue was stripped,and the tumor volume was calculated.The changes of ATR/CHK pathway were detected by WB.Result:1.Compared with the control group,the mRNA level of TIMELESS knockdown stable cell was significantly decreased(P<0.001).2.Compared with the control group,the protein level of TIMELESS knockdown stable cell line was significantly decreased(P<0.001).3.In the nude mice model,the tumor volume of nude mice in the TIMELESS and cisplatin combined group was significantly reduced in vivo.The growth rate of subcutaneous transplanted tumor was significantly lower than that of the control group(P<0.001).3.ATR/CHK pathway related proteins were significantly inhibited(P<0.05).Conclusion:We have successfully constructed an ovarian cancer stable cell line(A2780)that can knock down the expression of TIMELESS at the mRNA and protein levels,and TIMELESS plays an important role in cisplatin resistance.In the xenograft tumor model,when cisplatin induced DNA damage,the TIMELESS knockdown group cells were inhibited,and the repair function was impaired,thus promoting the sensitivity of ovarian cancer cells to cisplatin in vivo.