ABSTRACT
Objective To investigate the microenvironment alteration in epithehiae-mesenchymce transition(EMT)metastasis in gastric carcinoma induced by transforming growth factor β(TGF-β). Methods The gastric carcinoma cell line NCI-N87 was treated with TGF-βto induce cells to undergone EMT,real-time quantitative PCR(RT-qPCR)and immunofluorescence staining were used to examine expression of EMT markers and wound-healing assays,and transwell migration and invasion assays were performed to determine the potential of cell migration and invasion. Further,alteration of cytokines in tumor microenvironment was detected using real-time quantitative PCR and ELISA. Moreover,the activity of its downstream signaling molecules was detected by Western blot. Results TGF-β induced gastric carcinoma cells NCI-N87 to undergone EMT as well as promote cell migration and invasion. Further,TGF-βinduced upregulation of epidermal growth foctor(EGF)and vascular endothelial growth facfor(VEGF)expression,as well as downregulation of Dickkopf-1(DKK1)and secreted frizzled receptor proein1(SFRP1),which activated PI3K/AKT and Wnt/β-catenin sequentially. Conclusion TGF-β promotes EMT by inducing microenvironment alteration in gastric carcinoma cell NCI-N87.
ABSTRACT
Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) may be a precursor lesion of inifltrating pancreatic ductal adenocarcinoma. The mutation of the phenotypic impact of K-ras G12D alone, silencing of p53 and p16 could promote this process. The role of Smad4 in this progression was poorly understood. In our previous studies, we investigated that RNA interference silence of Smad4 to promote the PanIN cell malignant transformation. In the present study, we investigate. The further explores the siRNA interference of Smad4 expression on PanIN cells could lead to proliferation and metastasis in vitro and in vivo. Methods:Smad4 knock-down PanIN cells (PanIN-S) were established by stable transfection with lentiviral-mediated Smad4 RNA interference. In vitro,silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. A soft agar assay was used to assess the anchorage-independent growth ability of cells. Cell migration and invasion assays were performed using transwell chambers with or without Matrigel. In xenograft model experiments, PCNA, VEGF and MMP-9 staining was separately used to evaluate cell proliferation and angiogenesis and migration (VEGF and MMP-9). Results:Effect of siRNA of Smad4 gene in PanIN cells was conifrmed by real-time RT-PCR and western blot. In vitro, silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. Soft agar assay showed that there were more colony cell numbers in PanIN-S cells compared with PanIN cells (P<0.05). Using the transwell assay, we observed that PanIN-S cells migrated faster than PanIN cells and similar results were obtained by Matrigel assay (P<0.05). Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with cell proliferation (PCNA reactivity) and angiogenesis and migration (VEGF and MMP-9), and the expressions of PCNA, VEGF and MMP-9 in PanIN-S group were signiifcantly increased (P<0.05). Conclusion:Silence of Smad4 in PanIN cells enhanced progression to invasive adenocarcinoma of the pancreas by promoting cell growth, migration and invasion. Smad4 might be a new diagnostic marker in pancreatic cancer and prove to be a feasible and novel target for therapeutic intervention.