microRNA-22对调控软骨细胞自噬的影响及其在骨关节炎发病中的作用机制  

Role of microRNA-22 regulating chondrocyte autophagy in pathogenesis of osteoarthritis

作　　者：颜世举 董文静 李志锐[1] 赵燕鹏[3] 韩涛[1] 魏均强[1] 王俊良[1] 林峰[1] YAN Shiju;DONG Wenjing;LI Zhirui;ZHAO Yanpeng;HAN Tao;WEI Junqiang;WANG Junliang;LIN Feng(Department of Orthopedics,Hainan Hospital of Chinese PLA General Hospital,Sanya 572013,China;Department of Gerontology,Hainan Hospital of Chinese PLA General Hospital,Sanya 572013,China;Department of Orthopedics,the Fourth Medical Center of Chinese PLA General Hospital,Beijing 100048,China)

机构地区：[1]解放军总医院海南医院骨科,三亚572013 [2]解放军总医院海南医院老年医学科,三亚572013 [3]解放军总医院第四医学中心骨科,北京100048

出　　处：《武警医学》2022年第11期982-986,共5页Medical Journal of the Chinese People's Armed Police Force

基　　金：海南省自然科学基金青年基金项目(819QN379);国家自然科学基金面上项目(82172407)

摘　　要：目的探讨microRNA-22对软骨细胞自噬的影响及其在骨关节炎(OA)发病中作用机制。方法原代培养人健康、OA软骨细胞,荧光定量PCR检测microRNA-22在健康、OA软骨细胞中的表达水平;Western blot检测健康、OA软骨细胞中自噬相关蛋白LC3B-Ⅱ、Ⅰ的表达水平;软骨细胞转染microRNA-22模拟物、抑制物,平板克隆形成实验检测microRNA-22对软骨细胞活性的影响,Western blot检测软骨细胞自噬相关蛋白LC3B-Ⅰ、LC3B-Ⅱ及Ⅱ型胶原(CollagenⅡ)的表达水平。结果荧光定量PCR检测结果显示,OA软骨细胞(14例)中microRNA-22表达含量(2.50±0.39)显著增高(P<0.01),约为健康软骨细胞(12例)(0.98±0.25)的2.5倍。Western blot检测结果显示,与健康软骨细胞相比,OA软骨细胞自噬相关蛋白LC3B的Ⅱ类亚型含量降低;OA软骨细胞(4例)LC3B-Ⅱ/LC3B-Ⅰ比值为1.25±0.13,健康软骨细胞(4例)LC3B-Ⅱ/LC3B-Ⅰ比值为1.85±0.21,差异具有统计学意义(P<0.01)。平板克隆形成实验检测结果显示,与空白对照组(107.33±14.3)相比,软骨细胞转染miR-22模拟物组细胞克隆形成数量(75.67±11.36)降低,软骨细胞转染miR-22抑制物组(128.5±9.95)增高,差异有统计学意义(P<0.05)。Western blot检测结果显示:与空白对照组细胞相比,miR-22模拟物组软骨细胞中LC3B-Ⅱ含量降低(1.50±0.20),LC3B-Ⅱ/LC3B-Ⅰ比值降低(1.45±0.13),Ⅱ型胶原CollagenⅡ的表达水平降低(0.51±0.09);miR-22抑制物组软骨细胞中LC3B-Ⅱ含量增高(2.36±0.19),LC3B-Ⅱ/LC3B-Ⅰ比值增高(2.30±0.08),同时Ⅱ型胶原CollagenⅡ的表达水平增高(1.31±0.03),以上差异均有统计学意义(P<0.05)。结论microRNA-22抑制软骨细胞自噬,从而促进骨性关节炎的发生与进展。Objective To investigate the effects of microRNA-22 on autophagy in human articular chondrocytes and its role in OA pathogenesis.Methods Primary human articular chondrocytes were obtained from cartilage tissue donated by patients.Real time PCR was used to detect the expression level of microRNA-22 in healthy chondrocytes and osteoarthritis(OA)chondrocytes.Western blot was used to detect the expression of LC3 B-Ⅱ、LC3 B-Ⅰin healthy chondrocytes and OA chondrocytes and the ratio of LC3 B-Ⅱ/Ⅰwas calculated.After chondrocytes were transfected with miR-22 mimics and miR-22 inhibitor,colony formation assay was performed to evaluate cell viability and Western blot was used to detect the expression of LC3 B-Ⅱ,LC3 B-Ⅰand CollagenⅡ.Results Real-time PCR suggested that the expression level of microRNA-22 in OA chondrocytes(2.50±0.39)was significantly higher compared with healthy chondrocytes(0.98±0.25).Western blot results showed that LC3 B-Ⅱwas inhibited in OA chondrocytes.The ratio of LC3 B-Ⅱ/Ⅰin OA chondrocytes(1.25±0.13)was significantly lower compared with healthy chondrocytes(1.85±0.21).Results from colony formation assay demonstrated that chondrocytes transfected with miR-22 mimics formed less cell colonies(75.67±11.36)than NC group(107.33±14.3)while miR-22 inhibitor transfection promoted chondrocytes formed more cell colonies(128.5±9.95).Western blot results showed that miR-22 mimics transfection significantly reduced the expression level of LC3 B-Ⅱ(1.50±0.20),the ratio of LC3 B-Ⅱ/Ⅰ(1.45±0.13)and the expression level of CollagenⅡ(0.51±0.09)in chondrocyte while miR-22 inhibitor transfection significantly promoted the expression level of LC3 B-Ⅱ(2.36±0.19),the ratio of LC3 B-Ⅱ/Ⅰ(2.30±0.08)and the expression level of CollagenⅡ(1.31±0.03).Conclusions MicroRNA-22 plays a crucial role in OA pathogenesis through inhibiting chondrocytes autophagy and viability and could be a novel therapeutic target of OA.

关 键 词：microRNA-22 自噬 骨关节炎 软骨细胞 

分 类 号：R684.3[医药卫生—骨科学]