The Role And Mechanism Of MiR-3064-5p In The Proliferation And Metastasis Of Prostate Cancer Cells

Prostate cancer(PCa)is one of the most common cancer in men,and which is one of the main causes of death in male cancer patients.Though the impact of of PCa surgery and drug treatment has been greatly improved in recent years,PCa is prone to relapse and metastasis,and patients with advanced PCa eventually develop almost incurable,castration resistant prostate cancer(CRPC),which is almost incurable.This seriously endangers the quality of life and health of PCa patients.Therefore,exploring new diagnostic markers and therapeutic targets of PCa is of great obviously for the diagnosis and treatment of PCa.Studies in past have proven that micro RNA(miRNA)plays a significant regulatory role in a variety of malignant cancers.Micro RNA-3064-5p(miR-3064-5p)is a small molecular RNA located on human chromosome 17q23.3.Several studies have found that miR-3064-5p regulates the proliferation,invasion,migration,angiogenesis,stem cell maintenance and metabolism of tumor cells in many kinds of malignant tumors,such as gastric cancer,ovarian cancer,breast cancer,bladder cancer,etc.The previous study of the research group found that the m RNA level of miR-3064-5p was reduced in PCa cells,and using miR-3064-5p mimics could inhibit the proliferation and metastasis of PCa cells.However,the mechanism of miR-3064-5p participating in the proliferation and metastasis of PCa and the regulatory mechanism of its reduced expression in PCa cells are still unclear.Objective The aims was to explore the molecular mechanism of miR-3064-5p participating in the regulation of cell proliferation and metastasis in human PCa cell line,and the mechanism of miR-3064-5p expression imbalance in PCa cell line.To provide new targets and experimental basis for the molecular regulatory mechanism of miR-3064-5p in the occurrence and development of PCa and for the diagnosis and treatment of PCa.Methods 1.The expression of miR-3064-5p in different kinds of PCa cells,s and normal prostate epithelial cells was detected by qRT-PCR;2.Transfect miR-3064-5p mimics and miR-3064-5p inhibitor into DU145 and PC3 cells respectively by transfection reagent;3.Cell morphology staining and Western blot determined the optimal concentration of TGF-β1 inducing epithelial mesenchymal transition(EMT)in human PCa cells;using qRT-PCR detected the impact of TGF-β1 induction on the expression of miR-3064-5p;4.The impact of miR-3064-5p mimics transfection on the expression of EMT marker protein induced by TGF-β1 in PCa cells was detect by Western blot;4.Western blot was used to detect the effect of miR-3064-5p mimics transfection on the expression of EMT marker protein in PCa cells induced by TGF-β1;The effect of miR-3064-5p mimics on the migration and invasion ability of PCa cells induced by TGF-β1 was determined by cell function test.The impact of overexpression miR-3064-5p transfection on the proliferation of PCa cells and the expression of cell cycle regulating genes(CDK6,Cyclin D1)was detected by Western blot;5.Using tubule formation test,qRT-PCR and Western blot detected the impact of overexpression miR-3064-5p on the angiogenesis of human umbilical vein endothelial cells;6.Use bioinformatics software to predict the potential target gene of miR-3064-5p,screen the CTGF gene related to cell proliferation and metastasis as the research object,and verify the targeting relationship between miR-3064-5p and target gene CTGF through double luciferase test and Western blot test;7.The effect of si-CTGF transfection on the EMT and metastasis ability of PCa cells induced by TGF-β1 and the proliferation ability of PCa cells were determined by cell function test,qRT-PCR and Western blot;8.To further prove the regulatory role of miR-3064-5p and CTGF in TGF-β1-induced invasion and migration of PCa cells and cell proliferation through cell rescue experiment;9.Use bioinformatics software to analyze the methylation island of the miR-3064-5p promoter region,and detect the methylation level of the miR-3064-5p promoter region through methylation-specific PCR(MSP);Decitabine(DAC),a demethylating drug,was used to treat 22RV1 and DU145 cells.MSP and qRT-PCR were used to detect the effect of DAC on the methylation level and expression of miR-3064-5p promoter.Western blot was used to detect the level of EMT and proliferating protein.The miR-3064-5p inhibitor was transfected into the cells.At the same time,treated the cells by DAC,Cell function test was used to