High-expression Of MELK In Cervical Cancer And Its Mechanism In DNA Damage Repair

Cervical cancer is the second most common malignant tumor in women,and it is the most common malignant tumor in the female reproductive system,which seriously endangers women's physical and mental health.Most of the early stage cervical cancer patients were treated with radical surgery and adjuvant chemoradiotherapy.The survival rate of advanced and recurrent cervical cancer did not improve significantly.How to enhance the sensitivity of cervical cancer to radiochemotherapy.It is of great significance to find new targeted therapy for cervical cancer,reduce recurrence and metastasis after radiochemotherapy,and improve survival rate.PART ONE : Correlation between high-expression of MELK and clinicopathological features in cervical cancerObjective: To explore the differential expression of MELK in normal cervical tissues,cervical intraepithelial neoplasia,cervical cancer and paracarcinoma tissues,and retrospectively analyze the clinical and pathological data of cervical cancer in all stages to explore the correlation between MELK expression and clinicopathological features,and the correlation with Ki67 expression.Methods:The expression of MELK in cervical cancer tissues and matched paracancerous tissues was detected by RT-qPCR and immunohistochemistry.MELK was detected by immunohistochemistry in MELK from different clinical specimens of normal cervix,CIN and cervical cancer.And the correlation of MELK and Ki67 expression in cervical cancer tissues of the same sample,retrospective analysis of clinical and pathological data of cervical cancer in each stage to explore the correlation between MELK expression and clinicopathological features.Results: 1.MELK gradually increased in the course of progression from normal cervix,CIN and cervical cancer(P=0.001);2.The high expression rate of MELK in cervical cancer was 56.92%(37/65);3.The expression level of MELK in cervical cancer tissues was significantly higher than that in the matched paracancerous tissues.4.There was no significant correlation between age of patients(P=0.544),serum squamous cell-associated antigen(P=0.668),pathological stage(P=0.456),depth of tumor invasion(P=0.397),and pathological type(P=0.602).MELK was found in patients with early cervical metastasis.The expression rate was higher in patients with early metastasis than early non-metastasis(P=0.034);5.The high expression rate of Ki67 was 86.49% higher in patients with high expression of MELK.The ratio of high expression is relatively low50.00% in patients with high expression of MELK(P = 0.02).Conclusion: The expression level of MELK in cervical cancer is significantly higher than that in the paracancerous tissues.The expression level of MELK is gradually increased from the normal stage of cervical,CIN and cervical cancer.The abnormal expression of MELK in cervical cancer,It has an impact on biological behaviors such as proliferation and metastasis of cervical cancer cells.PART TWO:Functional study of MELK in cervical cancer cellsObjective: This study examined the expression of MELK in four cervical cancer cell lines,and the effects of MELK knockdown and MELK inhibitors on proliferation,apoptosis and colony formation of cervical cancer cells.Methods: Bioinformatics analysis was used to detect MELK expression in cervical cancer,RT-qPCR and Western blot were used to detect MELK expression levels in four cervical cancer cell lines Si Ha,He La,C33 A and Cas Ki;knockdown of MELK in vitro using siRNA-mediated RNAi technology,Western blot was used to detect the transfection efficiency of MELK siRNA.The proliferation and clonality of cervical cancer cell lines were detected by CCK8 and clone formation assays respectively.The siRNA knockdown and inhibition were detected by Western blot.The expression of apoptosis-related proteins P53 and Cleaved Caspase-3 after the action of the agent.Results: 1.The differential expression of MELK m RNA between cervical cancer tissues and normal cervical tissues was determined by analyzing the Oncomine microarray gene expression dataset.The results showed that the expression level of MELK in cervical cancer tissues was significantly higher than that in the corresponding normal cervical tissues(P< 0.001).2.Cell proliferation assay showed that cell proliferation was significantly inhibited 72 h to 120 h after transfection of MELK siRNA;The inhibitor OTSSP167 after 72 h to 120 h,cell proliferation