Mechanism And Role Of MiR-140 In The Epithelial-mesenchymal Transition Of Colorectal Cancer

Metastasis is the biological feature and leading cause of death in malignant tumor.It consists of sequential and interrelated steps,and many of them are favored by epithelial-mesenchymal transition(EMT).Obtaining mesenchymal state endows cells with enhanced migration and invasion,induces stemness,and prevents apoptosis and senescence,allowing their subsequent roles in the initiation of metastasis.The EMT process has been reported in a variety of epithelial malignancies including colorectal cancer(CRC).Colorectal cancer(CRC)is one of the most common malignant gastrointestinal carcinomas with a rising morbidity and mortality,which is one of the most common tumors of Chinese and became a serious threat to human health.CRC is the third leading cause of death from cancer and it has been estimated to be responsible for 50,000 deaths per year in the USA.Although surgery combination with adjuvant therapy has increased the prognosis of CRC,still approximately half of all CRC patients have metastasis at the time of diagnosis or after intended curative treatment,giving rise to a poor 5-year survival rate of slightly greater than 10%.A well explanation of metastasis of CRC would be helpful for the development of effective therapeutic strategies.Micro RNAs(miRNAs)are noncoding small RNA molecules that post-transcriptionally regulate protein expression via binding to the 3′-untranslated region(UTR)of the target genes.Some studies have shown the importance of miRNAs in the regulation of EMT either as inhibitor or promoter.SMAD family member 3(Smad3)is one of the key downstream factors of TGF-β signaling pathway,which is a critical inducer of the EMT in the malignant tumor,such as CRC.TGF-β binds with its two transmembrane receptors,type I(TGFBRI)and type II(TGFBRII),leading to the phosphorylation of Smad2 and Smad3,which translocate to the nucleus with Smad4 where it can regulate the downstream EMT-related genes expression.Importantly,Smad3 has been confirmed as the direct target of micro RNA-140-5p(miR-140).MiR-140 was firstly shown to be specifically expressed in the cartilage of developing zebrafish and mouse embryos during both long and flat bones development.MiR-140 can suppress colorectal cancer stem cell(CSC)survival and its metastatic potential by inhibiting Smad2 and autophagy.Downregulation of miR-140 induces EMT and promotes esophageal cancer cell invasion via targeting Slug.However,the impact and comprehensive mechanism of miR-140 in the invasion and metastasis of CRC still needs to be elucidated.Part I miR-140 target Smad3 and regulates its expressionObjective: we confirm Smad3 is indeed a direct target of miR-140.Methods: MiR-140 mimics(miR-140),miR-140 specific inhibitor(140i)or siRNA against Smad3(si Smad3)were transfected into human CRC cell line HCT116 and RKO respectively using Oligofectamine or Lipofectamine2000.Real-time q RT-PCR was used to measure the expression levels of miR-140 and Smad3 m RNA.Smad3 and p-Smad3 protein was analyzed by Western blot.Cell proliferation level was detected by CCK-8.The holocolony forming ability was detected by Colony formation assayResults: The results of real time qRT-PCR showed that HCT116 and RKO cells transfected with miR-140 mimics expressed higher level of miR-140 compared to the negative control(P < 0.01),indicating that the transfection of miR-140 succeeded.There was no significant difference in Smad3 m RNA expression by miR-140 treatment(P > 0.05).Ectopic transfection of miR-140 dramatically reduced the expression of Smad3 and p-Smad3 protein(P < 0.01)and inhibits the growth of tumor cells and the ability of cell clone formation(P < 0.01)whencompared to the negative control by Western blot analysis.On the contrary,the expression level of Smad3 and p-Smad3 protein in HCT116 and RKO cells transfected with the inhibitor of miR-140 increased dramatically(P < 0.01)and promote the growth of tumor cells and the ability of cell clone formation(P < 0.01)when compared to the negative control.Co-transfection of miR-140 inhibitor and Smad3 si RNA had no significant