MiR-140Suppresses The Migration And Invasion Of Colorectal Cancer Cell Through Targeting Smad3

Background: microRNAs (miRNAs) are a class of endogenous, single-strand,non-coding small RNA molecules, about18~25nucleotides long, that suppress theexpression of their target genes by translational arrest or mRNA degradation viacomplete or incomplete base pairing with the3’-untranslated regions (UTRs) of thetarget mRNAs. Recent studies have shown that miRNAs not only regulate cellularprocesses including cell proliferation, differentiation, metabolism and apoptosis, butalso function as tumor suppressors or oncogenes to be involved in the development andmetastasis of tumor. microRNA-140(miR-140) has been demonstrated to play criticalroles in the cartilage proliferation and development and its decreased expression caninduce the osteoarthritis. Currently little is known about the function of miR-140intumor development and metastasis, and some results are controversial. Smad3(SMADfamily member3) is a major transcript factor of TGF-β (Transforming Growth Factor-β)signaling pathway, which can significantly enhance the invasive and metastatic potentialof breast cancer and ovary cancer. Smad3was confirmed to be the direct target ofmiR-140. Therefore, we hypothesized that miR-140may be involved in the tumorinvasion and metastasis through regulating Smad3.Objective: Firstly we confirm Smad3is indeed a direct target of miR-140. Thenwe demonstrate the effects of miR-140on the migrating and invasive potential ofcolorectal cancer (CRC) cell RKO in vitro and the possible mechanism. Our results willprovide the candidate target of tumor invasion and metastasis diagnosis and therapy. Methods: MiR-140mimics (miR-140), miR-140specific inhibitor (Anti-miR-140)or siRNA against Smad3(Smad3siRNA) were transfected into human CRC cell lineRKO respectively using Oligofectamine or Lipofectamine2000. Real-time qRT-PCRwas used to measure the expression levels of miR-140and Smad3mRNA. Smad3protein was analyzed by Western blot. The in vitro cell migration and invasion potentialwere determined by wound-healing and Transwell chamber assays after up-regulating ordown-regulating miR-140or knocking down Smad3. The statistical analysis wasdetermined by SPSS19.0software.Results:1. The results of real time qRT-PCR showed that RKO cells transfected withmiR-140mimics expressed higher level of miR-140compared to the negative control(P <0.05), indicating that the transfection of miR-140succeeded. There was nosignificant difference in Smad3mRNA expression by miR-140treatment (P>0.05).Ectopic transfection of miR-140dramatically reduced the expression of Smad3protein(P <0.05) when compared to the negative control by Western blot analysis.2. The wound-healing assay indicated that ectopic expression of miR-140inhibitedthe migration of RKO cell. Transwell chamber assays showed that the cells goingthrough the membrane decreased significantly compared to the negative control (P <0.05). Knock-down of Smad3by siRNA had the similar effect with the miR-140transfection and no statistical significance was found (P>0.05).3. On the contrary, the expression level of Smad3protein in RKO cells transfectedwith the inhibitor of miR-140decreased dramatically (P <0.05), and meantimedown-regulation of miR-140enhanced the migrating and invasive potential of RKO cellvia wound-healing and Transwell chamber assays (P <0.05) when compared to thenegative control. Co-transfection of miR-140inhibitor and Smad3siRNA had nosignificant effect on the Smad3protein expression and the capacity of cell migrationand invasion either (P>0.05).Conclusions:1. Up-regulation of miR-140can reduce Smad3protein expression without significant alteration of Smad3mRNA, whereas inhibition of miR-140can increaseSmad3protein expression. We concluded that miR-140directly targets Smad3in thepost-transcriptional level.2. Up-regulation of miR-140or knock-down of Smad3respectively inhibited themigration and invasion of CRC cell. Down-regulation of miR-140decreased theexpression of Smad3protein and enhanced the migration and invasion potential of CRCcell. Co-transfection of miR-140inhibitor and Smad3siRNA had no significant effecton the Smad3protein expression and the capacity of cell migration and invasion.Therefore, miR-140suppresses the migration and invasion potential of CRC cell,possibly through the down-regulation of Smad3.3. The findings of this study suggest that miR-140may have a unique potential as anovel candidate for tumor metastasis diagnosis and therapy, and provide a new methodto improve the prognosis and decrease the recurrence of malignant tumor.