Study On The Effect Of SIRT7 On The Biological Behavior Of Prostate Cancer Cells And Its Mechanism

Objective:To investigate the expression of SIRT7 gene in prostate cancer and analyze its correlation with clinicopathological stage and survival of the patients.In addition,knockdown SIRT7 expression in prostate cancer cell lines in vitro and in vivo to investigate the effect of SIRT7 on proliferation,metastasis,autophagy,chemotherapy and radiotherapy sensitivity of prostate cancer and determine its possible mechanism.Methods:1.First,we determined the expression of SIRT7 in prostate cancer by analyzing the data in Oncomine database and results of immunohistochemistry assay.And then,the correlation between SIRT7 expression and clinical pathological data was analyzed.Thirdly,the levels of SIRT7 m RNA and protein in normal prostate epithelial cells and prostate cancer cell lines were detected by real-time quantitative PCR(RT-q PCR)and Western blot,respectively.Finally,gene expression profile interactive analysis tool(GEPIA)was used to analyze the effect of SIRT7 on the overall survival(OS)and disease-free survival(DFS)of patients with prostate cancer.2.First,the prostate cancer cell models with down-regulation and overexpression of SIRT7 were constructed in vitro.Then,the effect of SIRT7 on the proliferation of prostate cancer cells was detected by CCK8 assay,Ed U assay and colony forming assay.The effect of SIRT7 on the migration and invasion of prostate cancer cells was detected by cell scratch test,Transwell cell migration and invasion assay.The effect of SIRT7 on the androgen-induced autophagy of prostate cancer cells was determined by Transmission electron microscope(TEM),RFP-GFP-LC3 adenovirus infection assay and Western blot.Finally,flow cytometry apoptosis detection and CCK8 toxicity test were used to detect the effect of SIRT7 on the tolerance of prostate cancer cells to docetaxel and radiotherapy.3.As to in vivo experiment,the effect of SIRT7 on proliferation and autophagy of prostate cancer cells was detected by subcutaneous tumorigenesis experiment and the proliferation and autophagy markers of transplanted tumor were detected by immunohistochemistry.The effect of SIRT7 on migration and invasion was verified by tail vein injection metastasis experiment.4.Firstly,the m RNA expression levels of SIRT7 and AR in prostate cancer were analyzed by bioinformatics data and the protein expression levels of SIRT7 and AR were detected by tissue microarray.Then,the changes of AR expression in SIRT7down-regulated prostate cancer cells were detected by cellular immunofluorescence staining to clarify the correlation between SIRT7 and AR.Inhibition of AR activity and recovery test were conducted to verify the role of AR in the regulation of prostate cancer proliferation and autophagy by SIRT7.5.The interaction between SIRT7,AR and SMAD4 was detected by coimmunoprecipitation,and the changes of SMAD4 protein level in SIRT7 downregulated prostate cancer cells were detected by Western blot to clarify the mechanism of SIRT7 regulating AR through post-transcriptional regulation of SMAD4.Results: 1.Bioinformatics analysis,immunohistochemistry of tissue microarray and cytological experiments showed that SIRT7 protein and m RNA level were relatively high expression in prostate cancer but low expression in normal prostate tissues.High SIRT7 expression was associated with clinical stage and Gleason score.Survival analysis suggested that high expression of SIRT7 was associated with poor prognosis in prostate cancer patients.2.The SIRT7 down-regulated stable prostate cancer cell models were successfully constructed.The proliferation and the level of androgen-induced autophagy were inhibited after down-regulating SIRT7 in LNCap and 22Rv1 cells.In Transwell assay,migration and invasion were decreased in SIRT7 down-regulated prostate cancer cells.And the apoptosis rate of down-regulated SIRT7 prostate cancer cells was increased after docetaxel treatments or radiotherapy.3.The in vivo experiment showed that after knocking down SIRT7,the volume and the quality of transplanted tumor decreased significantly.The metastasis signal of Luciferase in the down-regulated SIRT7 group was significantly lower than those in the control group.Immunohistochemical staining showed that the expression of Ki-67 and LC3II decreased in tumor tissues with knocking down SIRT7.4.The results of bioinformatics analysis,tissue microarray and cytological experiments showed that there was a positive correlation between the expression of SIRT7 and AR,and the effect of SIRT7 on the proliferation and autophagy of 22RV1 cells could be eliminated when AR was inhibited.5.The results of co-immunoprecipitation suggested that SIRT7 could interact with SMAD4,a negative regulator of AR.After knocking down SIRT7,the acetylation level of SMAD4 in prostate cancer cells increased and the degradation of SMAD4 protein decreased.Conclusion:1.The overexpression of SIRT7 is closely related to the progression of prostate cancer,suggesting that SIRT7 is a potential marker for predicting the prognosis of prostate cancer.2.Down-regulation of SIRT7 can lead to inhibition of the proliferation,migration,invasion and autophagy of prostate cancer cells,and increase the sensitivity of prostate cancer cells to docetaxel and radiotherapy,suggesting that SIRT7 plays an important role in the occurrence and development of prostate cancer.3.AR plays an important role in the regulation of proliferation and autophagy of prostate cancer by SIRT7 and down-regulation of SIRT7 can inhibit the expression and activity of AR.4.Down-regulation of SIRT7 can increase the level of SMAD4 acetylation and reduce the degradation of SMAD4 protein,resulting in down-regulating the expression of AR.