| 摘要: |
| 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SW1353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-34bmimic）和miR-34b抑制物（miR-34binhibitor）。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）。SW1353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）。TargetsCan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-34bmimic和miR-34inhibitor后，与A组比较，C组Smad3mRNA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。 |
| 关键词: 微小 RNA-34b Smad3 骨关节炎 软骨细胞基质 |
| DOI：10.12056/j.issn.1006-2785.2017.40.4.2017-413 |
| 分类号: |
| 基金项目:嘉兴市科技局计划项目（2013AY21058） |
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| Molecular mechanism of mirR-34b in promoting degeneration of articular cartilage matrix |
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Jiaxing Second Municipal Hospital
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| Abstract: |
| 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 groupC (P＜0.05), and decreased in groupD (P＜0.05), compared to group A. Conclusion MiR-34b may promote the degeneration of human articular cartilage matrix in osteoarthritis by targeting Smad3. |
| Key words: miR-34b Smad3 Osteoarthritis Cartilage extracellular matrix |
