Objective To explore the molecular mechanism of miR-34b in promoting the degeneration of articular cartilage matrix.Methods Cartilage specimens from 10 osteoarthritis patients (OA group) and 7 traumatic amputees (control group) were collected.The levels of miR-34b in cartilage specimens was measured by using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR).Bioinformatics software Targetscan and luciferase assay were used to test whether Smad3 was the target gene of miR-34b.The cultured SW1353 cells were transfected with equivalent liposomes (groups A),scrambled oligonucleotides(group B),miR-34b mimic(group C) and miR-34b inhibitor(group D),respectively.The expression of miR-34b,Smad3,type Ⅱ collagen and matrix metalloproteinase-3 (MMP-13) were detected by RT-qPCR.Results Expression level of miR-34b was significantly higher in OA cartilages than that in normal cartilages (P＜0.05).The expression of miR-34b was significantly up-regulated in group C(P＜0.05) and inhibited in group D(P＜0.05),compared to group A;while there was no significant difference between group A and group B (P ＞0.05).The results of bioinformatics software Targetscan and luciferase assay showed that Smad3 was one of the target genes of miR-34b.After transfection of miR-34b mimic and miR-34b inhibitor,the mRNA expression level of Smad3 decreased in group C (P＜0.05) and increased significantly in group D (P＜0.05),compared to group A.While there was no significant difference between group A and group B (P ＞0.05).After transfection,the mRNA expression level of type Ⅱ collagen decreased in group C (P＜0.05),and increased in group D (P＜0.05),compared with group A.The expression of MMP-13 increased in group C (P＜0.05),and decreased in group D (P＜0.05),compared to group A.Conclusion MiR-34b may promote the degeneration of human articular cartilage matrix in osteoarthritis by targeting Smad3.%目的 探讨微小RNA-34b(miR-34b)促进关节软骨细胞基质退变的分子机制.方法 提取10例骨关节炎(OA)患者和7例创伤性截肢者的膝关节软骨组织,实时定量反转录聚合酶链反应(RT-qPCR)法检测组织标本中miR-34b表达水平.利用Targetscan基因信息软件预测得到miR-34b靶基因为Smad3,构建Smad3野生型及突变型3'非编码区(3'-UTR)-荧光素酶报告载体,利用荧光素酶报告基因实验检测miR-34b对Smad3基因野生型及突变型3'-UTR荧光素酶活性的影响.体外培养SW 1353细胞,分为A、B、C和D组,分别加入等量脂质体、无义序列、miR-34b模拟物(miR-34b mimic)和miR-34b抑制物(miR-34b inhibitor).RT-qPCR法检测miR-34b、Smad3、Ⅱ型胶原和基质金属蛋白酶-13(MMP-13)的表达水平.结果 OA软骨组织中miR-34b表达水平较正常膝关节软骨组织明显上调(P＜0.05).SW 1353细胞转染后,与A组比较,C组miR-34b表达水平明显升高(P＜0.05),D组明显降低(P＜0.05);A组与B组比较,差异无统计学意义(P＞0.05).Targets Can软件预测miR-34b的潜在靶基因为Smad3,荧光素酶报告基因实验证实miR-34b直接靶向抑制靶基因Smad3;转染miR-34b mimic和miR-34inhibitor后,与A组比较,C组Smad3 mRNA表达水平明显降低(P＜0.05),D组明显升高(P＜0.05);A组与B组比较,差异无统计学意义(P＞0.05).转染后,与A组比较,C组Ⅱ型胶原mRNA表达水平明显降低(P＜0.05),D组明显升高(P＜0.05);C组MMP-13mRNA明显升高(P＜0.05),D组明显降低(P＜0.05).结论 miR-34b可能通过抑制其靶基因Smad3的表达进而诱导人关节软骨细胞基质退变,从而促进OA的发生、发展.
展开▼
