The Effect And Mechanism Of Capn4 In The Progress And The Chemosensitivity Of Cisplatin In Ovarian Cancer

Ovarian cancer characterized by insidious onset,strong metastasis,and poor prognosis.They are one of the common malignant tumors in the female reproductive system,and the mortality rate is the highest in gynecologic malignancies.In recent years,new advances in the diagnosis and treatment of ovarian cancer have significantly improved the long-term survival rate of patients whose malignancies were found early.However,because the clinical symptoms are not obvious,most patients with ovarian cancer have been diagnosed with pelvic metastasis or lymphatic metastasis,and the prognosis is very poor,the mortality rate is high,and the 5-year survival rate is still less than 40%.At present,cytoreductive surgery for ovarian carcinoma and postoperative basal chemotherapy(cisplatin plus paclitaxel-based)are standard treatments for ovarian cancer,but with the reduction of tumor drug chemosensitivity and drug resistance,the effect of current ovarian cancer clinical chemotherapy is still not ideal.Many scholars are constantly searching for more effective treatments and therapeutic drugs.However,the survival rate of patients with ovarian cancer has not been effectively improved.Therefore,it is urgent to explore the mechanism of its occurrence and development from the molecular biology level to find effective treatment methods and innovative therapeutic drugs.Calpains are a highly conserved family of calcium-dependent non-lysosomal cysteine proteases that selectively catalyze the hydrolysis of specific substrate proteins involved in various cellular processes including motion,proliferation and apoptosis.To date,15 calpain phenotypes have been identified in mammals.Among them,Capn4 is a small regulatory subunit of the calpain proteolytic system and plays an extremely important role in maintaining the stability and activity of calpain-1 and calpain-2.Many studies have confirmed that it is highly expressed in a variety of malignant tumors,and is closely related to the degree of malignancy and prognosis of cancer patients.However,before the implementation of this study,scholars didn’t have a comprehensive and in-depth study of the specific mechanism of Capn4 expression in ovarian cancer and its role.The role that Capn4 may play in the development and prognosis of ovarian cancer has not been reported yet.New therapeutic targets for early biomarkers or early diagnosis and early treatment of ovarian cancer are unclear.Therefore,our study aimed to investigate the expression of Capn4 in ovarian cancer tissues and its relationship with clinicopathological features and prognosis of ovarian cancer patients and to explore its role in the proliferation,apoptosis,invasion and migration of ovarian cancer cells.The molecular mechanism and further research on the role of cisplatin chemotherapy sensitivity in ovarian cancer.In order to clarify the role of Capn4 in improving the sensitivity of ovarian cancer cisplatin chemotherapy drugs and improving the prognosis of patients,it finally provides new experimental basis for targeted therapy of Capn4.Part Ⅰ: Expression of Capn4 in ovarian cancer and its correlation with prognosisOBJECTIVE: To detect the expression level of Capn4 gene in ovarian cancer tissues,and to analyze the relationship between the expression of Capn4 in ovarian cancer tissues and the clinicopathological parameters of ovarian cancer patients,and to investigate its correlation with the prognosis of patients with ovarian cancer.METHODS: A total of 113 cases of tissue specimens diagnosed in the First Affiliated Hospital of Nanchang University and 35 cases of normal tissues adjacent to ovarian cancer were used as control group.Real-time quantitative PCR was used.qRT-PCR,Western-blot and immunohistochemistry were used to detect the m RNA and protein expression levels of Capn4 and positive expression.The chi-square test was used to analyze the correlation between Capn4 expression and clinicopathological parameters.A 5-year overall survival follow-up was performed in patients with ovarian cancer.Kaplan-Meier survival curves were used to analyze the relationship between Capn4 expression and disease-free survival and overall survival.Univariate and multivariate Cox regression analysis were used to determine the relationship.Can Capn4 expression be a risk factor for the overall survival of patients with ovarian cancer?RESULTS: Compared with ovarian normal tissues,the m RNA and protein expression levels of Capn4 in ovarian cancer tissues were significantly increased(P < 0.05).The expression of Capn4 in ovarian cancer tissues was significantly higher than that in low expression groups(P = 0.0054).The results of chi-square test showed that the expression level of Capn4 was positively correlated with the clinical pathological parameters of FIGO,tumor metastasis and tumor grade(P < 0.05).The Kaplan-Meier survival curve analysis confirmed the negative