effect on the Smad3 and p-Smad3 protein expression and the growth of tumor cells and the ability of cell clone formation(P > 0.05)Conclusion: Up-regulation of miR-140 can reduce Smad3 and p-Smad3 protein expression without significant alteration of Smad3 m RNA,and inhibits the growth of tumor cells;whereas inhibition of miR-140 can increase Smad3 and p-Smad3 protein expression and promote the growth of tumor cells.We concluded that miR-140 directly targets Smad3 in the post-transcriptional level and inhibits the growth of tumor cells and the ability of cell clone formation.Part II The mechanism of miR-140 regulation of EMT functionObjective: We demonstrate the effects of miR-140 rgulate the EMT on the migrating and invasive potential of colorectal cancer(CRC)cell HCT116 and RKO in vitro and the possible mechanism.Our results will provide the candidate target of tumor invasion and metastasis diagnosis and therapy.Methods: MiR-140 mimics(miR-140),miR-140 specific inhibitor(140i)or si RNA against Smad3(si Smad3)were transfected into human CRC cell line HCT116 and RKO respectively using Oligofectamine or Lipofectamine2000.The in vitro cell migration and invasion potential were determined by wound-healing and Transwell chamber assays after up-regulating or down-regulating miR-140 or knocking down Smad3.The expression of EMT-associated protein E-cadherin and Vimentin was detected by Western Blot and immunofluorescence.Results: The wound-healing assay indicated that ectopic expression of miR-140 inhibited the migration of HCT116 and RKO cell.Transwell chamber assays showed that the cells going through the membrane decreased significantly compared to the negative control(P < 0.01),EMT related molecular E-cadherin expression increased,Vimentin expression decreased(P<0.01).Knock-down of Smad3 by si RNA had the similar effect with the miR-140 transfection and no statistical significance was found(P > 0.05).On the contrary,down-regulation of miR-140 enhanced the migrating and invasive potential of HCT116 and RKO cell via wound-healing and Transwell chamber assays(P < 0.01),EMT related molecular E-cadherin expression decreased,Vimentin expression increased(P<0.01),when compared to the negative control.Co-transfection of miR-140 inhibitor and Smad3 si RNA had no significant effect on the capacity of cell migration and invasion(P > 0.05).Conclusion: Up-regulation of miR-140 or knock-down of Smad3 respectively inhibited the migration and invasion of CRC cell and inhibited the progression of EMT.Down-regulation of miR-140 increase the expression of Smad3 and p-Smad3 protein and enhanced the migration and invasion potential of CRC cell and promoted the progression of EMT.Co-transfection of miR-140 inhibitor and Smad3 si RNA had no significant effect on the Smad3 and p-Smad3 protein expression and the capacity of cell migration and invasion and the progression of EMT.Therefore,miR-140 suppresses the migration and invasion potential of CRC cell,possibly through the down-regulation of Smad3.Part III The inhibition effects of miR-140 on HCT116 colorectal cancer cell transplanted to nude miceObjective: To investigate the inhibition effects of over-expression of miR-140 in xenografts in vivoMethods: Mice were injected with lentivirus miR-140-transduced cells 、 lentivirus control-transduced and lentivirus sh RNA against Smad3-transduced cells.Each group was injected subcutaneously on the back of mice or through tail vein.Immunohistochemical staining and western blotting analysis were used to evaluate the expression of related proteins.Results: The tumor volumes in miR-140 over-expression or Smad3 depression group were small,grow slowly and the metastatic rate is low.The expression levels of Smad3,p-Smad3,Vimentin and Zeb1 were reduced in the miR-140 over-expression or Smad3 depression groups related to the control groups.The expression levels of E-cadherin wasincreased in the miR-140 over-expression or Smad3 depression groups related to the control groupsConclusion: Over-expression of miR-140 can inhibit tumor growth in nude mice,and the inhibition effects may because of miR-140 inhibits the expression of Smad3 protein.