The Role And Mechanism Of WTAP Gene In The Progression And Development Of Prostate Cancer

Prsotate cancer is a common genitourinary malignacy. The incidence of prostate cancer was higher than lung cancer and has become the first harmful factor that endanger the health of men in USA. The incidence of prostate cancer is rising rapidly according to the popularity of the screen of PSA, the change of lifestyle and the improvement of biopsy technique. Combined treatment can usually lead to a relatively good prognosis for the patients in early stage of prostate cancer. However, there is still a considerable part of the prostate cancer patients get diagnosed after the distant metastasis, which leading to a poor prognosis. Patients with advanced prostate cancer can be treated with endocrine therapy to control and improve the condition, but most of them will still progress to castration resistant prostate cancer, which means a very poor prognosis. Therefore, to explore the molecular pathways and related genes in the development of prostate cancer, to elucidate the mechanism of carcinogenesis and development and to find the molecular mechanism of metastasis and drug resistance are of great significance to improve the efficiency of early diagnosis of prostate cancer,the exploration of new therapeutic targets and the judgment of the prognosis.The expression and function of genes in cancer have always been the focus of the research on the mechanism of tumor development and metastasis, and also the focus of the early diagnosis, targeted therapy and prognosis of cancer. Recent studies have found that the WTAP gene plays an important role in the normal physiological processes of human and the development of tumor. WTAP can be combined with the WT1 gene specifically in vivo and in vitro. WTAP can also be involved in m6A methylation modification, RNA alternative splicing, cell cycle regulation and other physiological processes of the body. WTAP plays an important role in the occurrence and development of many kinds of malignant tumors such as glioblastoma, cholangiocarcinoma, acute myeloid leukemia and colorectal cancer. However, the expression and role of WTAP in prostate cancer are not clear.The purpose of this study is to explore the expression and role of WTAP in prostate cancer, to explore the molecular mechanism of the occurrence and progression of prostate cancer, and to provide new ideas for the early diagnosis and treatment of prostate cancer.Part Ⅰ Expression of WTAP in prostate cancer and its clinical significanceObjective:To explore the correlation between the expression level of WTAP gene in prostate cancer and its correlation with clinicopathological parameters and prognosis of patients. Materials and methods:The expression level of WTAP 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 WTAP was detected by immunohistochemical method in prostate cancer. Statistical analysis of the relationship between WTAP expression and clinical parameters and prognosis. Results:The results of qRT-PCR detection and Blot Western showed that the expression level of WTAP 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 WTAP experessed level had a shorter time of relapse free survival than patients with low expression of WTAP(log-rank test, P< 0.05). The expression level of WTAP in PCa patients was significantly correlated with tumor distant metastasis and recurrence free survival rate(P<0.05). Multivariate analysis showed that WTAP expression and tumor pathological grade were independent prognostic factors for recurrence free survival in patients with PCa.Conclusion:WTAP is highly expressed in prostate cancer tissues, the high level expression of wtap in prostate cancer is related to distant metastasis of the tumor and biochemical recurrence. The expression level of WTAP were independent risk factors of PCa patients.Part Ⅱ Effect of WTAP on cell function of prostate cancer cells and its molecular mechanism Objective:To investigate the expression of prostate cancer cell line WTAP on cell proliferation, migration, invasion, cell cycle, animal tumor formation ability and the possible molecular mechanism.Materials and methods:In PC3, DU145 and LNCaP of prostate cancer cell line, the overexpression and knock down model of WTAP was constructed by slow virus transfection, and the transfection efficiency was detected by real-time fluorescence quantitative PCR (RT-PCR) and Weatern blot. CCK-8 kit was used to detect the knockdown or overexpression of WTAP 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 WTAP on cell cycle. PC3 cells or control cells with low or over expression of WTAP were injected subcutaneously in mice to observe the ability of cells to form tumors in vivo.Results:We constructed two WTAP knock down cell line shWTAP1 and shWTAP2, a WTAP 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 WTAP cell proliferation was significantly increased (P< 0.05), proliferation ability of shWTAP1, shWTAP2 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 WTAP in PCa cells (P< 0.01),PCa cell migration and invasion ability was significantly reduced after WTAP knockdown(P<0.05). Flow cytometry was used to detect cell cycle results showed that knockdown wtap, compared with the normal control group (NC, prostate cancer cell line DU145 and PC3 cells in GO/G1 phase ratio significantly increased, corresponding the proportion of S phase and G2/M phase decreased significantly (P< 0.05), whereas overexpression of WTAP was on the contrary. Nude mice subcutaneous tumor test results showed that 28 days after inoculation of tumor cells in the control group, the weight of the low WTAP group was240±29mg,120±10mg,70±12mg (mean±standard deviation, P< 0.05). We found that vimentin and β-catenin is low expression in PC3, DU145, LNCaP cell lines which silenced by lentivirus (P<0.05); while the level of expression were high on the other hand (P>0.05). The results of western blot also reveled that the levels of WT1 and TBL1 were high in PC3, DU145 and LNCaP cell lines with WTAP overexpression (P<0.05); while the levels were low in WTAP knockdown prostate cell lines (P< 0.05).Conclusion:Experiments in vitro showed that WTAP could affect the proliferation, migration, invasion, cell cycle of PCa cell line, and affect the ability of tumor formation through EMT and. WTAP-WT1-TBL1 pathway.