miR-4319通过靶向TNFAIP3调控强直性脊柱炎成纤维细胞成骨分化

MiR-4319 regulates osteogenic differentiation of ankylosing spondylitis fibroblasts by targeting TNFAIP3

目的探讨miR-4319对强直性脊柱炎(AS)成纤维细胞骨向分化的影响机制。方法原代分离培养AS成纤维细胞和正常成纤维细胞;脂质体法将pcDNA组(转染pcDNA)、pcDNA-肿瘤坏死因子-α诱导蛋白3(TNFAIP3)组(转染pcDNA-TNFAIP3)、miR-NC组(转染miR-NC)、miR-4319组(转染miR-4319mimics)、miR-4319+pcDNA组(共转染miR-4319 mimics和pcDNA)、miR-4319+pcDNA-TNFAIP3组(共转染miR-4319 mimics和pcDNA-TNFAIP3)转染至AS成纤维细胞。实时荧光定量反转录聚合酶链反应(RT-qPCR)检测细胞中miR-4319、TNFAIP3的表达;Western blot检测细胞TNFAIP3蛋白;双荧光素酶报告实验、RNA结合蛋白免疫沉淀(RIP)实验、RNA下拉(RNA pull down)实验检测miR-4319与TNFAIP3的靶向关系;碱性磷酸酶(ALP)检测试剂盒、羟脯氨酸检测试剂盒、放免法骨钙素检测试剂盒检测成骨标志物ALP、胶原、骨钙素的含量。结果与正常成纤维细胞相比,AS成纤维细胞中miR-4319表达显著升高,TNFAIP3表达显著降低(P<0.05),并且miR-4319靶向结合TNFAIP3。过表达TNFAIP3能上调AS成纤维细胞ALP、胶原、骨钙素的含量,而过表达miR-4319则可抑制AS成纤维细胞ALP、胶原、骨钙素的含量。过表达TNFAIP3还可部分逆转过表达miR-4319对AS成纤维细胞ALP、胶原、骨钙素含量的调控。结论 miR-4319可抑制AS成纤维细胞的骨向分化,其机制与miR-4319靶向TNFAIP3有关。

Objective To investigate the effect of miR-4319 on bone differentiation of ankylosing spondylitis(AS)fibroblasts.Methods Primary AS fibroblasts and normal fibroblasts were isolated and cultured.The pcDNA group(transfected with pcDNA),pcDNA-TNFα-inducible protein 3 antibody(TNFAIP3)(transfected pcDNA-TNFAIP3),miR-NC(transfected miR-NC)),miR-4319 group(transfected miR-4319 mimics),miR-4319+pcDNA group(cotransfected miR-4319 mimics and pcDNA),miR-4319+pcDNA-TNFAIP3 group(co-transfected miR-4319 mimics and pcDNA-TNFAIP3)was transfected into AS fibroblasts.Real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR)method was used to detect the expression of miR-4319 and TNFAIP3 in cells;Western blot was used to detect the TNFAIP3 protein;dual luciferase report experiment,RIP experiment and RNA pull down experiment were used to detect the targeting relationship between miR-4319 and TNFAIP3;the alkaline phosphatase(ALP)detection kit,hydroxyproline detection kit,and radioimmunoassay osteocalcin detection kit were used to detect the osteogenic markers ALP,collagen,and osteocalcin.Results Compared with normal fibroblasts,the expression of miR-4319 in AS fibroblasts was significantly increased,the expression of TNFAIP3 was significantly reduced(P<0.05),and miR-4319 targeted to bind to TNFAIP3.Overexpression of TNFAIP3 can increase the content of ALP,collagen,and osteocalcin in AS fibroblasts,while overexpression of miR-4319 can inhibit the content of ALP,collagen,and osteocalcin in AS fibroblasts.Overexpression of TNFAIP3 can also partially reverse the regulation of overexpression of miR-4319 on the content of ALP,collagen,and osteocalcin in AS fibroblasts.Conclusion miR-4319 can inhibit bone differentiation of AS fibroblasts,and the mechanism is related to miR-4319 targeting TNFAIP3.