miR-34b促进关节软骨细胞基质退变的分子机制研究

Molecular mechanism of mirR-34b in promoting degeneration of articular cartilage matrix

目的探讨微小RNA-34b(mi R-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-q PCR)法检测组织标本中mi R-34b表达水平。利用Ta rg e ts c a n基因信息软件预测得到mi R-34b靶基因为Sma d 3,构建Sma d 3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mi R-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SW1353细胞,分为A、B、C和D组,分别加入等量脂质体、无义序列、mi R-34b模拟物(mi R-34b mimic)和mi R-34b抑制物(mi R-34b inhibitor)。RT-q PCR法检测mi R-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mi R-34b表达水平较正常膝关节软骨组织明显上调(P<0.05)。SW1353细胞转染后,与A组比较,C组mi R-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mi R-34b的潜在靶基因为Smad3,荧光素酶报告基因实验证实mi R-34b直接靶向抑制靶基因Smad3;转染mi R-34b mimic和mi R-34inhib itor后,与A组比较,C组Sma d 3 m RNA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m RNA表达水平明显降低(P<0.05),D组明显升高(P<0.05);C组MMP-13m RNA明显升高(P<0.05),D组明显降低(P<0.05)。结论 mi R-34b可能通过抑制其靶基因Sma d 3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。

Objective To explore the molecular mechanism of miR-34 b 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-34 b 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-34 b. The cultured SW1353 cells were transfected with equivalent liposomes(groups A), scrambled oligonucleotides(group B), miR-34 b mimic(group C) and miR-34 b inhibitor(group D), respectively. The expression of miR-34 b, Smad3, type Ⅱ collagen and matrix metalloproteinase-3(MMP-13) were detected by RT-qPCR. Results Expression level of miR-34 b was significantly higher in OA cartilages than that in normal cartilages(P<0.05). The expression of miR-34 b 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-34 b. After transfection of miR-34 b mimic and miR-34 b 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 ofMMP-13 increased in group C(P<0.05), and decreased in group D(P<0.05), compared to group A.Conclusion MiR-34 b may promote the degeneration of human articular cartilage matrix in osteoarthritis by targeting Smad3.