Effects Of TLR4 Gene Silencing On Proliferation And Apotosis In Hepatocarcinoma Cells And The Primary Study Of The Mechanism

Objective: To explore the influence of TLR4 gene silencing on proliferation and apotosis in hepatocarcinoma cells in vitro and further discussion the mechanism.Methods: Three TLR4 si RNA named TLR4-si RNA-1, TLR4-si RNA-2, TLR4-si RNA-3, one random negative control si RNA and one FAM-labeled negative control si RNA were constructed. FAM-labeled negative control si RNA transfected into Hep G2 cells with lipo2000. Ratios of si RNA to lipo2000 used were 0.5:1,1:1,1.5:1,2:1, and 2.5:1(μl/μl) to optimize the transfection conditions. Three TLR4 si RNA were transiently transfected into Hep G2 cells with lipefectamine 2000. TLR4 knockdown was confirmed using RT-PCR and Westen Blot. The effect of the TLR4 si RNA on tumor cell proliferation was monitored by MTT assay and cell apoptosis was observed by flow cytometry(FCM). The expression of TLR4-related proteins(My D88, TRIF, IRF3, IκBα, p-IκBα, NF-κB, ERK, and JNK) were detected by Westen Blot.Resμlts: Transfection efficiency coμld observed using fluorescence microscope. When the ratio of si RNA to lipo2000 used was 1:1,the transfection efficiency was the highest up to 90.48%±1.27%. TLR4-si RNA-1 had the strongest knockdown effect and it coμld inhibited TLR4 m RNA and protein expression. TLR4 knockdown with TLR4-si RNA-1 reduced cell proliferation and promoted cell apoptosis. My D88, TRIF, IRF3, IκBα, JNK and ERK were substantially suppressed in the cells transfected with TLR4 si RNA, However, nuclear expression of the NF-κB and p-IκBα increased in Hep G2 cells with TLR4 gene knockdown.Conclusions: The resμlts presented in this paper provide evidence that TLR4 knockdown coμld inhibit the human liver cancer cell proliferation and promote cell apoptosis in vitro through inhibition of ERK and JNK pathway and activate the NF-kappa B pathway in Hep G2.