文章摘要

MiR-467b调控JAK/STAT信号通路在脂多糖诱导ATDC5细胞增殖及炎症反应中的作用

作者: 1刘旭, 2王海龙, 2陈洪涛, 2那次克道尔吉, 2李龙, 1曹力
1 新疆医科大学第一附属医院关节外科,乌鲁木齐 830000
2 新疆医科大学第六附属医院运动损伤科,乌鲁木齐 830000
通讯: 曹力 Email: Xjbone@sina.com
DOI: 10.3978/j.issn.2095-6959.2021.03.002
基金: 国家自然科学基金(81602597)。

摘要

目的:探讨miR-467b调控JAK/STAT信号通路在脂多糖(lipopolysaccharide,LPS)诱导ATDC5细胞增殖及炎症反应中的作用。方法:将ATDC5细胞分为4组:对照组、LPS组、NC mimics组、miR-467b mimics组。于转染培养48 h后收集细胞并进行后续实验:采用ELISA检测各组细胞TNF-α、IL-1β水平;采用qRT-PCR法检测各组细胞miR-467b的表达水平;采用MTT法检测各组细胞的增殖能力;采用蛋白质印迹和qRT-PCR法检测各组细胞JAK/STAT信号通路相关基因的蛋白质及mRNA表达水平。结果:与对照组相比,LPS组和NC mimics组ATDC5细胞TNF-α、IL-1β的表达水平均显著升高、miR-467b的表达水平显著降低(P<0.05);MTT实验结果表明:LPS组在24、48、72 h的吸光度值显著降低(P<0.05)。蛋白质印迹结果发现:与对照组相比,LPS组和NC mimics组ATDC5细胞STAT1、STAT3、JAK2、p-STAT1、p-STAT3、p-JAK2蛋白表达水平及STAT1、STAT3、JAK2 mRNA表达水平均显著升高,差异具有统计学意义(P<0.05)。与NC mimics组相比,miR-467b mimics组TNF-α、IL-1β的表达水平均显著降低而miR-467b的表达水平显著升高(P<0.05);过表达miR-467b后,细胞在24、48、72 h的吸光度值显著升高(P<0.05),而STAT1、STAT3、JAK2、p-STAT1、p-STAT3、p-JAK2蛋白表达水平及STAT1、STAT3、JAK2 mRNA表达水平均显著降低(P<0.05)。结论:LPS刺激ATDC5细胞后可通过激活JAK/STAT3信号通路引起细胞炎性损伤,过表达miR-467b后可抑制JAK/STAT3信号通路的活化,降低ATDC5细胞的炎症水平。
关键词: miR-467b;ATDC5;JAK/STAT信号通路;增殖;炎症反应

Role of miR-467b in regulating JAK/STAT signaling pathway in LPS induced proliferation and inflammatory response of ATDC5 cells

Authors: 1LIU Xu, 2WANG Hailong, 2CHEN Hongtao, 2Nacikedaoer ji, 2LI Long, 1CAO Li
1 Department of Joint Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, China
2 Department of Sports Injury, Sixth Affiliated Hospital of Xinjiang Medical University, Urumqi 830000, China

CorrespondingAuthor: CAO Li Email: Xjbone@sina.com

Foundation: This work was supported by the National Natural Science Foundation of China (81602597).

Abstract

Objective: To explore the role of miR-467b in regulating JAK/STAT signaling pathway in lipopolysaccharide (LPS) induced proliferation and inflammatory response of ATDC5 cells. Methods: ATDC5 cells were divided into four groups: a control group, a LPS group, a NC mimics group, and a miR-467b mimics group. The cells were collected for the next experiment after they were transfected and cultured for 48 h. The levels of TNF-α and IL-1β were detected by ELISA. The expression of miR-467b in each group was detected by qRT-PCR. MTT method was used to detect the proliferation of cells in each group. Western blotting and qRT-PCR were used to detect the expression level of genes of protein and mRNA related to JAK/STAT signal pathway of cells in each group. Results: Compared with the control group, the expression levels of TNF-α and IL-1β in ATDC5 cells in LPS group and NC mimics group were significantly higher, and the expression level of miR-467b was significantly lower (P<0.05). The results of MTT showed that the absorbance value of LPS group decreased significantly at 24, 48 and 72 h (P<0.05). Western blotting showed that the expression level of STAT1, STAT3, JAK2, p-STAT1, p-STAT3, p-JAK2 protein and the expression level of STAT1, STAT3, JAK2 mRNA in ATDC5 cells of LPS group and NC MICs group were significantly higher than those of control group with statistic significance (P<0.05). Compared with NC mimics group, the expression level of TNF-α and IL-1β in miR-467b mimics group decreased significantly, while the expression level of miR-467b increased significantly (P<0.05). After the overexpression of miR-467b, the absorbance value of cells increased significantly at 24, 48 and 72 h (P<0.05), while the expression levels of STAT1, STAT3, JAK2, p-STAT1, p-STAT3, p-JAK2 protein and the expression levels of STAT1, STAT3, JAK2 mRNA decreased significantly (P<0.05). Conclusion: LPS can cause inflammatory damage by activating JAK/STAT signaling pathway after stimulating ATDC5 cells. The overexpression of miR-467b can inhibit the activation of JAK/STAT signaling pathway and reduce the inflammatory level of ATDC5 cells.
Keywords: miR-467b; ATDC5; JAK/STAT signaling pathway; proliferation; inflammatory response