The Role Of CAPN2 Gene In The Occurrence And Development Of Prostate Cancer And Its Regulatory Mechanism

In China,with the increase of tumor incidence and mortality,malignant tumors have become the main cause of death,tumor has become the major public health problems of China.Prostate cancer is one of the common malignant tumors of the urinary and male reproductive system.It is a common malignancy in older men.More than 95%of the diagnosed cases are between 45 and 89 years old,with an average age of 72 years.Prostate cancer morbidity and mortality vary by region,ethnicity,the highest incidence of black,followed by the United States whites,the lowest for the Orient.However,in China,due to increased awareness of physical examination and the aging of the population,the incidence of prostate cancer showed a rapid increase in the trend.Prostate cancer treatment,including surgery,radiotherapy,endocrine therapy,chemotherapy and targeted therapy and other comprehensive treatment measures,early prostate cancer patients usually have a good prognosis,but there is still a considerable part of prostate cancer patients after distant metastasis was diagnosed,leading to prognosis bad.For the study of how the prostate epithelium is transformed into a malignant tumor at the molecular level has made great progress in recent years,it has only recently begun to identify some carcinogenic and tumor suppressor genes that cause prostate cancer.These genes may be related to the occurrence and development of prostate cancer,but its specific mechanism of action and in the development of prostate cancer plays a role in the process there is no definite conclusion.Tumorigenesis and development are complex processes of multiple factors.Therefore,the study of prostate cancer should focus on exploring and discovering new molecular pathways of related genes and their roles,and further identifying potential therapeutic targets to improve early diagnosis and treatment of eancer,and provide new molecular markers for prognosis judgment.Recent studies have found that the calpain(CAPN)gene plays an important role in the normal physiological processes of human and the development of tumor.CAPN family contains more than ten subtypes,CAPN 1 and CAPN2 expression of the most extensive,and therefore attract the most attention.The study confirmed that CAPN plays an important role in the development and progression of various tumors such as hepatocellular carcinoma,pancreatic cancer,cholangiocarcinoma,endometrial carcinoma and breast cancer.But so far,CAPN2 in prostate cancer expression and role is not clear.The aim of this study was to investigate the expression and role of CAPN2 in prostate cancer,to explore the molecular mechanism of the development and progression of prostate cancer,and to provide new ideas for the early diagnosis and treatment of prostate cancerPart ?Expression of CAPN2 in prostate cancer and its clinical significance Objective:To explore the correlation between the expression level of CAPN2 gene in prostate cancer and its correlation with clinicopathological parameters and prognosis of patients.Materials and methods:The expression level of CAPN2 gene in prostate cancer tissues and paired normal tissues was detected by real-time fluorescence quantitative PCR(qRT-PCR)method and Blot Western method.The expression of CAPN2 was detected by immunohistochemical method in prostate cancer.Statistical analysis of the relationship between CAPN2 expression and clinical parameters and prognosis.Results:The results of qRT-PCR detection and Blot Western showed that the expression level of CAPN2 gene in prostate cancer tissue was higher than that in normal prostate tissues(P<0.05).Kaplan-Meier analysis showed that patients in PCa with high C APN2 experessed level had a shorter time of relapse free survival than patients with low expression of CAPN2(log-rank test,P<0.05).The expression level of CAPN2 in PCa patients was significantly correlated with tumor distant metastasis and recurrence free survival rate(P<0.05).Multivariate analysis showed that CAPN2 expression and tumor pathological grade were independent prognostic factors for recurrence free survival in patients with PCa.Conclusion:CAPN2 is highly expressed in prostate cancer tissues,the high level expression of CAPN2 in prostate cancer is related to distant metastasis of the tumor and biochemical recurrence.The expression level of CAPN2 were independent risk factors of PCa patients.Part ?Effect of CAPN2 on cell function of prostate cancer cells and its molecular mechanismObjective:To investigate the expression of prostate cancer cell line CAPN2 on cell proliferation,migration,invasion,cell cycle,and the possible molecular mechanism.Materials and methods:In PC3,DU145 and 22RVI of prostate cancer cell line,the overexpression and knock down model of CAPN2 was constructed by slow virus transfection,and the transfection efficiency was detected by real-time fluorescence quantitative PCR(RTpPCR)and Weatern blot.CCK-8 kit was used to detect the knockdown or overexpression of CAPN2 cell proliferation.The result was verified by cell cloning experiments.Transwell method was used to detect the migration and invasion ability.PI staining flow cytometry was used to detect the effects of low or over expression of CAPN2 on cell cycle.Results:We constructed a CAPN2 knock down cell line shCAPN2,a CAPN2 overespressed cell line and negative control SCH stable cell lines.Ability of cell proliferation of stabletransfection cell was detected by CCK-8.The results show that compared with the NC group,expression of CAPN2 cell proliferation was significantly increased(P<0.05),proliferation ability of shCAPN2 cell was decreased(P<0.05).This result was further verified in cell cloning experiment.Migration and invasion of the experimental results showed that compared with the control group NC,migration and invasion capacity was significantly enhanced after overexpressed CAPN2 in PCa cells(P<0.01),PCa cell migration and invasion ability was significantly reduced after CAPN2 knockdown(P<0.05).Flow cytometry was used to detect cell cycle results showed that knockdown CAPN2,compared with the normal control group(NC,prostate cancer cell line DU 145 and PC3 cells in G0/G1 phase ratio significantly increased,corresponding the proportion of S phase and G2/M phase decreased significantly(P<0.05),whereas overexpression of CAPN2 was on the contrary.The expression of CAPN2 in PC3,DU145 and 22RV1 cells was detected by Western blot.The results showed that CAPN2 could significantly down-regulate the expression of MMP9 and MMP2 protein,and overexpression of CAPN2 could significantly up-regulate the expression of MMP9 and MMP2,Suggesting that CAPN2 may promote the migration and invasion of prostate cancer by regulating the expression of MMP-related proteins.Detection of key protein expression levels of AKT/mTOR pathway showed that the expression levels of phosphorylated AKT and mTOR were significantly decreased in PC3 and DU 145 cells after knockdown of CAPN2.In 22RV1 cells overexpressing CAPN2,phosphorylated AKT and mTOR expression levels were significantly increased,the results show that CAPN2 may participate in AKT/mTOR signaling pathway to promote prostate cancer cell metastasis and proliferation.Conclusion:Experiments in vitro showed that CAPN2 could affect the proliferation,migration,invasion,cell cycle of PCa cell line,and affect the ability of tumor formation through MMP and.CAPN2-pAKT-mTOR pathway.