miR-122-5p对类风湿关节炎滑膜细胞增殖与凋亡的影响及其机制

EFFECT OF MIR-122-5P ON THE PROLIFERATION AND APOPTOSIS OF RHEUMATOID ARTHRITIS SYNOVIAL CELLS AND ITS MECHANISM

目的 研究miR-122-5p对类风湿关节炎(RA)滑膜细胞增殖、凋亡的影响及其机制。方法 运用实时荧光定量聚合酶链反应(qRT-PCR)分别检测正常关节滑膜细胞、RA滑膜细胞中miR-122-5p以及盘状结构域受体激酶(DDR2)的表达。实验分为阴性对照(miR-NC)组(转染mimic NC序列)、miR-122-5p寡核苷酸模拟物(miR-122-5p)组(转染miR-122-5p模拟物mimics)、miR-122-5p特异性寡核苷酸抑制剂阴性对照(anti-miR-NC)组、miR-122-5p特异性寡核苷酸抑制剂(anti-miR-122-5p)组、miR-122-5p+pcDNA组(共转染miR-122-5p mimics和空载体(pcDNA))、miR-122-5p+pcDNA-DDR2组(共转染miR-122-5p mimics和DDR2过表达载体(pcDNA-DDR2)),用脂质体法转染至RA滑膜细胞。采用Western blot方法检测细胞中DDR2、P21和Survival的蛋白表达,MTT法检测细胞增殖,流式细胞术检测细胞凋亡,双荧光素酶报告基因检测实验检测细胞中miR-122-5p与DDR2的结合力。结果 与正常滑膜细胞相比较,RA滑膜细胞中miR-122-5p的表达显著降低,DDR2的表达显著升高(t=27.714~48.853,P<0.001)。过表达miR-122-5p可抑制RA滑膜细胞增殖,促进其凋亡(t=16.716~121.500,P<0.001)。miR-122-5p可抑制野生型DDR2细胞的荧光活性。过表达DDR2可以逆转miR-122-5p对RA滑膜细胞的增殖抑制和凋亡促进作用(t=106.335~327.990,P<0.001)。结论 miR-122-5p可抑制RA滑膜细胞的增殖,促进其凋亡,其机制可能与靶向DDR2有关。

Objective To investigate the effect of miR-122-5 p on the proliferation and apoptosis of rheumatoid arthritis(RA) synovial cells and it mechanism. Methods Quantitative real-time PCR was used to measure the expression of miR-122-5 p and DDR2 in normal synovial cells and RA synovial cells. The experiment was divided into negative control(miR-NC) group(transfected with mimic NC sequence), miR-122-5 p oligonucleotide analogue group(transfected with miR-122-5 p mimics), miR-122-5 p specific oligonucleotide inhibitor negative control(anti-miR-NC) group, miR-122-5 p specific oligonucleotide inhibitor(anti-miR-122-5 p) group, miR-122-5 p+pcDNA group(co-transfected with miR-122-5 p mimics and empty vector pcDNA), and miR-122-5 p+pcDNA-DDR2 group(co-transfected with miR-122-5 p mimics and DDR2 overexpression vector pcDNA-DDR2), and RA synovial cells were transfected by lipofection. Western blot was used to measure the protein expression of DDR2, P21, and Survi-val;MTT assay was used to measure cell proliferation;flow cytometry was used to measure cell apoptosis;dual-luciferase reporter assay was used to measure the binding force of miR-122-5 p and DDR2. Results Compared with the normal synovial cells, the RA synovial cells had a significant reduction in the expression of miR-122-5 p and a significant increase in the expression of DDR2(t=27.714-48.853,P<0.001). Overexpression of miR-122-5 p inhibited the proliferation and promoted the apoptosis of RA synovial cells(t=16.716-121.500,P<0.001), and miR-122-5 p could inhibit the fluorescence activity of wild-type DDR2 cells. Overexpression of DDR2 reversed the effect of miR-122-5 p in inhibiting the proliferation and promoting the apoptosis of RA synovial cells(t=106.335-327.990,P<0.001). Conclusion This experiment shows that miR-122-5 p can inhibit the proliferation and promote the apoptosis of RA synovial cells, possibly by targeting DDR2.