摘要
目的 探讨lncRNA X染色体失活特异转录因子(XIST)靶向miR-302a-3p对白细胞介素-1β(IL-1β)诱导软骨细胞损伤的调控机制。方法 SW1353细胞分为Control组、IL-1β组、IL-1β+si-NC组、IL-1β+si-XIST组、IL-1β+si-PDK1组、IL-1β+si-XIST+miR-NC inhibitor组、IL-1β+si-XIST+miR-302a-3p inhibitor组。检测XIST、miR-302a-3p以及3-磷酸肌醇依赖性蛋白激酶1(PDK1)mRNA表达;噻唑蓝(MTT)实验测定细胞活力;流式细胞术检测细胞凋亡;qRT-PCR检测炎症因子(TNF-α、IL-1β、IL-4、IL-6、IL-10)水平;双荧光素酶报告基因检测实验证实miR-302a-3p与XIST或PDK1的靶向关系;免疫印迹法检测凋亡蛋白(Bcl-2、Bax)及PDK1蛋白表达。结果 与IL-1β组相比,IL-1β+si-XIST组XIST表达下调,细胞活力升高,凋亡率降低,Bax表达及TNF-α、IL-1β、IL-6水平下降,Bcl-2表达及IL-4、IL-10水平升高(P<0.05)。XIST直接靶向下调miR-302a-3p表达,miR-302a-3p下调可恢复si-XIST介导的促增殖、抗凋亡和炎症反应作用(P<0.05)。PDK1是miR-302a-3p的靶基因,沉默PDK1可抑制细胞的炎症反应(P<0.05)。结论 敲低XIST可提高IL-1β诱导软骨细胞的细胞活力,减轻OA样软骨细胞损伤,其作用机制与靶向上调miR-302a-3p进而抑制PDK1表达有关。
Objective To investigate the regulatory mechanism of lncRNA X inactive specific transcript(XIST) on interleukin-1β(IL-1β)-induced chondrocyte injury by targeting miR-302 a-3 p. Methods The human chondrosarcoma cell line SW1353 in vitro was induced by 10 ng/mL IL-1β to simulate osteoarthritis(OA)-like chondrocyte injury. SW1353 cells were separated into Control group, IL-1β group, IL-1β+si-NC group, IL-1β+si-XIST group, IL-1β+si-PDK1 group, IL-1β+si-XIST+miR-NC inhibitor group, IL-1β+si-XIST+miR-302 a-3 p inhibitor group. To measure the expression of XIST, miR-302 a-3 p and 3-phosphoinositide-dependent protein kinase 1(PDK1) mRNA;methyl thiazolyl tetrazolium(MTT) experiment was performed to determine the viability of cells;flow cytometry was performed to determine cell apoptosis;qRT-PCR was performed to determine the expression levels of inflammatory factors(TNF-α, IL-1β, IL-4, IL-6, IL-10);a dual-luciferase reporter gene assay was performed to confirm the targeting relationship of miR-302 a-3 p to XIST or PDK1;Western blot was performed to determine the expression of apoptotic protein(Bcl-2, Bax) and PDK1. Results Compared with IL-1β group, the expression of XIST in the IL-1β+si-XIST group was down-regulated, the cell viability was increased, the apoptosis rate was decreased, the expression of Bax and the levels of TNF-α, IL-1β and IL-6 was decreased, the expression of Bcl-2 and the levels of IL-4 and IL-10 was increased(P<0.05). XIST directly targeted and down-regulated the expression of miR-302 a-3 p, downregulation of miR-302 a-3 p was able to restore the pro-proliferative, anti-apoptotic and inflammatory responses mediated by si-XIST(P<0.05). PDK1 was a target gene of miR-302 a-3 p, the silencing of PDK1 inhibited the inflammatory response of cells(P<0.05). Conclusion Knockdown of XIST can improve the cell viability of IL-1β-induced chondrocytes, alleviate the damage of OA-like chondrocytes, and its mechanism is related to the inhibition of PDK1 expression by targeting the up-regulation of miR-302 a-3 p.
作者
黄涛
方红育
周少怀
卞峰
任敏
李宏亮
俞诗威
严锦曦
钱慧
李嘉琼
HUANG Tao;FANG Hongyu;ZHOU Shaohuai;BIAN Feng;REN Min;LI Hongliang;YU Shiwei;YAN Jinxi;QIAN Hui;LI Jiaqiong(Department of orthopedics,Wuhan Third Hospital,Wuhan 430060,China)
出处
《中国骨质疏松杂志》
CAS
CSCD
北大核心
2022年第10期1416-1421,1427,共7页
Chinese Journal of Osteoporosis
基金
武汉市医学科研项目(WX19D14)。
