Mechanism Study Of MiRNA-181b In Regulation Of TGF-? Induced Epithelial To Mesenchymal Transition Of Gastric Cancer Cells

Background/Aims:Gastric cancer(gastric cancer,GC),as a malignant digestive tract tLumor,seriously endangers human life and health.In order to early diagnosis and treatment of gastric cancer,mechanism study of the pathogenesis of gastric cancer is of great significance.Epithelial to mesenchymal transition(EMT)plays an important role in the development and progression of gastric cancer,EMT can weaken the adhesion of gastric cancer cells,promote the shedding of gastric cancer cells and endow them with migration,thus causing the distant metastasis of gastric cancer cells.As we know,the transforming growth factor ?(TGF-?)plays an important role in tumor growth,which restricts the proliferation of epithelial cells and tumor cells in early tumors,but activates EMT in advanced tumors and accelerates tumor progression and metastasis.The miRNA-181 family members play an important role in the proliferation,differentiation,apoptosis,invasion,metastasis and chemotherapy resistance of multiple malignancies(such as breast,lung,liver,hematological,pancreatic,gastric,and glioma).Among them,miRNA-181b was highly expressed in gastric tumors,and was also associated with multiple drug resistance via targeting BCL2.However,the potential mechanism of the role of TGF-?-induced miRNA-181b in the pathogenesis and metastasis of gastric cancer has not been fully elucidated.And the direct target gene of miRNA-181b is still not recognized in the EMT process induced by TGF-?.This study aimed to confirm the relationship of miRNA-181b and the TGF-?-Smad2/3/4 pathway with the induction of the epithelial to mesenchymal transition(EMT)in gastric cancer.Methods:This study investigated the ability of TGF-? to induce migration by wound healing and transwell invasion assays in human gastric cancer cell lines(MKN-28,SGC-7901,HGC-27 and AGS).MiRNA expression was altered by using miRNA-181b mimic and inhibitor in the same system.The morphological changes of gastric cancer cells were evaluated by immunofluorescence and laser confocal detection of cytoskeleton protein f-actin.Expression of miRNA-181b,the hypothetical target gene Timp3 and EMT-related markers were analyzed by real-time quantitative PCR.Western Blotting was used to investigate the levels of phospho-Smad2 and Smad4.A lentiviral vector containing Smad4-specific siRNA sequences was constructed and the gastric cancer cell lines with Smad4 stable knockout were screened.Dual-luciferase reporter assays were performed to confirm the direct binding of miRNA-181b to Timp3.We also established the Timp-3 overexpressed gastric cancer cell lines to perform rescue experiment.Finally,the effect of miRNA-181b-Timp3 pathway on the proliferation and migration of gastric cancer cells was validated by Balb/c NOD xenograft mice in vivo.Results:TGF-? stimulation can significantly promote the migration(HGC-27:P=0.014;AGS:P=0.021),invasion(HGC-27:P=0.002;AGS:P=0.011)and EMT of gastric cancer cells,and significantly increase the expression of precursor(MKN-28:P=0.033 SGC-7901:P=0.008 HGC-27:P=0.004;AGS:P=0.003)and mature(MKN-28:P=0.041;SGC-7901:P=0.003;HGC-27:P=0.009;AGS:P=0.005)miRNA-181b in gastric cancer cell lines.Overexpression of miRNA-181b mimic induced an in vitro EMT-like change to a phenotype similar to that following TGF-? treatment alone and was reversed by miRNA-181b inhibitor(HGC-27:P=0.036;AGS:P=0.024).Inhibition of TGF-?-Smad2/3 signaling with SD-208 significantly attenuated the upregulation of precursor(HGC-27:P=0.037;AGS:P=0.006)and mature(HGC-27:P=0.017;AGS:P=0.012)miRNA-181b.Knockdown of Smad4 in gastric cancer cells strongly attenuated the upregulation of precursor(HGC-27:P=0.042;AGS:P=0.036)and mature(HGC-27:P=0.039;AGS:P=0.021)miRNA-181b.Moreover,miRNA-181b was found to directly target the 3'-untranslated region(3 'UTR)of Timp3 mRNA affecting TGF-?-induced EMT.It was found that the miRNA-181b-timp3 pathway had no significant effect on the proliferation of gastric cancer cells,but significantly promoted the migration ability of gastric cancer cells in vivo(P=0.021).Conclusions:Our results elucidate a novel mechanism through which the TGF-?pathway regulates the EMT of gastric cancer cells by increasing the levels of miRNA-181b to target Timp3 via the Smad2/3/4-dependent pathway.These findings provide insights into the cellular and environmental factors regulating EMT,which may guide future studies on therapeutic strategies targeting human gastric cancer.