| Purpose: The aim of this study was to explore the effect of Hedgehogsignaling in epithelial-mesenchymal transition of NSCLC induced byTGF-β1.Methods: To assess the role of TGF-β1in EMT, A549cells weretreated with1,5, or10ng/ml TGF-β1for48h and morphological changeswere assessed by phase-contrast microscopy. The Gli1mRNA expressionlevel was detected by Real-Time PCR. The Gli1、E-cadherin and vimentinproteins expression level were assessed by western blot analysis. Theexpression of Gli1protein was also assessed by immunofluorescenceanalysis. The migratory capacity of A549after TGF-β1treatment wasdetected by wound-healing assays. Treatment of H460and SK-MES-1cellswith5ng/m TGF-β1for48h, the Gli1mRNA expression level wasdetected by Real-Time PCR. The Gli1、E-cadherin and vimentin proteinsexpression level were assessed by western blot analysis. The invasion capacity of the cells were assessed by transwell invasion assays.Last, in order to investigated the effect of Hedgehog signaling onepithelial-mesenchymal transition of NSCLC induced by TGF-β1. weemployed small-interfering RNA (siRNA) and Smo inhibitor Cyclopamineor the Gli1inhibitor GANT61to deplete Gli1mRNA and protein inNSCLC cells, then, the NSCLC cells were treated with5ng/ml TGF-β1for48h. Using Real-Time PCR and western blot analysis to assess theexpression of Gli1in the NSCLC cells. And immunofluorescence analysisto assess the expression of Gli1in A549cells. Western blot analysisperformed to analyze the expression of E-cadherin and vimentin in NSCLCcells, and immunofluorescence analysis to assess the expression ofE-cadherin and vimentin in A549cells.And then we performed transwellinvasion assays to analyze the migratory capacity of NSCLC cells.Results: After A549cells were treated with1,5, or10ng/ml TGF-β1for48h,We observed that while the untreated A549cells exhibited a classicepithelial morphology, cell morphology dramatically changed after48h ofTGF-β1treatment. The treated A549acquired a mesenchymal phenotype,elongated, spindle shaped cells with reduced cell-cell contact. And TGF-β1treated led to reduced expression of the epithelial marker E-cadherin andincreased expression of the mesenchymal marker vimentin in aconcentration-dependent meaner in A549cells,these results were confirmedin additional NSCLC cell lines H460and SK-MES-1. In a wound healing assay, we observed that TGF-β1treatment led to highly migratory behavioraccelerated wound over the course of48h compared to untreated cells inA549cells. And then we performed transwell invasion assays usingTGF-β1treated H460and SK-MES-1cells. Treatment with5ng/mlTGF-β1led to increased invasion of both cell lines.Using Real-Time PCR and western blot analysis, we found thatTGF-β1stimulation significantly increased Gli1mRNA and protein levelsin NSCLC cells, compared to untreated cells. Similar induction of Gli1wasobserved by immunofluorescence analysis of TGF-β1treated A549cells.We employed small-interfering RNA (siRNA) and Smo inhibitorCyclopamine or the Gli1inhibitor GANT61to deplete Gli1mRNA andprotein in NSCLC cells, afterwards, the NSCLC cells were treated with5ng/ml TGF-β1for48h. Using Real-Time PCR and western blot analysis toassess the expression of Gli1in the NSCLC cells. Gli1mRNA and proteinexpression was suppressed by Gli1-specific siRNA and Cyclopamine orGANT61. The same result was observed by immunofluorescence analysisin A549cells. Western blot and immunofluorescence analysis demonstratedthat, compared to cell transfected with Gli1siRNA or pharmacologicalinhibition of Gli1and smo, E-cadherin expression was elevated in cells,while vimentin protein levels were decreased. Compared to cellstransfected with Gli1siRNA or pharmacological inhibition of Gli1and smo,had significantly reduced the invasion and migration capacity in a transwell assay.Conclusions: Our findings suggest that Hedgehog signaling restrainsTGF-β1-induced EMT in NSCLC, and may imply that Hedgehog signalingplays an important role in EMT of NSCLC. |
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