骨关节炎诱导软骨细胞凋亡和细胞外基质降解的机制

Mechanism of osteoarthritis induced chondrocyte apoptosis and extracellular matrix degradation

背景:研究发现长链非编码RNA IFNG-AS1在类风湿关节炎中发挥调控作用,但是其在骨关节炎中的作用仍不明确。目的:探究长链非编码RNA IFNG-AS1调控miR-376b-3p/AKT丝氨酸/苏氨酸激酶3(AKT serine/threonine kinase 3,AKT3)轴对骨关节炎软骨细胞增殖、凋亡和细胞外基质降解的影响。方法:采用实时荧光定量PCR检测骨关节炎和非骨关节炎患者软骨组织中IFNG-AS1、miR-376b-3p和AKT3的表达;CCK-8、流式细胞术和ELISA法分析细胞增殖、凋亡和细胞外基质相关因子基质金属蛋白酶3,9,13的表达;采用双荧光素酶报告法和RNA pull-down分析miR-376b-3p与IFNG-AS1或AKT3间的互作用关系。结果与结论:①相对于正常软骨组织,IFNG-AS1和AKT3在骨关节炎软骨组织中的表达水平升高,miR-376b-3p表达水平降低(均P<0.05);②敲减IFNG-AS1能促进软骨细胞增殖,抑制细胞凋亡和细胞外基质降解;③IFNG-AS1与miR-376b-3p存在靶向关系,下调miR-376b-3p可以部分逆转敲减IFNG-AS1对软骨细胞的影响(均P<0.05);④进一步分析发现IFNG-AS1能够作为ceRNA吸附miR-376b-3p进而调节AKT3的表达,AKT3上调部分挽救了敲减IFNG-AS1对软骨细胞的影响(均P<0.05);⑤提示长链非编码RNA IFNG-AS1通过调节miR-376b-3p/AKT3轴抑制软骨细胞增殖,促进细胞凋亡和细胞外基质降解,IFNG-AS1有望成为骨关节炎治疗的潜在靶点。

BACKGROUND:It has been found that long non-coding RNA(LncRNA)IFNG-AS1 plays a regulatory role in rheumatoid arthritis,but its role in osteoarthritis remains largely unknown.OBJECTIVE:To investigate the effects of LncRNA IFNG-AS1 on proliferation,apoptosis and extracellular matrix degradation of chondrocytes in osteoarthritis via regulating miR-376b-3p/AKT serine/threonine kinase 3(AKT3)axis.METHODS:Real-time fluorescence quantitative PCR was used to detect the expression of IFNG-AS1,miR-376b-3p,and AKT3 in cartilage tissue of osteoarthritis and non-osteoarthritis patients.Cell proliferation,apoptosis,and the expression of extracellular matrix-related factors matrix metalloproteinase-3,matrix metalloproteinase-9 and matrix metalloproteinase-13 were analyzed by cell counting kit-8,flow cytometry and enzyme-linked immunosorbent assay.The interaction between miR-376b-3p and IFNG-AS1 or AKT3 was analyzed by dual luciferase reporter assay and RNA pull-down assay.RESULTS AND CONCLUSION:Compared with normal cartilage tissue,the expression of IFNG-AS1 and AKT3 was increased in osteoarthritis cartilage tissue,while the expression of miR-376b-3p was decreased.Knockdown of IFNG-AS1 promoted cell proliferation but inhibited apoptosis and extracellular matrix degradation in chondrocytes.There was a targeted relationship between IFNG-AS1 and miR-376b-3p,and down-regulation of miR-376b-3p partially reversed the effect of IFNG-AS1 knockdown on chondrocytes(all P<0.05).Further analysis showed that IFNG-AS1 that acted as ceRNA adsorbed miR-376b-3p and regulated the expression of AKT3,and up-regulation of AKT3 partially saved the effect of IFNG-AS1 knockdown on chondrocytes(all P<0.05).To conclude,LncRNA IFNG-AS1 can inhibit chondrocyte proliferation but promote apoptosis and extracellular matrix degradation by regulating miR-376b-3p/AKT3 axis.IFNG-AS1 is expected to be a potential target for osteoarthritis therapy.