MiR-132 directly inhibits the expression of ZEB2 through its 3′UTR and regulates the EMT of NSCLC cells.

<p>(A) The miR-132 binding site predicted in the 3′UTR of ZEB2 mRNA. (B) Mutant was generated at the seed region of ZEB2 3′ UTR as indicated by the underline. A 3′ UTR fragment of ZEB2 mRNA containing wild-type or mutant of the miR-132 binding sequence was cloned into the downstream of the luciferase gene in pMIR vector. (C) L9981 cells were transfected with pMIR reporter vectors containing either wild-type or mutant ZEB2 3′UTR (indicated as pMIR-ZEB2-3′ UTR-wt and pMIR-ZEB2-3′ UTR-mut) with either pcDNA3.1 (indicated as NC) or pcDNA3.1-miR-132 vector (indicated as miR-132). Luciferase activity was determined 48 h after transfection. (D) ZEB2 mRNA was detected by qRT-PCR in cell lines transfected with pcDNA3.1 (indicated as NC) or pcDNA3.1-miR-132 vector (indicated as miR-132). (E, F) The protein levels of ZEB2, E-cadherin or vimentin was examined by Western blot in cells transfected with different plasmids. Figure F shows the relative gray values of each band (normalized to β-actin). Protein bands from three independent Western blot assays were quantified using Quantity One software (Bio-Rad, USA). Data are reported as mean ±SD (**<i>P</i><0.01, Student’s <i>t</i>- test).