Institute of Biochemistry and Molecular Biology, School of Life Science, National Yang Ming Chiao Tung University, Taipei 112, Taiwan.
General Surgery Division, Department of Medical Research, Far Eastern Memorial Hospital, New Taipei City 220, Taiwan.
Int J Mol Sci. 2022 Jul 29;23(15):8419. doi: 10.3390/ijms23158419.
Chronic inflammation caused by liver damage or infection plays an important role in the development and progression of hepatocellular carcinoma (HCC). The activation of Toll-like receptors 4 (TLR4) is involved in HCC tumorigenesis. Moreover, high TLR4 expression in HCC has been linked to poor prognosis. Although the expression of TLR4 in HCC is relatively low compared to hematopoietic cells, it is important to explore the molecular mechanism leading to the elevation of TLR4 in HCC. In this study, we aimed to investigate the positive regulating loop for TLR4 expression in HCC in response to chronic inflammation. Our results confirm that the mRNA expression of TLR4 and proinflammatory cytokines, including interleukin 6 (IL6) and C-C motif chemokine ligand 2 (), positively correlate in human HCC samples. High TLR4 expression in HCC is more susceptible to lipopolysaccharide (LPS); TLR4 activation in HCC provides growth and survival advantages and thus promotes tumorigenesis. It has been shown that the LIN28/let-7 microRNA (miRNA) axis is a downstream effector of the TLR4 signal pathway, and let-7 miRNA is a potential post-transcriptional regulator for TLR4. Thus, we investigated the correlation between TLR4 and LIN28A mRNA and let-7g miRNA in HCC clinical samples and found that the expression of TLR4 was positively correlated with LIN28A and negatively correlated with let-7g miRNA. Moreover, by culturing PLC/PRF5 (PLC5) HCC cells in low-dose LPS-containing medium to mimic chronic inflammation for persistent TLR4 activation, the mRNA and protein levels of TLR4 and LIN28A were elevated, and let-7g miRNA was decreased. Furthermore, the 3' untranslated region (3'UTR) of TLR4 mRNA was shown to be the target of let-7g miRNA, suggesting that inhibition of let-7g miRNA is able to increase TLR4 mRNA. While parental PLC5 cells have a low susceptibility to LPS-induced cell growth, long-term LPS exposure for PLC5 cells leads to increased proliferation, cytokine expression and stemness properties. In conclusion, our studies demonstrate positive feedback regulation for chronic TLR4 activation in the modulation of TLR4 expression level through the LIN28A/let-7g pathway in HCC and suggest a connection between chronic inflammation and TLR4 expression level in HCC for promoting tumorigenesis.
肝损伤或感染引起的慢性炎症在肝细胞癌 (HCC) 的发生和发展中起重要作用。Toll 样受体 4 (TLR4) 的激活参与 HCC 肿瘤发生。此外，HCC 中高 TLR4 表达与预后不良相关。尽管与造血细胞相比，HCC 中 TLR4 的表达相对较低，但探索导致 HCC 中 TLR4 升高的分子机制很重要。在这项研究中，我们旨在研究慢性炎症下 HCC 中 TLR4 表达的正调控环。我们的结果证实，人类 HCC 样本中 TLR4 和促炎细胞因子（包括白细胞介素 6 (IL6) 和 C-C 基序趋化因子配体 2 () 的 mRNA 表达呈正相关。HCC 中高 TLR4 表达对脂多糖 (LPS) 更敏感；TLR4 在 HCC 中的激活提供了生长和存活优势，从而促进了肿瘤发生。已经表明，LIN28/let-7 微小 RNA (miRNA) 轴是 TLR4 信号通路的下游效应物，而 let-7 miRNA 是 TLR4 的潜在转录后调节物。因此，我们在 HCC 临床样本中研究了 TLR4 和 LIN28A mRNA 与 let-7g miRNA 之间的相关性，发现 TLR4 的表达与 LIN28A 呈正相关，与 let-7g miRNA 呈负相关。此外，通过在含有低剂量 LPS 的培养基中培养 PLC/PRF5 (PLC5) HCC 细胞来模拟持续 TLR4 激活的慢性炎症，TLR4 和 LIN28A 的 mRNA 和蛋白水平升高，而 let-7g miRNA 降低。此外，TLR4 mRNA 的 3'非翻译区 (3'UTR) 被证明是 let-7g miRNA 的靶标，表明抑制 let-7g miRNA 能够增加 TLR4 mRNA。虽然亲本 PLC5 细胞对 LPS 诱导的细胞生长的敏感性较低，但长期 LPS 暴露导致 PLC5 细胞增殖、细胞因子表达和干性增加。总之，我们的研究表明，在 HCC 中，通过 LIN28A/let-7g 通路对慢性 TLR4 激活的正反馈调节调节 TLR4 表达水平，并提示慢性炎症与 HCC 中 TLR4 表达水平之间存在促进肿瘤发生的联系。
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