detect the proliferation and migration of cells.Results 1.The results of qRT-PCR showed that the expression of miR-3064-5p in PCa cells 22RV1,DU145 and PC3 was significantly lower than that in normal human prostate epithelial cells(P<0.001);2.PCa cell models with overexpression of miR-3064-5p and low expression of miR-3064-5p were successfully constructed by transient transfection technology;3.The results of cell morphology staining and Western blot presented that PCa cells could undergo significant EMT after being induced by TGF-β1 with a concentration of 10ng/m L;At the same time,the expression level of EMT marker protein also changed correspondingly(P<0.05);The results of qRT-PCR showed that the expression level of miR-3064-5p decreased in a dose-dependent manner with the increase of TGF-β1 concentration(0,5,10 ng/m L)(P<0.05);4.The results of Western blot,scratch healing test and Transwell test proved that overexpression of miR-3064-5p could obviously reverse the protein expression changes of EMT-related markers induced by TGF-β1(P<0.05),and obviously inhibit the metastasis of PCa cells induced by TGF-β1(P<0.05);The results of qRT-PCR and Western blot showed that overexpression of miR-3064-5p significantly inhibited the protein expression level of G1 phase cycle regulation genes CDK6,Cyclin D1 and proliferation marker PCNA(P<0.05);5.The results of tubule formation test,qRT-PCR and Western blot showed that overexpression of miR-3064-5p significantly inhibited the tubule formation ability of umbilical vein endothelial cells(HUVECs),and the expression m RNA level of VEGF-A in PCa cells was obviously inhibited(P<0.05);6.Bioinformatics database prediction showed that CTGF gene related to EMT and proliferation of tumor cells was a potential target gene of miR-3064-5p.The results of double luciferase report experiment proved that miR-3064-5p.The results of double luciferase report experiment showed that miR-3064-5p significantly inhibited the luciferase activity of CTGF wild-type group(P<0.01),and the results of Western blot showed that miR-3064-5p could significantly inhibit the expression of CTGF protein in DU145 and PC3 cells(P<0.01);7.Western blot proved that the protein expression level of CTGF in three PCa cell lines 22RV1,DU145 and PC3 was obviously higher than that of normal cell line RWPE-1(P<0.05);The results of scratch healing test,Transwell test,qRT-PCR and Western blot showed that si-CTGF transfection could significantly inhibit the EMT and metastasis of PCa cells induced by TGF-β1(P<0.05);The results of cell clone formation test,CCK8 test,qRT-PCR and Western blot showed that transfected si-CTGF could significantly inhibit the proliferation of PCa cells(P<0.05),and the expression of proliferating gene Cyclin D1 and PCNA was also significantly decreased(P<0.05);8.The results of rescue experiment showed that siCTGF transfection could significantly reverse the promotion of miR-3064-5p inhibitor on the metastasis and proliferation of PCa cells(P<0.05);9.Bioinformatics analysis showed that there was a methylation island in the 1977 bp-2075 bp of the miR-3064-5p promoter region.The MSP detection results showed that the methylation level of the miR-3064-5p promoter region of 22RV1 and DU145 cells was significantly higher than that of the RWPE-1 cells;After DAC treatment,the methylation degree of miR-3064-5p promoter region and the protein expression of methyltransferase 3B in 22RV1 and DU145 cells were significantly decreased,while the expression of miR-3064-5p was significantly increased.At the same time,DAC treatment promoted the expression of E-Cadherin and inhibited the expression of Vimentin and PCNA(P<0.05).The results of rescue test showed that DAC could reverse the inhibitory effect of low expression of miR-3064-5p on the proliferation and migration of PCa cells(P<0.05).Conclusion 1 Mi R-3064-5p showed low expression in PCa cells,and overexpression of miR-3064-5p could inhibit TGF-β1 induced cell EMT,migration and invasion,and angiogenesis;2.miRNA-3064-5p can significantly inhibit the EMT,migration and invasion of PCa cells induced by TGF-β1 and inhibit the proliferation of PCa cells by inhibiting the expression of Cyclin D1 by targeting CTGF and regulating the expression of CTGF;3.The low expression of miR-3064-5p in prostate cancer cells is related to the hypermethylation of the promoter DNA.DAC treatment can reduce the DNA methylation level of the promoter region of miR-3064-5p,reactivate the expression of miR-3064-5p,and restore its anti-cancer effect.