Effect Researches Of MiR-146b-5p Regulats Proliferation,Apoptosis,Migration And Invasion Of Gallbladder Cancer Cells Via Targeting TLR4

Object:Explore the expression of miR-146b-5p in gallbladder cancer and its effect on the biological behavior of gallbladder cancer cells,and preliminarily reveal the possible mechanism of action.Methods:First,the expressions of miR-146b-5p in gallbladder cancer tissues and cell lines were detected by RT-qPCR technique.Second,transfer miR-146b-5p mimic and inhibitor in gallbladder cancer cells.CCK-8,EdU staining and clone formation experiments were used to detect the effect of miR-146b-5p on cell proliferation activity;flow cytometry and TUNEL assay were used to observe the cell apoptosis rate;scratch and Transwell chamber experiments were used to detect the cell migration and invasion ability;Western blot was used to detect cell proliferation-related proteins PCNA,apoptosis-related proteins Cleaved caspase-3,Cleaved caspase-9,Bax,Bcl-2,and migration,invasion and epithelial-mesenchymal transition(EMT)related proteins COX-2,MMP-2,MMP-9,E-cadherin,N-cadherin,Vimentin.Use dual luciferase report to verify the binding of miR-146b-5p and TLR43’-UTR,and analyze the correlation between miR-146b-5p and TLR4;RT-qPCR and Western blot were used to detect the expressions of TLR4 mRNA and protein in gallbladder cancer tissues and cell lines,and to detect the regulatory effect of miR-146b-5p on TLR4 expression.Construct a TLR4 overexpression plasmid,and verify whether miR-146b-5p participates in the regulation of the biological behavior of gallbladder cancer cells by targeting TLR4.Finally,construct a nude mouse transplanted tumor model and a hepatic metastasis model of human gallbladder cancer cells to detect the volume of the transplanted tumor,use HE staining to detect the pathological damage of transplanted tumors and hepatic tissues,use TUNEL to detect cell apoptosis rate in transplanted tumor,use immunohistochemical staining to detect the expression of TLR4 in transplanted tumors and hepatic tissues,use Western blot to detect the expressions of Cleaved caspase-3,Cleaved caspase-9,MMP-2 and MMP-9 in transplanted tumors and hepatic tissues.Results:RT-qPCR results showed that the expressions of miR-146b-5p in gallbladder cancer tissues and cells were significantly lower than that in adjacent tissues and gallbladder epithelial cells;Cell functional experiments showed that miR-146b-5p mimics can inhibit proliferation,migration and invasion of gallbladder cancer cells and promote their apoptosis,while miR-146b-5p inhibitors showed the opposite effect;Western blot results showed that miR-146b-5p overexpression can significantly reduce the expressions of PCNA,Bcl-2,COX-2,MMP-2,MMP-9,N-cadherin and Vimentin in gallbladder cancer cells,and increase the expressions of Cleaved caspase-3,Cleaved caspase-9,Bax and E-cadherin,while transfection of miR-146b-5p inhibitors has the opposite effect on the expressions of these proteins.Dual luciferase report showed that miR-146b-5p can bind to TLR4 3’-UTR,and there was a significant negative correlation between TLR4 and miR-146b-5p(r2=0.9587);RT-qPCR and Western blot all showed that the expression of TLR4 in gallbladder cancer tissues and cell lines was significantly higher than that in adjacent tissues and gallbladder epithelial cells,and miR-146b-5p can regulate the expressions of TLR4 mRNA and protein in gallbladder cancer cells.Rescue experiments confirmed that the rescue of TLR4 expression weakened the effect of miR-146b-5p on the biological behavior of gallbladder cancer cells,and the expression level of related proteins also changed significantly.The results of in vivo experiments showed that miR-146b-5p mimics can inhibit the growth of transplanted tumors,reduce the number of hepatic metastatic nodules and improve hepatic tissue pathological damage;TUNEL assay and immunohistochemical staining showed that miR-146b-5p mimics can increase the apoptosis rate of cancer cells in transplanted tumors,and reduce the expression of TLR4 in transplanted tumors and hepatic tissues.In addition,miR-146b-5p mimics can increase the expressions of Cleaved caspase-3 and Cleaved caspase-9 in transplanted tumors,and reduce the expressions of MMP-2 and MMP-9 in hepatic tissues.Conclusions:(1)miR-146b-5p is lowly expressed in gallbladder cancer and its overexpression can inhibit malignant progression and hepatic metastasis in vivo of gallbladder cancer.(2)miR-146b-5p plays the role of anti-gallbladder cancer by regulating TLR4 expression.