LncRNA ZFAS1影响骨关节炎软骨细胞增殖和凋亡的机制研究

Mechanism of lncRNA ZFAS1 affecting proliferation and apoptosis of osteoarthritis chondrocytes

目的探讨长链非编码RNA(lncRNA)锌指蛋白反义链1(ZFAS1)在骨关节炎(OA)中的作用及其潜在分子机制。方法将于恩施州中心医院进行膝关节置换术的OA患者软骨组织样本作为OA组,将同期外伤急诊截肢患者的正常膝关节组织样本作为对照(Control)组,RT-qPCR实验检测OA软骨组织中lncRNA ZFAS1和miR-590的表达。将Vector和pcDNA-ZFAS1转染到原代软骨细胞后,MTT实验和流式细胞术分别检测OA软骨细胞增殖和凋亡情况。双荧光素酶报告基因实验分别验证ZFAS1和miR-590以及miR-590和转化生长因子-β受体2(TGFβR2)的靶向关系。将miR-590 mimic或NC mimic与Vector和pcDNA-ZFAS1共转染到原代软骨细胞,检测细胞增殖和凋亡,RT-qPCR实验检测软骨细胞中TGFβR2 mRNA表达水平,Western blot检测软骨细胞中TGFβR2蛋白的表达。结果与Control组比较,OA组软骨组织中的lncRNA ZFAS1表达显著下调(P<0.05),miR-590表达显著上调(P<0.05)。与Vector组比较,pcDNA-ZFAS1组软骨细胞中lncRNA ZFAS1表达水平显著升高,miR-590表达水平显著降低,软骨细胞增殖能力增强,细胞凋亡率降低,差异均有统计学意义(P<0.05)。MiR-590与ZFAS1存在互补的结合位点,TGFβR2与miR-590也存在互补的结合位点。与NC mimic组比较,miR-590 mimic和3 UTR-ZFAS1-WT共转染组的细胞荧光素酶活性显著降低(P<0.05),而miR-590 mimic和3 UTR-ZFAS1-MUT共转染组的细胞荧光素酶活性无统计学差异(P>0.05)。此外,与NC mimic组比较,miR-590 mimic和3 UTR-TGFβR2-WT共转染组的细胞荧光素酶活性显著降低(P<0.05)。与NC mimic组比较,miR-590 mimic组细胞中TGFβR2 mRNA和蛋白表达水平均显著降低(P<0.05)。与pcDNA-ZFAS1+NC mimic组比较,pcDNA-ZFAS1+miR-590 mimic组软骨细胞中miR-590表达水平显著升高,软骨细胞增殖能力减弱,细胞凋亡率升高,TGFβR2 mRNA和蛋白的表达水平显著降低,差异均有统计学意义(P<0.05)。结论LncRNA ZFAS1通过调控miR-590/TGFβR2轴促进OA中软骨细胞增殖,并抑制细胞凋亡。

Objective To explore the role of long non-coding RNA(lncRNA)zinc finger protein antisense strand 1(ZFAS1)in osteoarthritis(OA)and its underlying molecular mechanism.Methods Cartilage tissue samples of OA patients who underwent knee replacement in Enshi Central Hospital were taken as the OA group,and normal knee joint tissue samples of trauma patients who underwent emergency amputation during the same period were taken as the Control group.The expression of lncRNA ZFAS1 and miR-590 in OA cartilage were detected by RT-qPCR assay.After Vector and pcDNA-ZFAS1 were transfected into primary chondrocytes,MTT assay and flow cytometry were used to detect the proliferation and apoptosis of OA chondrocytes.Dual luciferase reporter gene assay was used to verify the targeting relationship of ZFAS1 and miR-590,as well as miR-590 and transforming growth factor-beta receptor type 2(TGFβR2),respectively.MiR-590 mimic or NC mimic was co-transfected with Vector and pcDNA-ZFAS1 into primary chondrocytes to detect cell proliferation and apoptosis;RT-qPCR assay was used to detect the expression level of TGFβR2 mRNA in chondrocytes,and Western blot was used to detect the expression of TGFβR2 protein in chondrocytes.Results Compared with the Control group,the expression of lncRNA ZFAS1 in cartilage tissue in the OA group was significantly down-regulated,and the expression of miR-590 was significantly up-regulated(P<0.05).Compared with the Vector group,the expression level of lncRNA ZFAS1 in chondrocytes in the pcDNA-ZFAS1 group was significantly increased,the expression level of miR-590 was significantly decreased,the proliferation ability of chondrocytes was enhanced,and the apoptosis rate was decreased,with statistically significant differences(P<0.05).There were complementary binding sites between miR-590 and ZFAS1,and there were complementary binding sites between TGFβR2 and miR-590.Compared with the NC mimic group,the cell luciferase activity in the miR-590 mimic and 3’UTR-ZFAS1-WT co-transfection group was significantly reduced(P<0.05),while there was no statistically significant difference in the cell luciferase activity in the miR-590 mimic and 3’UTR-ZFAS1-MUT co-transfection group(P>0.05).Compared with the NC mimic group,the cell luciferase activity in the miR-590 mimic and 3’UTR-TGFβR2-WT co-transfection group was significantly reduced(P<0.05).Compared with the NC mimic group,the expression levels of TGFβR2 mRNA and protein in the miR-590 mimic group were significantly decreased(P<0.05).Compared with the pcDNA-ZFAS1+NC mimic group,the expression level of miR-590 in chondrocytes in the pcDNA-ZFAS1+miR-590 mimic group was significantly increased,the proliferation ability of chondrocytes was weakened,the apoptosis rate was increased,and the expression levels of TGFβR2 mRNA and protein were significantly reduced,with statistically significant differences(P<0.05).Conclusion LncRNA ZFAS1 promotes proliferation of OA chondrocytes and inhibits the cell apoptosis by regulating the miR-590/TGFβR2 axis.