was significantly inhibited,and the inhibition became stronger with time and concentration.3.The clonality of cervical cancer C33 A and He La cells transfected with MELK siRNA was significantly decreased,and the ability of colony formation was also significantly decreased after the inhibitor of concentration of OTSSP167,and the decrease of colony formation ability was more significant with the increase of inhibitor concentration,the difference was statistically significant(P<0.05).4.The expression of apoptosis-related protein P53 and Cleaved Caspase-3 was significantly increased after siRNA knockdown.The expression of apoptosis-related protein P53 and Cleaved Caspase-3 was also significantly increased by using different concentrations of inhibitor OTSSP167.Within a certain concentration range(0n M-5n M),the expression rates of P53 and Cleaved Caspase-3 increased with the increase of inhibitor concentration,and the difference was statistically significant(P<0.05).Conclusion: MELK is expressed in all four cervical cancer cell lines;MELK knockdown and inhibitor OTSSP167 can inhibit the proliferation of cervical cancer C33 A,He La,Cas Ki and Si Ha cells;MELK knockdown and inhibitor OTSSP167 can inhibit The clonality of cervical cancer C33 A and He La cells;MELK knockdown and inhibitor OTSSP167 can promote apoptosis of cervical cancer C33 A cells.PART THREE:Molecular mechanism of MELK-mediated DNA damage repair in cervical cancer cellsObjective: This section explores the molecular mechanism of targeting MELK-mediated DNA damage repair in cervical cancer.First,under the action of siRNA and inhibitor,DNA damage repair proteins ?-H2 AX,p-ATM,p-CHK2,Mre11,Rad50 combined with chemotherapy drugs Cisplatin,observed changes in DNA damage repair proteins,and the role of MELK in the DNA damage repair pathway homologous recombination repair pathway and non-homologous end joining repair pathways.Methods: The sensitivity of each group of cells to cisplatin chemotherapy was detected by CCK8 method.The expression of ?-H2 AX,p-ATM and p-CHK2 was detected by Western blot after siRNA knockdown.The different concentrations of inhibitor OTSSP167 acted on cervical cancer cell C33 A,DNA Expression of damage protein?-H2AX;detection of ?-H2 AX foci in C33 A and He La cells by MELK knockdown group by immunofluorescence assay;Western blot analysis of DNA damage protein ?-H2 AX in siRNA knockdown combined with DDP group expression of P53,Caspase-3,Rad50,Mre11 and Ku70/80.Results: 1.CCK8 method was used to detect the sensitivity of each group to cisplatin.With the increase of cisplatin concentration,the cell proliferation of siRNA knockdown group was inhibited,and the semi-inhibitory concentration(IC50)of cisplatin was significantly different among each group.2.After MELK siRNA knockdown,the expression level of ?-H2 AX in cervical cancer cell C33 A was increased.After the inhibitor OTSSP167 acted on cervical cancer cell C33 A,the expression level of ?-H2 AX increased gradually,in the concentration range of 0-10 n M/ml,the expression of ?-H2 AX increased with the increase of inhibitor concentration.The results of immunofluorescence assay confirmed that there were more ?-H2 AX foci formation in C33 A and He La nucleus of MELK knockdown group compared with Ctrl siRNA transfected cells.3.Western blot analysis showed that compared with the control group,the expression of DNA damage breakpoint marker protein ?-H2 AX,apoptosis-related protein P53 and Caspas-3 increased in MELK siRNA knockdown combined with DDP group,the difference was statistically significant(P<0.05);4.Cervical cancer cell line C33 A was transfected with Ctrl siRNA,siRNA#1,and siRNA#2,respectively.Compared with the control group,the expression levels of p-ATM and p-CHK2 in the MELK knockdown group were increased,ATM-CHK2 pathway suggests that MELK knockdown leads to replication stress;5.The expression levels of HR repair proteins Rad50 and Mre11 in MELK siRNA combined with DDP group were significantly lower than those in DDP group,but there was no significant correlation between NHEJ repair protein Ku70/80 and MELK knockdown combined with chemotherapeutic drug DDP.Conclusion: Down-regulation of MELK significantly enhanced the sensitivity of C33 A cells to DDP.Down-regulation of MELK could cause DNA damage in cervical cancer cells.MELK knockdown could induce activation of ATM/CHK2 signaling pathway.MELK may participate in DNA damage repair through homologous recombination repair pathway.