TGF-β Enhances The Potential Of Migration And Invasion Of Esophageal Squamous Cell Carcinoma Cell EC9706 Through Upregulation Of TAGLN2

Background Esophageal carcinoma(EC) ranks fourth among all malignant tumors in terms of morbidity and mortality, and newly diagnosed EC accounts for about half of new cases each year in China. Esophageal squamous cell carcinoma(ESCC) is the main histological subtype, which is accounting for more than 90% of all EC in China. The prognosis of EC is poor mainly due to distant metastases at presentation. Epithelial mesenchymal transition(EMT) is closely related to the invasion and metastasis of malignancies, which involves deregulation of multiple signal transduction. TGF-β is a multifunctional cytokine that can induce the development of EMT in a variety of tissue cells. TGF-β has a dual function in initiation and development of tumor. It functions as a tumor suppressor in early stage of tumor whilst a tumor promoter in late stage. Advanced tumor tissue can produce large amounts of TGF-β, which induces the development of EMT in tumor cells, thereby promoting cancer cell invasion and metastasis. TAGLN2 is a member of the calcium binding protein calponin family, which is highly homologous with TAGLN, and is mainly involved in cell stress fibers and actin stabilizing maintenance. In addition, TAGLN2 is closely related to multiple biological processes, such as cell differentiation, migration, podosoma formation, cell infiltration and extracellular matrix remodeling. The abnormal expression of TAGLN and TAGLN2 was found in a variety of tumors and we found that TAGLN2 was significantly up-regulated in ESCC. The mechanism of TAGLN2 overexpression in ESCC, however, remains unclear. Numerous studies showed that TGF-β induced the expression of TAGLN or TAGLN2 dependent on microenvironmental cues. Therefore, the present study was undertaken to investigate TAGLN2 expression and its biological function in process of EMT induced by TGF-β in ESCC cell lines EC9706.Aims ESCC cell lines EC9706 was infected by lentivirus carrying TAGLN2 specific silencing sequence or unrelated control sequence and then stable subline EC9706 sh TAGLN2 and negative control EC9706 sh Control were established. The present study aimed to determine the correlation between TGF-β and TAGLN2, and the roles of TAGLN2 in TGF-β signaling pathway in esophageal carcinogenesis.Methods 1 EC9706 cells were cultured in incubator at 37℃ with 5% CO2 and 95% air. The RPMI-1640 medium was supplemented with 10% fetal bovine serum. 2 EC9706 cells was infected by lentivirus carrying TAGLN2 silencing sequence or irrelevant control sequence followed by puromycin selection for stable sublines of EC9706 sh TAGLN2 and EC9706 sh Control. 3 Cells were treated a series of different concentrations of TGF-β for 72 h, or 40 ng/m L TGF-β for different time, followed by cell collection, protein extraction and subsequent experiments. 4 For Western blot analysis, protein was separated by SDS-PAGE in 12% separation gel and 4% stacking gel and then electroblotted to PVDF membrane. The membrane was blocked with 5% non-fat milk and incubated with corresponding first and secondary antibody followed by ECL detection of protein bands. 5 For assays of potentials of migration and invasion, the EC9706 sh TAGLN2 and EC9706 sh Control were seeded in inserts for migration or invasion, incubated in incubator for 48 to 72 h, stained with crystal violet. Five random microscopic views were selected for quantitative evaluation and statistical analyses. 5 Statistical analysis: The SPSS17.0 software was used for statistical analysis. T-test was used for significant difference between groups. The measurement data were compared with single factor analysis of variance and repeated measurement data test. P < 0.05 was regarded as statistical significance.Results 1 Concentration-dependent effects of TGF-β on EC9706 sh Control and EC9706 sh TAGLN2 After exposure of EC9706 sh Control cell with a series concentration of 5~80 ng/m L TGF-β, TAGLN2 expression was concentration dependent with a maximal expression at 40 ng/m L and 80 ng/m L. In EC9706 sh TAGLN2 cells, however, 5~20 ng/m L TGF-β failed to induce the expression of TAGLN2 while 40 ng/m L and 80 ng/m L TGF-β can significantly induce TAGLN2 expression. In the concentration-dependent effects, the stimulation of TGF-β had no effect on the proliferation of EC9706 sh Control and EC9706 sh TAGLN2. 2 Time-dependent effects of TGF-β on EC9706 sh Control and EC9706 sh TAGLN2 TAGLN2 expression was time-dependent with 40 ng/m L TGF-β exposure with maximal responses at 48 h and 72 h on EC9706 sh Control. In contrast, EC9706 sh TAGLN2 did not show up-regulation of TAGLN2 expression after TGF-β exposure for 72 h. In the time-dependent effects, the stimulation of TGF-β had no effect on the proliferation of EC9706 sh Control and EC9706 sh TAGLN2. 3 Induction of EMT by TGF β in EC9706 sh Control Using different concentrations of 5 to 80 ng/m L TGF-β to treat EC9706 sh Control, the expressions of mesenchymal markers such as N-cadherin and Vimentin were significantly increased and the expression of epithelial markers such as E-cadherin was significantly decreased with gradual increase of TAGLN2 expression increased. TAGLN2 knock-down inhibited the increase of mesenchymal markers(N-cadherin, Vimentin) and the decrease of epithelial markers(E-cadherin) which induced by TGF-β. 4 TAGLN2 silencing inhibited the capabilities of migration and invasion induced by TGF-β The cells of migration or invasion were significantly increased after TGF-β treatment in EC9706 sh Control(P <0.05), while TGF-β did not increase the number of migration or invasion cells in EC9706 sh TAGLN2. 5 TAGLN2 silence suppressed the expression of p-Smad2/3 which is a molecule in downstream of, and the activity of MMP-2/9. TGF-β can significantly induce the expression of p-Smad2/3 in EC9706 sh Control cells. TAGLN2 knock-down inhibited the phosphorylation of p-Smad2/3 of TGF-β signaling pathway. The constitutive expression of MMP-2 and its expression induced by TGF-β were remarkably decreased after TAGLN2 knock-down, while the expression of MMP-9 was not appreciably changed.Conclusions 1 TGF-β may contribute to the malignant progression of esophageal squamous cell carcinoma through TAGLN2 up-regulation. 2 TAGLN2 up-regulation can promote the invasion and metastasis of ESCC, which may be one of the key molecules in the malignant progression of ESCC. 3 TGF-β may promote the malignant evolution of ESCC through TAGLN2 and TAGLN2 may be one of potential molecular targets for the treatment of esophageal squamous cell carcinoma.