摘要
目的探讨微小RNA-135b-5p(miR-135b-5p)是否通过靶向血管生成素样蛋白2(ANGPTL2)影响白介素-1β(IL-1β)诱导的软骨细胞损伤。方法使用IL-1β诱导SD大鼠膝关节软骨细胞损伤。使用10 ng/ml IL-1β处理软骨细胞48 h,论为IL-1β组,同时以未经任何处理的软骨细胞设为对照(Con组)。在软骨细胞中转染miR-135b-5p并使用IL-1β损伤软骨细胞。实时荧光定量PCR(qPCR)检测miR-135b-5p和ANGPTL2 mRNA表达,酶联免疫吸附法(ELISA)检测白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、γ干扰素(IFN-γ)水平,流式细胞术检测细胞凋亡,蛋白质印迹法(Western blot)检测ANGPTL2、B细胞淋巴瘤/白血病-2(Bcl-2)抗体、Bcl-2相关X蛋白(Bax)蛋白表达,Targetscan预测结合双荧光素酶报告实验分析miR-135b-5p与ANGPTL2的靶向关系。共转染miR-135b-5p和pcDNA-ANGPTL2,并使用IL-1β损伤软骨细胞,观察ANGPTL2过表达对miR-135b-5p过表达诱导的IL-1β损伤软骨细胞凋亡、炎症的影响。结果与Con组比较,IL-1β诱导的软骨细胞中miR-135b-5p表达量、Bcl-2蛋白表达量明显减少(P<0.05),ANGPTL2 mRNA、ANGPTL2蛋白表达量、IL-6、TNF-α、IFN-γ水平、细胞凋亡率、Bax蛋白水平显著升高(P<0.05)。miR-135b-5p过表达明显降低IL-1β诱导软骨细胞ANGPTL2蛋白表达量、IL-6、TNF-α、IFN-γ水平、凋亡率、Bax蛋白表达量(P<0.05),显著提高Bcl-2蛋白水平(P<0.05)。miR-135b-5p靶向调控ANGPTL2的表达。ANGPTL2过表达逆转了miR-135b-5p过表达抑制IL-1β损伤的软骨细胞凋亡、IL-6、TNF-α、IFN-γ水平、Bax蛋白表达的作用,和促进Bcl-2蛋白表达的作用。结论miR-135b-5p通过靶向调控ANGPTL2表达保护IL-1β诱导的软骨细胞损伤。
Objective To investigate whether miR-135b-5p can affect IL-1β-activated chondrocyte injury by targeting ANGPTL2.Methods IL-1βwas used to induce injury of knee joint chondrocytes in SD rats,and miR-135b-5p was transfected into chondrocytes,which was taken as observation group,moreover,the chondrocytes without any treatment were taken as control group.The expressions levels of miR-135b-5p and ANGPTL2 mRNA were detected by qPCR.The levels of IL-6,TNF-αand IFN-γwere detected by ELISA,and cell a poptosis was detected by flow cytometry.The expression levels of ANGPTL2,Bcl-2 and Bax protein were detected by Western Blot.The targeted correlation between miR-135b-5p and ANGPTL2 was analyzed by Targetscan prediction combined with dual luciferase reporter assay.Moreover the effects of overexpression of ANGPTL2 on the cell apoptosis and inflammation of IL-1β-injuried chondrocytes induced by overexpression miR-135b-5p were analyzed.Results As compared with those in control group,the expression levels of miR-135b-5p and Bcl-2 protein in observation group were significantly decreased(P<0.05),however,the levels of ANGPTL2 mRNA,ANGPTL2 protein,and the expression levels of IL-6,TNF-α,IFN-γ,Bax protein and cell apoptosis rate were significantly increased(P<0.05).Moreover the overexpression of miR-135b-5p significantly decreased the expression levels of ANGPTL2 protein,IL-6,TNF-α,IFN-γ,Bax protein and cell apoptosis rate in observation group(P<0.05),but,which significantly increased the levels of Bcl-2 protein(P<0.05).In addition miR-135b-5p targetedly regulated the expression of ANGPTL2,and the overexpression of ANGPTL2 reversed the inhibition effects of miR-135b-5p overexpression on IL-1β-stimulated chondrocyte apoptosis,and the expression levels of IL-6,TNF-α,IFN-γ,Bax protein,as well as its promotion effects on Bcl-2 protein expression.Conclusion The miR-135b-5p can protect IL-1β-induced chondrocyte injury by regulating targetedly ANGPTL2 expression.
作者
赵晓东
王磊
ZHAO Xiaodong;WANG Lei(Department of Surgery,Zaozhuang Hospital of Zaozhuang Mining Industry Group,Shandong,Zaozhuang 277100,China)
出处
《河北医药》
CAS
2021年第8期1136-1141,共6页
Hebei Medical Journal
