The MiRNA-based Mechanisms Underlying Desregulated SMAD3/4 Moderating TGF-?-induced EMT And Cell Invasion In Non-small-cell Lung Cancer

[Background and Objectives]:Non-small-cell lung cancer(NSCLC)accounts for about 80% of all types of lung cancers,which is the primary cause of cancer related deaths around the world.Most NSCLC patients are in the advanced stage at diagnosis,that is to say,tumor cells have already been metastasized or owned invasive and metastatic character.Epithelial-mesenchymal transition(EMT)has become the focus of attention in recent years,which plays an important role not only in embryonic development but also in tumor cell invastion and metastasis.Increasing evidence indicates that TGF-? signaling mediated EMT plays an essential role during the invasion and metastasis in human cancers.SMAD3 and SMAD4,as key members of TGF-? signaling pathway,were activated by phosphrylation of TGF-? receptor and then form complex to translocate to nucleus,therein,they modulate expression of genes,including some genes involving EMT.micro RNAs(miRNAs)are a family of 19-24 nucleotides small non-coding RNA molecules,which post-transcriptionnally regulate gene expression by targeting m RNA transcripts and lead to transcriptional repression and/or degradation of target m RNAs.Most human genes are poteintially direct targets of miRNAs.Recent studies demonstrate that miR-203 and miR-145 inhibit the cell invasion in NSCLC and nasopharyngeal cancer,respectively.Meanwhile,decreased expression of miR-205 was detected in lung cancer,indicating a tumor suppressing character of miR-205.Computational algorithms predicted that SMAD3 is the direct target of miR-203 and miR-145,and SMAD4 is the target of miR-205.However,it is poorly understood whether the miRNAs described above are involved in TGF-?-induced EMT,migration and invasion in NSCLC.[Methods]:1)With the use of real-time quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR)and Western blot,expression of genes and miRNAs in cells and tissues were detected.2)The targeting effects between miRNAs and 3'-untranslated region(3'-UTR)of gene transcripts were detected using the dual-luciferase reporter gene system.3)Mi RNA mimics were designed and synthesized to transiently transfected to NSCLC cells.4)The cell migratory and invasive ability was tested using the cell transwell assay.[Results]:Part 1 miR-145 and miR-203 repress TGF-?-induced EMT and invasion by inhibiting SMAD3 in non-small-cell lung cancer cells1)SMAD3 expression is up-regulated in NSCLC tissues and cell lines,whereas expression of miR-145 and miR-203 was decreased in NSCLC tissues and cell lines.2)miR-145 and miR-203 repress SMAD3 expression by targeting SMAD3 3'-UTR in NSCLC cells.3)miR-145 and miR-203 inhibite TGF-?-induced EMT and invasion in NSCLC cells.4)Knockdown of SMAD3 suppress TGF-?-induced EMT and migration and invasion of NSCLC cells.Part 2 Repression of SMAD4 by miR-205 inhibits TGF-?-induced epithelial-mesenchymal transition in non-small cell lung cancer1)Knockdown of SMAD4 inhibits TGF-?-induced EMT,invasion and migration in NSCLC cells.2)miR-205 directly targets SMAD4 3'-UTR and inhibits SMAD4 expression in NSCLC cells.3)miR-205 moderates the EMT,invasion and migration induced by TGF-?/SMAD4 signaling in NSCLC cells.[Conclusions]:In NSCLC cells,miR-145 and miR-203 inhibit TGF-?-induced EMT and invasion through repression of SMAD3,and miR-205 also has the ability to inhibit TGF-?-induced EMT and invasion through repression of SMAD4.Out findings provides insights into the miRNA-based mechanism for controlling TGF-?-induced EMT of NSCLC cells and a strategy for targeted therapy of NSCLC.