The Influence And Molecular Mechanism Of Mir-145-5p On Proliferation And Migration Of Prostate Cancer Cells

BackgroundProstate cancer(PCA)is a malignant tumor that occurs in the prostate epithelium,which is associated with genetic factors and increases with age.Prostate cancer is more frequent in European and American countries,the fatality rate is second only to lung cancer.In recent years,the incidence and mortality of prostate cancer in China have been increasing year by year.Compared with developed countries in Europe and the United States,the proportion of newly diagnosed prostate cancer patients in China with advanced stage of prostate cancer is significantly higher.At present,the pathogenesis and development mechanism of prostate cancer are not yet clear.MicroRNA(miRNA)is closely related to the occurrence and development of PCa,about 50%get annotations of miRNAs in genome associated with tumors located in fragile site,similar to the function of tumor suppressor genes and oncogenes.Researchs showed that miR-145 played an anti-cancer role in neuroblastoma,osteosarcoma,cervical carcinoma,and prostate cancer.However,its regulatory mechanism needs to be further clarified.ObjectTo study the effects of miR-145-5p on the proliferation and migration of prostate cancer cells,and possible molecular mechanisms,to provide new ideas for revealing the pathogenesis of prostate cancer,and to provide possible new targets for prostate cancer prevention and treatmentPart 1 Influence of miR-145-5p on the proliferation and migration of prostate cancer cellsMethodThe lentivirus expression vector of miR-145-5p was constructed,and the stable cell line(LNCaP)that overexpressed miR-145-5p was established,and the blank control group(control),negative control group(negative control)and experimental group(miR-145-5p)were set up.The inhibition rate of cell proliferation was detected by WST-8.Early apoptosis rate was detected by flow cytometry.The clone formation test was used to detect the cell clone formation ability,and the ability of transwell to detect cell invasion and migration.The relative expression level of protein was detected by western-blotting.The effect of miR-145-5p overexpression on the growth of transplanted tumor was observed by constructing a nude mouse model of tumor formation in prostate cancer.ResultsIn this study,miR-145-5p lentiviral vector was successfully constructed,an d the virus titer was 1.25 ×105 TU/ml.We successf?lly established stable cell 1 ine(LNCaP)with miR-145-5p.A stable cell line overexpressing miR-145-5p w as successfully established.The inhibitory rate of overexpression of miR-145-5 p on LNCaP cells was 12.55±0.21%(24h),25.77±2.75%(48h)and 36.54±3.81%(72h)respectively.After overexpression of miR-145-5p for 48 hours,the e arly apoptosis rate of LNCaP cells was 7.81±0.25%(3.2±0.31%in control grou p,2.6±0.31%in NC group).The rate of clone formation was 36±0.55%(67±0.64%in control group,62±0.35%in NC group).The number of cell migration was 152117%(265±15%in control group,280±19%in NC group).The number of cell invasion was 42±5%(254±10%in control group,263±14%in NC group).After the stable cells at a certain concentration was injec ted into the nude mice for 29 days,the weight of the obtained tumor was 0.11±0.02g in overexpression miR-145-5p group,0.30±0.02g in the control group,and 0.28±0.02g in the NC group.The relative expression level of E-cadherin p rotein in tumor tissues was increased by western-blotting,while the expression1 evel of P-catenin,MMP2 and MMP9 protein decreased,which were no signifi cant change in the blank control group and NC group.Part 2:To study the molecular mechanism of miR-145-5p on the proliferation and migration of prostate cancer cells.MethodBioinformatics software targetscan was used to predict target genes regulated by miR-145-5p.Chipbase was used to predict the transcription factors of binding sites in the upstream promoter region of miR-145-5p.RT-PCR was used to detect the relative expression level of genes.The binding effect of miR-145-5p with target genes was verified by constructing a dual fluorescent reporter gene vector.The transcription initiation site of miR-145-5p was confirmed by 5'RACE.The binding of transcription factors to miR-145-5p was detected by chromatin immunoprecipitation.ResultThe results of bioinformatics analysis suggested that Sentrin-specific protease 1(SENP1)was the target gene regulated by miR-145-5p and there were multiple binding sites of transcription factor CDX2 in the miR-145-5p promoter region.Further verification revealed that the transcription initiation site of miR-145-5p was located at the upstream promoter region 1408bp Guanine(G).Transcription factor CDX2 could bind to the miR-145-5p promoter region and inhibit its transcription.miR-145-5p could bind to 3'UTR of SENP1 and inhibit SENP1 protein translation.Conclusion1.Overexpression miR-145-5p could significantly inhibit the proliferation,clonogenic formation,invasion and migration of LNCaP cell and also promote early apoptosis.In vivo tests confirmed that overexpression of miR-145-5p inhibited the growth of prostate cancer xenografts.2.CDX2 inhibits the expression of miR-145-5p,thereby releasing the inhibitory effect of SENP1 on the translation of miR-145-5p and affecting the invasion and migration of prostate cancer cells...