微小RNA-127-5p调节人软骨细胞中基质金属蛋白酶13的表达及白细胞介素-1β诱导的分解代谢效应。

MicroRNA-127-5p regulates matrix metalloproteinase 13 expression and interleukin-1β-induced catabolic effects in human chondrocytes.

作者信息

Park Su Jin, Cheon Eun Jeong, Lee Mi Hyun, Kim Hyun Ah

机构信息

Hallym University Sacred Heart Hospital, Kyunggi, Republic of Korea.

出版信息

Arthritis Rheum. 2013 Dec;65(12):3141-52. doi: 10.1002/art.38188.

DOI:10.1002/art.38188

PMID:24022470

Abstract

OBJECTIVE

MicroRNAs (miRNAs), small noncoding RNA molecules, are involved in the pathogenesis of various diseases such as cancer and arthritis. The aim of this study was to determine whether miR-127-5p regulates interleukin-1β (IL-1β)-induced expression of matrix metalloproteinase 13 (MMP-13) and other catabolic factors in human chondrocytes.

METHODS

Expression of miR-127-5p and MMP-13 by normal and osteoarthritic (OA) human cartilage was determined using real-time polymerase chain reaction. The effect of miR-127-5p on MMP-13 expression was evaluated using transient transfection of human chondrocytes or chondrogenic SW-1353 cells with miR-127-5p or its antisense inhibitor (anti-miR-127-5p). MMP-13 protein production was quantified by enzyme-linked immunosorbent assay, and the involvement of miR-127-5p in IL-1β-mediated catabolic effects was examined by immunoblotting. MicroRNA-127-5p binding with the putative site in the 3'-untranslated region (3'-UTR) of MMP-13 messenger RNA (mRNA) was validated by luciferase reporter assay.

RESULTS

There was a significant reduction in miR-127-5p expression in OA cartilage compared with normal cartilage. Up-regulation of MMP-13 expression by IL-1β was correlated with down-regulation of miR-127-5p expression in human chondrocytes. MicroRNA-127-5p suppressed IL-1β-induced MMP-13 production as well as the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA. In addition, mutation of the miR-127-5p binding site in the 3'-UTR of MMP-13 mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with anti-miR-127-5p remarkably increased reporter activity and MMP-13 production. Interestingly, the IL-1β-induced activation of JNK, p38, and NF-κB and expression of MMP-1 and cyclooxygenase 2 were significantly inhibited by miR-127-5p.

CONCLUSION

MicroRNA-127-5p is an important regulator of MMP-13 in human chondrocytes and may contribute to the development of OA.

摘要

目的

微小RNA（miRNA）是一类小的非编码RNA分子，参与癌症和关节炎等多种疾病的发病机制。本研究旨在确定miR - 127 - 5p是否调节白细胞介素 - 1β（IL - 1β）诱导的人软骨细胞中基质金属蛋白酶13（MMP - 13）及其他分解代谢因子的表达。

方法

使用实时聚合酶链反应测定正常和骨关节炎（OA）人软骨中miR - 127 - 5p和MMP - 13的表达。通过用miR - 127 - 5p或其反义抑制剂（抗miR - 127 - 5p）瞬时转染人软骨细胞或软骨生成性SW - 1353细胞，评估miR - 127 - 5p对MMP - 13表达的影响。通过酶联免疫吸附测定法定量MMP - 13蛋白产量，并通过免疫印迹检查miR - 127 - 5p在IL - 1β介导的分解代谢作用中的参与情况。通过荧光素酶报告基因测定法验证miR - 127 - 5p与MMP - 13信使核糖核酸（mRNA）3'非翻译区（3'-UTR）中假定位点的结合。

结果

与正常软骨相比，OA软骨中miR - 127 - 5p表达显著降低。IL - 1β诱导的人软骨细胞中MMP - 13表达上调与miR - 127 - 5p表达下调相关。miR - 127 - 5p抑制IL - 1β诱导的MMP - 13产生以及包含人MMP - 13 mRNA 3'-UTR的报告基因构建体的活性。此外，MMP - 13 mRNA 3'-UTR中miR - 127 - 5p结合位点的突变消除了miR - 127 - 5p介导的报告基因活性抑制。相反，用抗miR - 127 - 5p处理显著增加了报告基因活性和MMP - 13产量。有趣的是，miR - 127 - 5p显著抑制了IL - 1β诱导的JNK、p38和NF - κB激活以及MMP - 1和环氧化酶2的表达。