Huang Cuiyuan, Zhang Hong, Bai Ruidan, Wang Li, Lv Jian
Department of Pharmacy, Renmin Hospital of Wuhan University , Wuhan, China .
DNA Cell Biol. 2017 Nov;36(11):1029-1038. doi: 10.1089/dna.2017.3892. Epub 2017 Sep 25.
Toll-like receptor 4 (TLR4) appears to play an important role in the development and progression of hepatocellular carcinoma (HCC), but it is unclear whether single-nucleotide polymorphisms (SNPs) in the TLR4 gene influence HCC. In this study, we investigated the effects of TLR4 SNPs on HepG2 cell survival and proliferation, migration, and invasion. Plasmids carrying wild-type or mutant versions of the TLR4 gene (A896G and/or C1196T) were stably transfected into HepG2 cells, and cell viability and proliferation were analyzed using the Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, whereas apoptosis was assessed using flow cytometry. Migration and invasion were measured in a transwell chamber assay, and expression of inflammatory cytokines and downstream effectors was examined using real-time PCR and western blotting. Specific inhibitors of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), or phosphatidylinositol 3-kinase (PI3K) were added to the HepG2 cultures to explore the potential role of each pathway in TLR4 signaling. TLR4 SNPs did not affect expression levels in transfected cells. Compared with wild-type TLR4, mutant TLR4 was associated with lower cell proliferation, migration, invasion, and apoptotic threshold. In addition, the mutations were associated with significantly lower expression of nuclear factor κB (NF-κB), IL-6, and TGF-β1, even after stimulation with lipopolysaccharide. The expression of p-Akt was similar in the presence of wild-type or mutant TLR4. The 896G and 1196T SNPs in the TLR4 gene are associated with reduced TLR4-mediated signaling and, therefore, with lower survival, proliferation, and metastasis in HepG2 cells.
Toll样受体4（TLR4）似乎在肝细胞癌（HCC）的发生和发展中起重要作用，但尚不清楚TLR4基因中的单核苷酸多态性（SNP）是否会影响HCC。在本研究中，我们调查了TLR4 SNP对HepG2细胞存活、增殖、迁移和侵袭的影响。将携带野生型或突变型TLR4基因（A896G和/或C1196T）的质粒稳定转染到HepG2细胞中，并使用细胞计数试剂盒-8（CCK-8）和5-乙炔基-2'-脱氧尿苷（EdU）掺入试验分析细胞活力和增殖，而通过流式细胞术评估细胞凋亡。在Transwell小室试验中测量迁移和侵袭，并使用实时PCR和蛋白质印迹法检测炎性细胞因子和下游效应分子的表达。向HepG2培养物中添加c-Jun氨基末端激酶（JNK）、细胞外信号调节激酶（ERK）或磷脂酰肌醇3-激酶（PI3K）的特异性抑制剂，以探索各信号通路在TLR4信号传导中的潜在作用。TLR4 SNP不影响转染细胞中的表达水平。与野生型TLR4相比，突变型TLR4与较低的细胞增殖、迁移、侵袭和凋亡阈值相关。此外，即使在用脂多糖刺激后，这些突变也与核因子κB（NF-κB）、IL-6和TGF-β 的表达显著降低相关。在野生型或突变型TLR4存在下，p-Akt的表达相似。TLR4基因中的896G和1196T SNP与TLR4介导的信号传导减少相关，因此与HepG2细胞中较低的存活率、增殖和转移相关。
