Role Of Ski In Epithelial-Mesenchymal Transition Induced By TGF-β In 95C Cell Line

Background and objective: Lung cancer has been one of malignant tumors,which has the highest cancer mortality rate worldwide.Of all lung cancers,non-small cell lung cancer(NSCLC)accounts for approximately 85%.Since the leading cause of cancer deaths in patients with solid tumor is metastasis,elucidating the mechanisms underlyinglungcancermetastasisappears to be vital important.Epithelial-mesenchymal transition(EMT) is correlated with tumor metastasis and invasion, which is characterized by loss of epithelial marker(e.g. E-cadherin).During the process of EMT,epithelial cells lose polarity, gain more ability of invasion and migration. Transforming growth factor beta(TGF-β), playing a dual role of tumor suppressoror promoter in different periods,is ubiquitous and essential in cellular and physiologic processes,such as cellular proliferation, differentiation and apoptosis. In the early stage, TGF-β plays a role as a tumor-suppressor factor. However, in late-stage tumors, TGF-β promotes tumor metastasis because of tumor microenvironment changes. TGF-β participates in EMT mainly via Smad and non-Smad pathway.There is accumulating evidence showing that Ski is an important negative regulator ofTGF-β signaling, playing a dual role of tumor suppressoror promoter in different types of tumors as well.However,the mechanisms by which Ski affects TGF-β-induced EMT and migration in non-small cell lung cancer(NSCLC) remain poorly understood.Methods: qRT-PCR and western blot analysis were performed to analyze the expression of Ski in NSCLC cell lines and tissues; Generation of stable Ski-silenced 95 C cells(95C-sh-Ski); Western blot was employed to determine the expression of EMT markers(E-cadherin, N-cadherin),p-Smad and Smad3 in 95C-sh-Ski treated with or without TGF-β1 for 24 h. Transfect 95C-sh-Ski cells with PAI-1 promoter luciferase constructs. Inabsence or presence ofTGF-β1for 24 h, the 95C-sh-Ski cellswere subjected to luciferase assays.Wound healing assay was used toexamine the effect of Ski on cell migratory ability.Results: When compared with metastatic NSCLC cells, the expression level of Ski was high in non-metastatic NSCLC cells. Upon TGF-β1 stimulation,95C-sh-Ski cells had more obvious features of EMT. Moreover,95C-sh-Ski cells showed an increase in Smad3 phosphorylation level.Importantly,the result ofluciferase assays confirmed that Ski inhibited transcription via TGF-β/Smad signaling pathway. Wound healing assay showed that Ski inhibited the migratory ability of 95 C cells.Conclusion: Reduced expression of Ski was observed in metastatic NSCLC cells,which suggested that Ski might play a role in development and progression of NSCLC and was related to NSCLCinvasion and migration. In addition, we identified that Ski represses TGF-β-induced EMT and migration mostly through inhibiting Smad-dependent signaling in 95 C cell line.