| Background and Objectives:Lung cancer is one of the most common malignances with the high morbidity and mortality,of which non-small-cell lung cancer(NSCLC)accounts for about 85%.Although the current surgery and combination therapy have made great progress,the 5-year survival rate for NSCLC patients is still poor.According to statistical analysis,more than 90%of lung cancer deaths are due to the metastasis of cancer cells.Epithelial-mesenchymal transition(EMT)is a key event in cancer metastasis,which confers cancer cells with increased motility and invasiveness.The occurrence of EMT is related to various cytokines,signal transduction pathways and transcription factors.The transformation growth factor(TGF)?-signal pathway induced EMT is prove to play a pivotal role in the invasion progress of NSCLS cells.As a member of ZEB transcription factor family,Zinc finger e-box binding homeobox 1(ZEB1)is highly expressed in a variety of malignant cancers and is involved in the regulation of cancer metastasis and EMT process.As a transcriptional coactivator,MYOCD has been reported to be closely related to cancer,but its role in NSCLC has not yet been elucidated,the molecular mechanism is still unclear.In this study,we sought to elucidate the role of MYOCD in TGF-?-induced EMT and to illustrate the transcriptional regulatory mechanisms between ZEB1 and MYOCD in NSCLC,furthermore to supply new sights for the diagnosis and treatment of NSCLC.Methods:(1)Generation of stable cell lines overexpressing MYOCD using lentiviral vectors and construction of transiently overexpression plasmids with ZEB1.(2)the MYOCD or ZEB1 were silenced by transfection of small interference RNAs in NSCLC cells.(3)Real-time PCR and Western blot were used to detect the expression of MYOCD mRNA and protein in NSCLC cells treated with TGF-?.(4)MYOCD promoter luciferase construct was generated and co-transfected into A549 cells with ZEB1 siRNA or ZEB1 overexpressed plasmid,Luciferase assay was used to detect the activity changes of MYOCD promoter region after overexpressing or knockdown ZEB1(5)Chromatin immunoprecipitation assay was performed to verify whether ZEB1 can be recruited to the MYOCD promoter.(6)Transwell assay was performed to evaluate the migration and invasion ability of NSCLC cells.(7)Real-time PCR was used to detect the expression levels of MYOCD and ZEB1 in NSCLC tissues with different metastatic status,the correlation between ZEB1 and MYOCD expression in NSCLC tissues was analyzed by software.Results;(1)Overexpression of MYOCD enhanced TGF-?-induced EMT and invasion of NSCLC cells,and knockdown of MYOCD inhibited TGF-?-induced EMT and invasion of NSCLC cells.(2)The expression level of MYOCD was up-regulated in NSCLC cells treated with TGF-?.(3)ZEB1 positively regulated MYOCD expression in NSCLC cells,Overexpression of ZEB1 enhanced MYOCD expression,and knockdown of ZEB1 inhibited MYOCD expression.(4)ZEB1 was recruited to MYOCD promoter and regulated positively MYOCD promoter activity.(5)Overexpression of ZEB1 enhanced TGF-?-induced EMT and invasion of NSCLC cells,and knockdown of ZEB1 expression inhibited TGF-?-induced EMT and invasion of NSCLC cells.(6)The expression levels of MYOCD and ZEB1 mRNA were higher in metastatic NSCLC tissues compared with non-metastatic tissues,and the expression of them were positively correlated.Conclusion:In the present study,we found that transcription factors ZEB1 promoted TGF-?-induced EMT by transcriptional regulation of MYOCD in NSCLC,and provided an important way to interfere with the invasion and migration of NSCLC by regulating the TGF-? signaling pathway. |
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