MicroRNA-140-5p Inhibits Hepatocellular Carcinoma By Directly Targeting The Unique Isomerase Pin1 To Block Multiple Cancer-driving Pathways

Objective: Our laboratory group take the effect of Pin1 on development and treatment of HCC as the main science research direction,stable knockdown of Pin1 and experimental filter of microRNAs targeting Pin1 were performed.In order to investigate the effect of mi R-140-5p on hepatocellular carcinoma,we taken Huh7 and PLC/PRF/5 as our research object to observe the effect of miR-140-5p on the cell proliferation,colony formation,migration and invasion by targeting Pin1 in vitro and in vivo,and explored the clinical significance of miR-140-5p-mediated Pin1 regulation using hepatocellular carcinoma clinical specimens.Our studies might uncover novel molecular mechanisms leading to the development of HCC,as well as might provide a new reference for treatment of HCC.Methods:(1)We packaged stable knpckdown Pin1 and miR-140-5p overexpression lentivirus and infected Huh7,PLC/PRF/5 cells to select the stable infected cell lines.(2)Using phase contrast microscope to observe morphological changes of Huh7 cells stably infected with lentiviruses expressing miR-140-5p and shPin1,and the change of Pin1 and EMT related proteins were detected by Western blot.(3)We carried out cell proliferation assay,colony formation assay and transwell assay for testing the migration and invasion of Huh7 cells stably infected with lentiviruses expressing miR-140-5p,shPin1,miR-140-5p combined with miR-140-5p-resistant Pin1 and miR-140-5p combined with shPin1.(4)The protein levels of cyclin D1,CDK2,NF-kB,Akt,pAkt-473,ERK and pERK were detected by Western blot after stable Pin1 knockdown,miR-140-5p overexpression and miR-140-5p combined with miR-140-5p-resistant Pin1 in Huh7 cells.(5)Overexpression of mi R-140-5p in nude mouse models to elucidate its role in inhibiting HCC tumor growth by targeting Pin1.(6)Using human HCC tissues to explore the relations between Pin1 protein levels and mi R-140-5p levels,detected by Western blot and QPCR,respectively.Results:(1)The expression of miR-140-5p,which was confirmed by RT-PCR,lead to significant reduction of Pin1 protein expression without significant effects on Pin1 mRNA level,suggesting that miR-140-5p mainly regulates Pin1 at the translational level.(2)Moderate overexpression of miR-140-5p potently induces MET of cells.(3)Moderate overexpression of mi R-140-5p potently inhibits cell growth,colony formation,migration and invasion of human HCC cells.(4)Stable knockdown of Pin1 also inhibits cell growth,colony formation,migration and invasion of human HCC cells.(5)MiR-140-5P blocks multiple Pin1-dependent cancer pathways by targeting Pin1 simultaneously.(6)MiR-140-5p inhibits HCC tumor growth by targeting Pin1 in vivo.(7)MiR-140-5p is downregulated and correlated with Pin1 overexpression in human HCC cell lines and human HCC tissues.Conclusions: MicroRNA-140-5p inhibits hepatocellular carcinoma by directly targeting the unique isomerase Pin1 to block multiple cancer-driving pathways.Our results uncovered a novel molecular mechanisms leading to the development of HCC as well as might lead to new therapies for HCC.