LEF1Targeting EMT In Prostate Cancer Invasion Is Regulated By MiR-34a And Mediated By MiR-181a

Part one:LEF1associated miRNA high-throughput screening and verification in prostate cancer cell linesObjectives Screening and verifying LEF1associated miRNAs expression difference.Methods LNCaP(low LEF1expression), LNCaP-AI(high LEF1expression) and LNCaP-AILEF1(LEF1knocdown) three cell lines were used for miRNAs array analysis. TaqMan real time PCR assay was used for verifying miRNAs array data.Results Of687miRNAs when performing a cluster analysis between LNCaP-AI to LNCaP and LNCaP-AI to LNCaP-AILEF1shRNA,18miRNAs showed changes of at least2-fold with miR-34a showing6.79fold decrease and miR-181a as12.36fold increase in group of LNCaP-AI to LNCaP. We confirmed the expression levels of top9up-regulated and down-regulated miRNAs by TaqMan real time PCR assay, and the real time PCR results were consistent with above.Conclusions miR-34a and miR-181a have a negative and positive correlation with LEF1respectively, which was successfully selected for further study of the mechanism between LEF1and miRNAs. Part two:Study the effects of miR-34a and miR-181a on EMT and invasion in vitro in prostate cancerObjectives To evaluate the effects of miR-34a and miR-181a on EMT and invasion in vitro in prostate cancer.Methods Chemosynthetic microRNA oligonucleotides miR-34a or miR-181a mimic and inhibitor were used for miRNA overexpression and downexpression; Western-blot was used for detecting E-cadherin and N-cadherin expression level as EMT marker; BD matrigel and transwell chamber were used for invasion and migration evaluation.Results Reduced EMT phenotype and invasion capacity was observed with miR-34a overexpression or miR-181a downexpression in LNCaP-AI and C4-2B cell lines. Induced EMT phenotype and invasion capacity was observed with miR-34a downexpression or miR-181a overexpression in LNCaP and C4-2B.Conclusions miR-34a reduce EMT and invasion, miR-181a induce EMT and invasion in prostate cancer in vitro. Part three:Study the mechanism of LEF1associated miRNA of miR-34a and miR-181a on EMT and invasion in vitro in prostate cancerObjectives To determine the regulation mechanism of miR-34a, LEF1and miR-181a.Methods TaqMan real time PCR and Western blot were used for detecting mRNA and protein exrepssion level; dual luciferase reporter assay was used to confirm the specific miR-34a binding site on LEF13UTR mRNA. ChIP analysis was used to detect LEF1occupancy at each of four putative binding sites of has-miR-181a-2promoter region.Results miR-34a negatively regulated LEF1of mRNA and protein level via directly targeting LEF13’UTR, meanwhile, miR-34a negatively regulated miR-181a. LEF1transcriptional enforce the expression of miR-181a via directly targeting has-miR-181a-2upstream promoter region.Conclusions miR-34a-LEF1-miR-181a axis play an important role in regulating PCa EMT and invasion.