Downregulation Of MiR-195 In Castration-resistant Prostate Cancer By Targeting HMGA1

The incidence of prostate cancer was explosive growth,but the specific cause is still not completely clear.In the early stages of the disease,almost all of the hormone-dependent prostate cancer(ADPC),after surgery castration or drug castration,the condition will be effectively controlled.Unfortunately,almost all patients will eventually develop into castration-resistant prostate cancer(CRPC).The clinical treatment of castration-resistant prostate cancer is tricky,after a series of chemotherapy radiotherapy and other systemic treatment,the final occurrence of metastasis,and lead to death.The current research focus is focused on how to prevent patients from hormone-dependent prostate cancer into castration-resistant prostate cancer,which molecular and biological factors involved,how to find the regulation of this change in drugs and methods.MiRNAs are the basic research hotspots at present.They play an important role in the biological processes such as proliferation,differentiation,growth,apoptosis,migration and invasion of cells through transcriptional regulation of target gene expression.MiRNA gene deletion,mutation and other changes in the synthesis process abnormalities,will lead to its level of expression disorders,may disrupt the cell proliferation,differentiation and apoptosis and other regulatory processes involved in the development and progression of the tumor,in cancer play an oncogene or The role of tumor suppressor gene.The current study found that a variety of miRNAs in the occurrence and development of prostate cancer play an important role.The effect of miR-195 on the biological behavior of hormone-independent prostate cancer cell lines was studied by in vitro and in vivo experiments.The downstream genes affected by miR-195 were identified and the genes of the high mobility group protein A1(HMGA1)Learning function.To enrich the theoretical basis of castration resistance of prostate cancer can provide experimental basis for new drugs.PART I Study of clinical relevanceDifferences in the expression of miR-195 in hormone-dependent prostate cancer and castration resistant prostate cancerOBJECTIVE:To study the effect of miR-195 on the biological behavior of hormone-independent prostate cancer cell lines DU145 and PC3 in vitro and in vivo.To investigate the downstream genes affected by miR-195 and to study the effect of miR-A1(HMGA1)biological function.METHODS:miRNAs were detected by miRNAs chip detection and found to be differentially expressed in the early ADPC and late CRPC tissues,and were further verified by qRT-PCR.The expression of miR-195 in the prostate cancer was analyzed by comparing the miR-195 expression in the differentiated differentiation of prostate cancer.The relationship between the expression of miR-195 and the prognosis of the patients was analyzed by using the gene chip data obtained from the GSE21034 database on the miRNA expression of prostate cancer.The results of microRNAs showed that some mi-RNAs were abnormal in ADPC and CRPC tissues,and qRT-PCR showed that miR-195 was significantly lower in CRPC than ADPC(p<0.05).In the data of GSE21034 database,the results of miR-195 expression in prostate cancer were positively correlated with Gleson score and tumor-free survival in prostate patients.CONCLUSIONS:The expression of miR-195 in CRPC is significantly lower than that in ADPC,suggesting that it may be a potentially important tumor suppressor gene for prostate cancer.The secondary analysis of GSE21034 database data suggests that miR-195 can be used as a prognostic factor for prostate cancer.PART ? Functional analysisThe influence of miR-195 on cell cycle,apoptosis,proliferation,migration,invasion and in vivo tumorigenesis of prostate cancer were observed.OBJECTIVE:The first part of the study suggests that the expression of miR-195 in CRPC is significantly lower than that in ADPC,suggesting that it may be a potentially important tumor suppressor gene for prostate cancer.We hope to further verify the specific role of miR-195 in prostate cancer through related functional experiments.To investigate the effect of miR-195 on cell cycle,cell apoptosis and cell proliferation of DU 145 cells and PC3 in ovariectomized prostate cancer cells and the effect of subcutaneous tumorigenesis in nude mice.Methods:In vitro experiments,DU 145 and PC3 cell lines were transfected into miR-195 in vitro.The ability of cell proliferation was detected by MTT assay and cell colony formation assay.The cell cycle and apoptotic rate were measured.In subcutaneous tumor of nude mice,the subcutaneous tumor of PC3 cells transfected with miR-195 was examined.RESULTS:MTT assay showed that cell proliferation rates were significantly lower in patients with hormone-resistant prostate cancer cells DU145 and PC3 than in negative control group(NC).In vitro clonal formation experiments,miR-195 inhibited the number and size of prostate cancer cell colonies.In the flow cytometry,the percentage of cells in the miR-195 group was increased at G0/G1 phase and the apoptotic rate was significantly higher than that of the NC group.In nude mice,the size of subcutaneous tumors of PC3 cells transfected with miR-195 was significantly smaller than that of the control group.CONCLUSIONS:In hormone-independent prostate cancer cell lines,miR-195 regulates the proliferation,cycle and apoptosis of prostate cancer cells.PART ? Molecular mechanism researchMiR-195 directly acts on HMGA1 to promote apoptosis of prostate cancer cellsOBJECTIVE:To predict the potential target gene of miR-195 in prostate cancer by bioinformatics analysis,suggesting that the structural transcription factor HMGA1 may be a potential target gene for miR-195 in prostate cancer.We attempted to further validate the role of HMGA1 in prostate cancer by experiments,and further experiments confirmed that miR-195 was able to inhibit the expression of HMGA1 directly with HMGA1 3'-UTR.Methods:GSEA was enriched and analyzed,and the target gene of miR-195 was observed.The expression of DU145 and PC3 cells transfected with miR-195 was detected by RNA sequence,and these differential genes were analyzed by GSEA.The potential target gene HMGA1 and the luciferase reporter gene of miR-195 were predicted by miRanda and TargetScan,and the binding ability of miR-195 to the potential target gene 3'UTR was detected.The expression of HMGA1 was detected by Western Blotting.Similarly,we analyzed the GSE35988 database data,compared the expression of HMGA1 in different types of prostate tissue,and analyzed the correlation between the expression of HMGA1 and the survival rate of prostate cancer.The differentially expressed genes between low expression and high expression of HMGA1 were analyzed by GSEA to find the gene pathway that HMGA1 might control.The expression of HMGA1 was then inhibited by small interfering RNA(siRNA).The proliferation of DU145 and PC3 cells was detected by MTT assay and in vitro cloning assay.The effects of cell cycle and apoptosis were detected by flow cytometry.The results of the study:GSEA enrichment analysis,suggesting that miR-195 may be mainly through the regulation of cycle-related genes affect cell function.Predictive software predicts that HMGA1 may be the target gene of miR-195.The results of luciferase reporter gene show that miR-195 can significantly inhibit the fluorescence of wild-type reporter gene.Western blot was used to detect the expression of HMGA1 in DU145 and PC3 cells.The expression of HMGA1 was significantly decreased after transfection with miR-195 compared with the transfected cells.The secondary analysis of GSE35988 data showed that the expression of HMGA1 in castration resistant prostate cancer was higher than that in HMGA1 group.The survival time and overall survival time of HMGA1 overexpression group were shorter than that of HMGA1 group.GSEA enrichment analysis showed that HMGA1 regulated genes were associated with cell cycle.In vitro transfection of si-HMGA1,MTT assay showed that si-HMGA1 could inhibit the proliferation of prostate cancer cells in Du145 and PC3 cells.Flow cytometry showed that the percentage of cells in the Si-HMGA1 group was increased at GO/G1 phase,indicating that the apoptotic rate was significantly higher than that of the control group.In nude mice,the expression of HMGA1 in miR-195 group was significantly decreased by immunohistochemical staining.CONCLUSIONS:HMGA1 is a direct target gene for miR-195,and HMGA1 plays a role in cancer progression in prostate cancer.