探讨微小RNA-27b-3p(miR-27b-3p)与基质金属蛋白酶-13(MMP-13)在人软骨细胞表达及其靶向对应关系。

运用蛋白质印迹法(WB)与实时定量PCR技术(qRT-PCR)明确miR-27b-3p与MMP13在正常和骨关节炎(OA)人软骨细胞的表达。利用不同浓度的白介素(IL)1β干预原代人软骨细胞24 h，或利用不同时间点的IL-1β(10 ng/ml)干预原代人软骨细胞。利用原位杂交、转染及双荧光素酶报告技术确定miR-27b-3p与MMP13的靶向对应关系；结合运用核转录因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路抑制剂评估其作用机制。两组资料比较采用独立样本t检验，多组资料比较采用单因素方差分析，LSD法多重比较检验。

WB、qRT-PCR和原位杂交检测结果显示，与正常软骨相比，OA软骨中miR-27b-3p表达降低(t＝5.07，P<0.01)，MMP13表达升高(t＝－6.31，P<0.01)。IL-1β干扰后的结果显示miR-27b-3p表达降低(F＝129.54，P<0.05)，MMP-13表达升高(F＝394.50，P<0.05)。通过TargetScan数据库和荧光素酶报告基因检测结果分析，野生型-MMP13组荧光素酶活性降低(F＝55.27，P<0.001)，突变型-MMP-13荧光素酶活性变化没有统计学意义(P＝0.654)。利用特异性MAPK信号抑制剂和NF-kB抑制剂干预IL-1β诱导软骨细胞模型结果提示，与对照组相比，抑制剂组的MMP13表达水平降低(F＝28.43，P<0.001)，miR-27b-3p表达水平增高(F＝35.04，P<0.001)。

miR-27b-3p在OA软骨细胞呈现低表达，并负向调控MMP13的表达，其作用机制可能是通过NF-κB和MAPK信号通路，这结果提示这miR-27b-3p可能作为OA诊断与治疗的潜在靶点。

To investigate the expression of microRNA (miR)-27b-3p and matrix metalloproteinase (MMP)-13 in human chondrocytes and their targeting relationship.

Western blot (WB) and quantitative real time PCR(qRT-PCR)were used to determine the expression of miR-27b-3p and MMP13 in chondrocytes from normal human and osteoarthritis(OA). Primary human chondrocytes were treated with interleukin (IL)-1β at different concentrations for 24 h, and with IL-1β (10 ng/ml) at different time points. The targeting relationship of miR-27b-3p and MMP13 were determined by in situ hybridization, transfection and dual luciferase reporter; inhibitors of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were used to evaluate the mechanism of miR-27b-3p. The data of two groups were compared by independent sample t test, and multiple groups of data were compared by one-way ANOVA and LSD multiple comparison test.

The results of WB, qRT-PCR and in situ hybridization showed that compared with normal cartilage, the expression of miR-27b-3p in OA cartilage decreased (t=5.07, P<0.01), and the expression of MMP13 increased (t=-6.31, P<0.01). After treated by IL-1β, the expression of miR-27b-3p was low (F=129.54, P<0.05) while the expression of MMP-13 was high (F=394.50, P<0.05). Through TargetScan database and luciferase reporter gene detection, the results showed that the relative luciferase activity carrying MMP13 wild-type 3′-UTR decreased (F=55.27, P<0.001). There was no statistically significant change in the luciferase activity of the mutant MMP-13 plasmid (P=0.654). Specific MAPK signaling inhibitors and NF-kB inhibitors were applied to intervene in the chondrocyte model induced by IL-1β. The results showed that compared with the control group, the expression level of MMP13 in the inhibitor group was significantly reduced (F=28.43, P<0.001), and the expression level of miR-27b-3p was increased (F=35.04, P<0.001).

miR-27b-3p is low expressed in OA chondrocytes, and it negatively regulates the expression of MMP13, and is regulated by the NF-κB and MAPK signaling pathways, which may provide a potential target for the diagnosis and treatment of OA.