Regulatory Mechanisms Of TIF1? And Its Relationship With TGF-?-induced Epithelial-mesenchymal Transition In Non-small-cell Lung Cancer

Background and Objectives:Lung cancer is the most common cancer type(13.5% of all tumors,2016)and the leading cause of cancer deaths worldwide(26.5%,2016),and non-small-cell lung cancer(NSCLC)accounts for 85% of all cases of lung cancer.In fact,more than 90% of deaths from solid tumors,including NSCLC,are mainly attributed to metastasis.Epithelial-mesenchymal transition(EMT)is a key event in cancer metastasis,which confers cancer cells with increased motility and invasiveness.There is accumulating evidence that transforming growth factor(TGF)-? signaling is a potent inducer of EMT in various cancers.TIF1?(Transcription intermediary factor 1 gamma)is a novel regulator of TGF-? signaling.TIF1? expression was decreased in various tumors,and silencing TIF1? improved TGF-?-induced EMT in mammary epithelial cells.Our previous study showed that expression of TIF1? was frequently reduced in NSCLC.However,to date,little is known about the role that transcription-mediated events play in TIF1? inhibition and whether inactivation of TIF1? can promote TGF-?-induced EMT in NSCLC.In this study,we sought to elucidate the role of TIF1? in TGF-?-induced EMT and to illustrate the transcriptional regulatory mechanisms underlying down-regulation of TIF1? expression in NSCLC,further,to supply new sight for the pathogenesis of NSCLC.Methods:Part 1 Repression of TIF1? by SOX2 promotes TGF-?-induced epithelial–mesenchymal transition in non-small-cell lung cancer(1)A series of luciferase reporter constructs containing various lengths of TIF1? 5'-promoter were generated and transfected into A549 cells to assess the basal luciferase activities of TIF1?.(2)Electrophoretic mobility shift assay(EMSA)and chromatin immunoprecipitation(Ch IP)assays were performed to verify whether SOX2 can be recruited to the TIF1? promoter.(3)Generation of stable cell lines overexpressing SOX2 and establishment of TIF1?-silenced stable cell lines.(4)PAI-1 promoter luciferase construct was generated and transfected into cell lines underexpessing TIF1? or overexpressing SOX2,and then assessed the luciferase activities in the absence or presence of TGF-?1.(5)Real-time quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR)and Western blot were used to detect the expression levels of TIF1?,SOX2 and other proteins.(6)Transwell assay and in vivo model of metastasis assay were performed to evaluate the migration and invasion ability of cells in vitro and vivo.(7)Immunofluorescence analysis was performed to detect the expression of SMAD3 and SMAD4 in TIF1?-silenced A549 cells.Part 2 Circ PTK2 modulates TGF-?-induced epithelial–mesenchymal transition by regulating TIF1? expression in NSCLC(1)Real-time quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR),Immunohistochemistry(IHC)and western blot were used to detect the expression of circ PTK2,mi R-429,TIF1? and other proteins.(2)Interactions between mi R-429 and 3'-untranslated region(3'-UTR)of TIF1? gene or sequence of circ PTK2 were detected using dual-luciferase report gene plasmid technology.(3)The migration and invasion ability of cells were observed with the use of transwell assay.(4)Generation of stable A549 cell lines overexpressing circ PTK2.(5)mi R-429 mimics and anti-mi R-429 mimics were transfected into A549 cells to overexpress and silence the expression of mi R-429 respectively.(6)Correlation analysis of circ PTK2 and TIF1? expression in NSCLC tissues.Res ults:Part 1 Repression of TIF1? by SOX2 promotes TGF-?-induced epithelial–mesenchymal transition in non-small-cell lung cancer(1)SRY-related protein is involved in transcriptional inhibition of TIF1? expression.(2)SOX2 is recruited to TIF1? promoter and regulates negatively TIF1? promoter activity and overexpression of SOX2 inhibits TIF1? expression.(3)TIF1? m RNA expression is reduced and inversely correlated with SOX2 protein expression in NSCLC tissues.(4)Knockdown of TIF1? enhances TGF-?-induced EMT and invasion of NSCLC cells in vitro and promotes NSCLC cell metastasis in vivo.(5)Overexpression of SOX2 enhances TGF-?-induced EMT and invasion of NSCLC cells.(6)SOX2 knockdown inhibits TGF-?-induced EMT and invasion of NSCLC cells.(7)TIF1? knockdown and SOX2 overexpression enhances SMAD4-dependent PAI-1 promoter activity.Part 2 Circ PTK2 modulates TGF-?-induced epithelial–mesenchymal transition by regulating TIF1? expression in NSCLC(1)Expression of TIF1? is reduced in human NSCLC tissues and high expression of TIF1? is associated with higher survival rates.(2)Circ PTK2 expression is downregulated through TGF-?-induced EMT in A549 cells.(3)mi R-429 reduces TIF1? expression and enhances TGF-?-induced EMT and invasion of NSCLC cells.(4)Circ PTK2 can recruit mi R-429 and regulate the expression of TIF1?.(5)Overexpression of circ PTK2 supresses TGF-?-induced EMT and invasion of NSCLC cells.(6)Circ PTK2 expression is positively correlated with TIF1? m RNA expression in NSCLC tissues.Conclusion:Our findings in part one revealed a new mechanism by which SOX2-mediated transcription repression of TIF1? promoted TGF-?-induced EMT in NSCLC and TIF1? inhibited TGF-?-induced EMT through competing with SMAD4 in NSCLC cells.In part two,we found circ PTK2 could modulate TGF-?-induced epithelial–mesenchymal transition by regulating TIF1? expression in NSCLC.Moreover,this part of study elucidated a new transcriptional regulatory mechanism of TIF1? in NSCLC.Taken together,these two parts of studies provide an important way to interfere with the invasion and migration of NSCLC by regulating the TGF-? signaling pathway.