The Mechanism Study On MiR-33a Regulating Epithelial-mesenchymal Transition In NSCLC Cells

Background and objectiveMicroRNAs (miRNAs) are small non-coding RNAs and range in size about22nucleotides (nt) whichdownregulate gene expression by base-pairing to the3’-UTR of the target mRNA. In recent years, miRNAare reported to be involved in cancer metastasis. However, the specific molecular mechanisms of cellmetastasis still need to be found. Epithelial-mesenchymal transition (EMT), whose most significant sign isthe loss of epithelial markers and increasing expression of mesenchymal markers, can invert the epithelialcells into mesenchymal cells and has been reported not only involved in tissue development duringembryogenesis but also central mechanisms that enhance the invasive and metastatic ability of cancer cells.But the related signal pathway and molecular mechanisms remain unclear. There are more and more studiessuggesting that dysregulation of certain miRNAs may cause human disease including carcinogenesisproliferation and tumor metastasis in human. All of these suggest that the specific miRNAs may beinvolved in epithelial-mesenchymal transition. miR-33a，often appears abnormal expression in metabolismand metabolic disorders, but its role in EMT and metastasis has been little studied. Here we mainly focuson the role and the regulating mechanism of miR-33a in epithelial-mesenchymal transition in NSCLC cells.Methods（1） We performed a bioinformatics search to find that Twist1is a potential target ofmiR-33a.（2） According to the synthesis principle of miRNAdesign, miR-33a inhibitor, miR-33amimics, and the negative control were synthesized. To decrease or increase the miRNA level in cells,miR-33a inhibitor and mimics were transfected into cells using Lipofectamine2000according to themanufacturer’s protocol. And Real time RT-PCR was used for analysis the miR-33a expression aftertransfection in NSCLC cell lines.（3） Real time RT-PCR analysis and Western Blot were performed to detect the changesof Twist1mRNA and protein expression after miR-33a inhibitor and mimics being transfected in NSCLCcell lines.（4） To further confirm that Twist1is the target of miR-33a, the Dual LuciferaseAssaywas used.（5） After transfection of miR-33a inhibitor, miR-33a mimics and Twist1siRNA, EMTand metastasis potential of NSCLC cells was separately analysed by Western Blot, wound healing assayand Transwell invasion assay to evaluate the effect miR-33a and Twist1on EMT and metastasis in NSCLCcells.Results（1） Different NSCLC cells with different potential of metastasis，miR-33a was expressed differently.The expression of miR-33a was inversely proportional to the metastasis potential of NSCLC cells.（2） MiR-33a overexpression could induce the expression of epithelial marker CDH1, suppress theexpression of mesenchymal marker Vimentin and decrease the metastasis potential, indicating thatmiR-33a overexpression could block EMT and metastasis in NSCLC cells. （3） MiR-33a downregulation could suppress the expression of epithelial marker CDH1, induce theexpression of mesenchymal marker Vimentin and increase the metastasis potential indicating thatmiR-33a downregulation could promote EMT and metastasis in NSCLC cells.（4） Twist1downregulation could induce the expression of epithelial marker CDH1, suppress theexpression of mesenchymal marker Vimentin and decrease the metastasis potential indicating that Twist1downregulation could block EMT and metastasis in NSCLC cells.（5） MiR-33a was inversely proportional to Twist1expression and could down-regulated Twist1expression.（6） Twist1is a target of miR-33a.Conclusions（1） The expression of miR-33a is inversely proportional to the metastasis potential in NSCLCcells; miR-33a is involved in EMT and metastasis in NSCLC cells.（2） MiR-33a may inhibit the EMT and metastasis through targeting Twist1in NSCLC cells.（3） MiR-33a can function as an anti-metastasis miRNA, Twist1may be used as a potentialtherapeutic target for the treatment of the advanced NSCLC patients.