Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells

Liu,Yating; Li,Tao; Xu,Yuanhong; Xu,Enjun; Zhou,Min; Wang,Baolong; Shen,Jilong

doi:10.3892/ol.2016.4338

Effects of TLR4 gene silencing on the proliferation and apotosis of hepatocarcinoma HEPG2 cells

Authors:
- Yating Liu
- Tao Li
- Yuanhong Xu
- Enjun Xu
- Min Zhou
- Baolong Wang
- Jilong Shen
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Affiliations: Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P.R. China, ICU, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P.R. China, Department of Clinical Laboratory, The Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui 230022, P.R. China, Anhui Provincial Laboratory of Pathogen and Biology Zoonoses, Anhui Medical University, Hefei, Anhui 230022, P.R. China
Published online on: March 16, 2016 https://doi.org/10.3892/ol.2016.4338
Pages: 3054-3060
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Toll-like receptors (TLRs) are key factors in the innate immune system and initiate an inflammatory response to foreign pathogens, such as bacteria, fungi and viruses. TLR4-mediated signaling has been implicated in tumor cell proliferation and apoptosis in numerous cancers. The present study aimed to investigate the biological effect of TLR4 on the proliferation and apoptosis of human liver cancer cells and the mechanisms responsible for the regulation of cellular responses following TLR4 gene knockdown. Three TLR4 small interfering (si)RNA constructs, consisting of TLR4‑siRNA‑1, TLR4‑siRNA‑2 and TLR4‑siRNA‑3, were transiently transfected into HepG2 cells using Lipofectamine 2000. TLR4 knockdown was confirmed using reverse transcription‑polymerase chain reaction and western blotting. The effect of the TLR4 siRNA on tumor cell proliferation was monitored by methyl thiazolyl tetrazolium assay and cell apoptosis was observed by flow cytometry. The expression of TLR4‑associated proteins, consisting of myeloid differentiation primary response 88 (MyD88), Toll‑interleukin‑1R‑domain‑containing adapter‑inducing interferon‑β (TRIF), interferon regulatory factor‑3 (IRF3), nuclear factor (NF)‑κB, NF‑κB inhibitor α (IκBα), phosphorylated IκBα (p‑IκBα), extracellular signal‑regulated kinase (ERK) and c‑Jun N‑terminal kinase (JNK), was detected by western blot analysis. TLR4‑siRNA‑1 had the strongest knockdown effect and inhibited TLR4 messenger RNA and protein expression. TLR4 knockdown with TLR4‑siRNA‑1 reduced cell proliferation and promoted cell apoptosis. MyD88, TRIF, IRF3, IκBα, JNK and ERK were markedly suppressed in the cells transfected with TLR4 siRNA. However, nuclear expression of NF‑κB and p‑IκBα increased in HepG2 cells with TLR4 gene knockdown. The present study revealed that TLR4‑mediated signaling plays a key role in the proliferation and apoptosis of cultured hepatocarcinoma cells. Therefore, RNA interference-directed targeting of TLR4 may raise the potential of the application of TLR4 knockdown for liver cancer therapy.

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