The Action and Mechanism of Action of Toll-Like Receptor 4 (TLR4) on the Proliferation and Migration of Non-Small Cell Lung Carcinoma Cells

Abstract

Background: The overall cure and survival rates of non-small cell lung cancer (NSCLC) are still very low, and this disease has serious effects on patients’ quality of life. The aim of this study was to explore the role and possible mechanism of toll-like receptor 4 (TLR4) in NSCLC. Methods: This study screened the core differentially expressed genes (DEGs) related to cell adhesion molecules (CAMs), through bioinformatics methods. The TLR4 expression in A549 was knocked down by transfecting with shTLR4 (TLR4 shRNA). Scratch assay and flow cytometry were used to evaluate the effects of TLR4 knockdown on A549 cell migration and apoptosis, respectively. The binding of TLR4 and miR-20b-5p was proved by dual-luciferase reporter assay. The effect of TLR4 on tumor growth in vivo was investigated by establishing xenograft tumor mouse model. The quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect messenger ribonucleic acid (mRNA) and protein levels of CAMs-related genes. Results: miR-20b-5p and TLR4 are core differentially expressed miRNA (DEmiRNA) and DEG with targeted regulatory relationship in the interaction network through bioinformatics analysis. In vitro, shTLR4 transfection significantly inhibited A549 cell migration (p < 0.01) and promoted cell apoptosis (p < 0.001). In vivo, xenograft tumor growth in SH group mice was also significantly inhibited (p < 0.01). Dual luciferase reporter gene experiment also demonstrated the targeted regulation of miR-20b-5p on TLR4. In addition, qRT-PCR and Western blot also showed that TLR4 knockdown could significantly down-regulate the expression of syndecan-1 and intergrin β3, and up-regulate the expression of E-cadherin. These results above indicated that TLR4 was targeted and down-regulated by miR-20b-5p and promoted the proliferation, migration of NSCLC cells and inhibiting the apoptosis by regulating the levels of CAMs. Conclusions: TLR4 and miR-20b-5p can be used as meaningful biological targets to study the regulation of cell proliferation and migration by NSCLC.