环状核糖核酸0136474调控骨关节炎软骨细胞增殖和凋亡的分子机制研究

Molecular Mechanism Study of Circular Ribonucleic Acid 0136474 in Regulating Proliferation and Apoptosis of Osteoarthritis Chondrocytes

目的:探讨环状RNA-0136474(circ_0136474)调控微小RNA-127-5p(miR-127-5p)/Zest同源物2(EZH2)轴对骨关节炎(OA)软骨细胞增殖和凋亡的影响及其分子机制。方法:收集骨关节炎患者软骨组织并检测其中circ_0136474表达;IL-1β诱导人软骨细胞CHON-001,并分为对照组、IL-1β组、si-NC组、si-circ_0136474组、si-circ_0136474+anti-miR-NC组、si-circ_0136474+anti-miR-127-5p组、miR-NC组和miR-127-5p mimics组,CCK-8法检测细胞活力,流式细胞仪检测细胞周期和细胞凋亡,RT-qPCR检测circ_0136474、miR-127-5p及EZH2、增殖细胞核抗原(PCNA) mRNA表达,ELISA试剂盒检测IL-6、TNF-α、MMP-13含量,Western Blot检测增殖细胞核抗原、Bcl-2、Bax、EZH2蛋白表达,双荧光素酶报告基因实验验证miR-127-5p与circ_0136474、EZH2的靶向关系。结果:circ_0136474在骨关节炎患者软骨组织中高表达。经IL-1β诱导后,细胞活力、S期、PCNA表达、Bcl-2表达和miR-127-5p显著降低,G0~G1期细胞比例、细胞凋亡率、Bax、IL-6、TNF-α、MMP-13、circ_0136474、EZH2蛋白和EZH2 mRNA显著增加(P<0.05);双荧光素酶报告基因实验显示,circ_0136474、EZH2与miR-127-5p存在靶向关系,沉默circ_0136474可促进细胞增殖、延长S期,缩短G0~G1期,并抑制细胞凋亡,减少IL-6、TNF-α、MMP-13含量(P<0.05);抑制miR-127-5p表达可逆转沉默circ_0136474对细胞凋亡、IL-6、TNF-α、MMP-13的抑制作用(P<0.05)。结论:circ_0136474在骨关节炎软骨组织中高表达,沉默circ_0136474可通过调节miR-127-5p/EZH2轴促进软骨细胞增殖而抑制细胞凋亡。

Objective:To investigate the effect of circular RNA-0136474(circ_0136474) on the proliferation and apoptosis of osteoarthritis chondrocytes by regulating the micro RNA-127-5p(miR-127-5p)/Zest homolog 2(EZH2) axis and its molecular mechanism.Methods:The osteoarthritis patients were collected.The expression of circ_0136474 in cartilage tissue of osteoarthritis patients was detected;human chondrocytes CHON-001 were induced by IL-1β and divided into control group,IL-1β group,si-NC group,si-circ_0136474 group,si-circ_0136474+anti-miR-NC group,si-circ_0136474+anti-miR-127-5p group,miR-NC group,and miR-127-5p mimics group.The CCK-8 was applied to detect the cell viability,flow cytometer was applied to detect the cell cycle and apoptosis,RT-qPCR was applied to detect the expression of circ_0136474,miR-127-5p,EZH2,and proliferating cell nuclear antigen(PCNA) mRNA,ELISA kit was applied to detect the levels of IL-6,TNF-α,and MMP-13,Western Blot was applied to detect the expression of PCNA,Bcl-2,Bax,and EZH2 proteins,double luciferase reporter gene experiment was applied to verify the targeting relationship between miR-127-5p and circ_0136474,EZH2.Results:The circ_0136474 was highly expressed in cartilage tissue of osteoarthritis patients;after induction by IL-1β,the cell viability,S phase,PCNA expression,Bcl-2 expression,and miR-127-5p were obviously reduced,the proportion of G0-G1 phase cells,cell apoptosis rate,Bax,IL-6,TNF-α,MMP-13,circ_0136474,EZH2 protein,and EZH2 mRNA were obviously increased(P<0.05).The double luciferase reporter gene experiment showed that circ_0136474 and EZH2 had a targeting relationship with miR-127-5p,silencing circ_0136474 can promote cell proliferation,prolong the S phase,shorten the G0-G1 phase,inhibit cell apoptosis,reduce the levels of IL-6,TNF-α,and MMP-13(P<0.05),inhibiting the expression of miR-127-5p can reverse the inhibitory effects of silencing circ_0136474 on cell apoptosis,IL-6,TNF-α,and MMP-13(P<0.05).Conclusion:The circ_0136474 is highly expressed in osteoarthritis cartilage tissue,and silencing circ_0136474 can promote chondrocyte proliferation and inhibit cell apoptosis by regulating the miR-127-5p/EZH2 axis.