Hui Li1, Shi-Bing Guan1, Yi Lu2, Fei Wang3. 1. Department of Hand and Foot Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Ji'nan, 250021, PR China. 2. Department of Anesthesiology Surgery,Shandong Provincial Hospital Affiliated to Shandong University, Ji'nan, 250021, PR China. 3. Department of Joint Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, 050051, PR China. Electronic address: wangfei050051@163.com.
Abstract
OBJECTIVES: This study was aimed to investigate the effects of miR-140-5p on the proliferation and inflammatory cytokines secretion of rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Synovial tissue samples from 23 rheumatoid arthritis (RA) patients and 18 normal synovial tissue samples were collected. The RASFs were isolated and cultured. Then, miR-140-5p and TLR4 expression in both synovial tissue and RASFs were detected using the quantitative real-time PCR (qPCR) and western blot. Dual luciferase reporter gene assay was employed to evaluate the interaction between miR-140-5p and 3'UTR of TLR4. Western blotting and qPCR were used to examine TLR2 expression after upregulation or downregulation of miR-140-5p in RASFs. After RASFs co-infected with TLR4 overexpression lentivirus and lentivirus containing miR-140-5p or miR-control respectively, the cellular proliferation and secretion of IL-6 and IL-8 level were detected through the MTS assay and enzyme-linked immunosorbent assay, respectively. RESULTS: MiR-140-5p was significantly down-regulated, and TLR4 was significantly up-regulated in synovial tissue samples from 23 RA patients and RASFs. Dual luciferase activity assay showed that miR-140-5p could specifically bind to the 3'UTR of TLR4. Down-regulation or up-regulation of miR-140-5p not only significantly increased or decreased the expression of TLR4, but also could promote or inhibit RASF proliferation and secretion of IL-6, and IL-8 in RASFs. Furthermore, overexpression of TLR4 can reverse the inhibitory effects of miR-140-5p on proliferation and inflammatory cytokines release of RASFs. CONCLUSIONS: MiR-140-5p could inhibit the proliferation and secretion of IL-6 and IL-8 through regulation of TLR4 expression.
OBJECTIVES: This study was aimed to investigate the effects of miR-140-5p on the proliferation and inflammatory cytokines secretion of rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Synovial tissue samples from 23 rheumatoid arthritis (RA) patients and 18 normal synovial tissue samples were collected. The RASFs were isolated and cultured. Then, miR-140-5p and TLR4 expression in both synovial tissue and RASFs were detected using the quantitative real-time PCR (qPCR) and western blot. Dual luciferase reporter gene assay was employed to evaluate the interaction between miR-140-5p and 3'UTR of TLR4. Western blotting and qPCR were used to examine TLR2 expression after upregulation or downregulation of miR-140-5p in RASFs. After RASFs co-infected with TLR4 overexpression lentivirus and lentivirus containing miR-140-5p or miR-control respectively, the cellular proliferation and secretion of IL-6 and IL-8 level were detected through the MTS assay and enzyme-linked immunosorbent assay, respectively. RESULTS:MiR-140-5p was significantly down-regulated, and TLR4 was significantly up-regulated in synovial tissue samples from 23 RApatients and RASFs. Dual luciferase activity assay showed that miR-140-5p could specifically bind to the 3'UTR of TLR4. Down-regulation or up-regulation of miR-140-5p not only significantly increased or decreased the expression of TLR4, but also could promote or inhibit RASF proliferation and secretion of IL-6, and IL-8 in RASFs. Furthermore, overexpression of TLR4 can reverse the inhibitory effects of miR-140-5p on proliferation and inflammatory cytokines release of RASFs. CONCLUSIONS:MiR-140-5p could inhibit the proliferation and secretion of IL-6 and IL-8 through regulation of TLR4 expression.