Mingliang Qiu1,2, Lisha Mo2, Juxiang Li1, Hua Liang3, Weina Zhu4, Xiangjuan Zheng5, Xinwang Duan6, Weidong Xu7. 1. Department of Cardiovascular Medicine, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, China. 2. Department of Rheumatology, The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, 330006, China. 3. Department of Clinical Laboratory, The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, 330006, China. 4. Department of Pediatrics, The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, 330006, China. 5. Department of Chemistry, Nanchang University, Nanchang, 330031, China. 6. Department of Rheumatology, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, China. 13970085678@163.com. 7. Department of Rheumatology, The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, 330006, China. jindadong169@163.com.
Abstract
OBJECTIVE: miR-150-5p has been implicated in the regulation and onset of immune diseases. We investigated the effects of miR-150-5p on the functions of RA synovial fibroblasts (RASFs). METHOD: The binding site between suppressor of cytokine signaling 1 (SOCS1) and miR-150-5p was analyzed using European Bioinformatics Institute database, and the 3' UTR of SOCS1 mRNA, including the binding site, was amplified and ligated to the 3'-end of LUC2 gene in the pmirGL0 dual-luciferase vector. The pmirGL0 vector and corresponding mimics were subsequently co-transfected into 293T cells to compare the relative fluorescence intensity of LUC2 between the miR-150-5p mimics and the negative control (NC) mimics groups. Further, the RASF cell line MH7A was transfected with miR-150-5p or NC mimics and subjected to flow cytometric analysis, cell counting kit-8 assay, western blot analysis, qPCR, and enzyme-linked immunosorbent (ELISA) assay 48 h after transfection. RESULTS: miR-150-5p mimics resulted in a lower cell apoptotic rate and proportion of cells in the S phase. Using a dual-luciferase reporter gene assay, we then found that SOCS1 is a potential target of miR-150-5p. Compared with NC mimics, miR-150-5p mimics significantly decreased the protein and mRNA expression levels of SOCS1. ELISA assay showed that miR-150-5p mimics increased interleukin-6 level in the cell culture medium but did not influence tumor necrosis factor-alpha levels. CONCLUSIONS: Overall, the growth-promoting effect of miR-150-5p on MH7A cells may be attributed to the miR-150-5p-induced degradation of SOCS1 mRNA, suggesting a potential therapeutic target for RA.Key Points• SOCS1 is a potential target of miR-150-5p.• miR-150-5p promoted the growth of RASF cell line MH7A.• miR-150-5p increased the secretion of IL-6 but did not significantly affect TNF-α levels in MH7A cells.
OBJECTIVE:miR-150-5p has been implicated in the regulation and onset of immune diseases. We investigated the effects of miR-150-5p on the functions of RA synovial fibroblasts (RASFs). METHOD: The binding site between suppressor of cytokine signaling 1 (SOCS1) and miR-150-5p was analyzed using European Bioinformatics Institute database, and the 3' UTR of SOCS1 mRNA, including the binding site, was amplified and ligated to the 3'-end of LUC2 gene in the pmirGL0 dual-luciferase vector. The pmirGL0 vector and corresponding mimics were subsequently co-transfected into 293T cells to compare the relative fluorescence intensity of LUC2 between the miR-150-5p mimics and the negative control (NC) mimics groups. Further, the RASF cell line MH7A was transfected with miR-150-5p or NC mimics and subjected to flow cytometric analysis, cell counting kit-8 assay, western blot analysis, qPCR, and enzyme-linked immunosorbent (ELISA) assay 48 h after transfection. RESULTS:miR-150-5p mimics resulted in a lower cell apoptotic rate and proportion of cells in the S phase. Using a dual-luciferase reporter gene assay, we then found that SOCS1 is a potential target of miR-150-5p. Compared with NC mimics, miR-150-5p mimics significantly decreased the protein and mRNA expression levels of SOCS1. ELISA assay showed that miR-150-5p mimics increased interleukin-6 level in the cell culture medium but did not influence tumor necrosis factor-alpha levels. CONCLUSIONS: Overall, the growth-promoting effect of miR-150-5p on MH7A cells may be attributed to the miR-150-5p-induced degradation of SOCS1 mRNA, suggesting a potential therapeutic target for RA.Key Points• SOCS1 is a potential target of miR-150-5p.• miR-150-5p promoted the growth of RASF cell line MH7A.• miR-150-5p increased the secretion of IL-6 but did not significantly affect TNF-α levels in MH7A cells.
Authors: R Starr; T A Willson; E M Viney; L J Murray; J R Rayner; B J Jenkins; T J Gonda; W S Alexander; D Metcalf; N A Nicola; D J Hilton Journal: Nature Date: 1997-06-26 Impact factor: 49.962