The Influence And The Possible Mechanism Of MiR-145to The Oct4Genein In HepG2Cell

ObjectiveMechanism of epigenetic modifications played an important role in the occurrence ofliver carcinoma developing, its mechanism mainly involves DNA methylation, variousforms of histone modification, chromatin reconstruction and non coding RNA regulation.Micrornas (miRNA) through regulating oncogenes or tumor suppressor genes involved inmultiple signaling pathways which formed human liver cancer，then played a promoting orinhibiting effect.This study will investigate the effect and its possible molecular mechanismof miR-145on cancer stem cell differentiation gene Oct4in HepG2hepatocellularcarcinoma cell line, in order to clarify the mechanism of action of cell miRNA regulation,this paper reveals the mechanism of liver cancer cell growth, provide new ideas andmethods to seek for the effective treatment of liver cancer.Methods1) In vitro HepG2cell line, take logarithmic phase cells, the vaccination in cell culturebottle or culture plate, completely attached to cells, discard the original nutrientsolution.2) This experiment is divided into mimics group, transfection reagent group, controlgroup, and negative control group, then design and synthesis the double chain mimicsaccording to the humanized mir-145-5p series. Put mir-145-5p mimics to liposomeLipofectamine2000packages, and transfected into HepG2cell line.3) Detecte transfection effect48hours after transfection:Adopt the real-time fluorescentquantitative polymerase chain reaction (PCR) detection the expression level ofmiR-145.4) Through the analysis of the CCK-8hepatocellular carcinoma cell line in different time (24h,48h and72h) growth activity changes.5) Bioinformatics software forecast miR-145’s potential target genes.6)48hours after transfection,adopt qRT-PCR and western blot detection the mRNA andprotein expression level of intracellular target gene (Oct4, Wnt5b, β-Catenin).Results1) Compared with the control, in miR-145mimics group miR-145were obviouslyup-regulated;2) Over express miR-145can inhibit the growth of HepG2cell;3) Oct4is a potential target genes of miR-145predict by MiRanda software;4) OCT4, Wnt5b, β-Catenin protein expression levels were significantly lower than theother three groups in mimics group (P <0.05); but the differences of those mRNAexpression level among these four groups were no statistical significance (P>0.05).Conclusion1) MiR-145has influence in tumor stem cells differentiation genes Oct4in HepG2cell,Oct4gene is the target genes of miR-145.2) In HepG2cells, miR-145can down-regulated the expression level of OCT4proteinrather than mRNA,play negative regulatory role in translation level after transcriptionof the target gene.3) There are some interactions between Oct4and Wnt/beta catenin signal transductionpathways in HepG2cell lines.