correlation between the Capn4 expression and ovarian cancer survival and overall survival.The prognosis of patients with high expression of Capn4 was poor(P = 0.006).Cox regression analysis found that the expression of Capn4 could be an independent risk factor for prognosis of ovarian cancer patients(HR=2.157).,95% CI: 1.091-3.138,P = 0.014).Conclusion: The abnormal expression level of Capn4 in ovarian cancer tissues is positively correlated with the malignant degree of ovarian cancer,and it can be used as an independent risk factor for the prognosis of patients with ovarian cancer,and is closely related to the poor prognosis of patients.Part Ⅱ: The effect and mechanism of Capn4 in the progress of ovarian cancerOBJECTIVE: To observe and evaluate the effects of Capn4 on the biological behaviors such as proliferation,apoptosis,invasion and migration of ovarian cancer cells,and to explore its possible mechanism.METHODS: qRT-PCR was used to detect the expression of Capn4 in different grades of ovarian cancer cells(A278,SKOV3,and OVCAR-3)and normal ovarian epithelial cells.The transient transfection technique was used to interfere with Capn4 in SKOV3 and OVCAR-3 cell lines.Expression of Capn4 were detected in ovarian cancer cells after stable transfection of interfering RNA by qRT-PCR and Western-blot methods;MTS assay,colony formation assay,Transwell Migration Assay,Wound healing assay,flow cytometry The experimental methods were used to detect the expression of Capn4 after transient interference of Capn4-siRNA in ovarian cancer cell lines.The effects of Capn4 on the biological behaviors such as proliferation,invasion,migration and apoptosis of ovarian cancer cells were observed and evaluated.Western blot was further used.The method was used to detect the expression of proteins related to cell cycle,apoptosis,invasion and migration after Capn4 interference expression,in order to explore the possible molecular mechanism of its influence on effect.RESULTS: Compared with normal ovarian epithelial cells,Capn4 expression was significantly increased in different ovarian cancer cells(A278,SKOV3,OVCAR-3)(P < 0.05);transient transfection interference in ovarian cancer SKOV3,OVCAR-3 cells After Capn4-siRNA,the expression of Capn4 in Capn4-siRNA group was significantly down-regulated compared with the blank group(P < 0.05).In vitro cell experiments showed that Capn4 inhibited expression significantly reduced ovarian cancer after transient transfection of Capn4-siRNA interference sequence.Cell proliferation and cell cloning ability,and promote apoptosis of ovarian cancer cells;Capn4 inhibition of expression leads to cell cycle arrest in G0/G1 phase by decreasing key regulators Cyclin D1,CDK4 expression significantly,Capn4 inhibition can also affect the expression of matrix metalloproteinases MMP2,MMP9 and epithelial-mesenchymal transition-related proteins Vimentin and E-cadherin,which hinders the epithelial-mesenchymal transition process of ovarian cancer cells and inhibits their invasion and migration.Conclusion: Capn4 is highly expressed in ovarian cancer cells;interference with Capn4 expression can inhibit the proliferation,invasion and migration of ovarian cancer cells and promote the apoptosis of ovarian cancer cells.Part Ⅲ: Capn4 affects the chemotherapy sensitivity of cisplatin in ovarian cancer OBJECTIVE: To investigate the effect of Capn4 expression on cisplatin chemotherapy sensitivity of ovarian cancer and its possible mechanism.METHODS: The expression of Capn4 in ovarian cancer cells(SKOV3,OVCAR-3)was silenced by siRNA transient interference technique.The sensitivity of Capn4 to cisplatin chemotherapy in ovarian cancer was detected by MTS assay and the results were analyzed by flow cytometry.The effect of Capn4 on the susceptibility of cisplatin to chemotherapeutic drugs in ovarian cancer was examined and preliminarily investigated by western-blot method.RESULTS: After interfering with the expression of Capn4 in ovarian cancer cells(SKOV3,OVCAR-3),the proliferation of ovarian cancer cells was significantly decreased compared with the control group,and the IC50 value of cisplatin was significantly decreased(P < 0.05).Subsequent flow cytometry showed that the number of ovarian cancer apoptosis cells induced by cisplatin was significantly increased after Capn4 inhibited expression compared with the control group.In addition,the expression of Capn4 in cisplatin was dependent on concentration and time.Capn4 inhibition can lead to up-regulated expression of Caspase-3 and Bax proteins in tumor cell apoptosis,decreased expression of Bcl-2 and ERCC1,thereby increasing the chemosensitivity of cisplatin in ovarian cancer cells.Conclusion: The inhabitation of Capn4 increases the cisplatin drug sensitivity in ovarian cancer cells(SKOV3,OVCAR-3).The possible mechanism of action is that the expression of Capn4 affects tumor cell apoptosis related proteins Caspase-3,Bax,Bcl-2 and Nucleotide excision repair related protein ERCC1 expression.