微小RNA-106b通过调控核因子-κB受体活化因子配体/骨保护素对胶原诱导的小鼠关节炎骨质破坏的作用

MicroRNA-106b inhibitor attenuates joint destruction in collagen-induced arthritic mice by regulating receptor activator of nuclear factor-κB ligand/bone protection element signal pathway

目的观察微小RNA(miRNA,miR)-106b对胶原诱导的小鼠关节炎(CIA)骨关节破坏的影响.方法实验分为4组:对照组(Sham组),慢病毒载体组(NC组),miR-106bmimic组(Mimic组)和miR-106b inhibitor组(Inhibitor组).采用牛Ⅱ型胶原免疫DBA/1小鼠建立CIA模型.显微CT (Micro-CT)检查评估骨组织破坏程度,抗酒石酸酸性磷酸酶(TRAP)染色检测成熟破骨细胞;反转录-聚合酶链反应(RT-PCR)检测CIA小鼠膝关节中miR-106b的表达;酶联免疫吸附(ELISA)检测外周血中核因子-κB受体活化因子配体(RANKL)和骨保护素(OPG)的表达变化.应用SPSS 11.0统计软件分析,数据以均值±标准差(Mean±SD)表示,多样本间比较采用单因素方差分析,组间比较采用Tukeypost-hoc pairwise分析,统计运算前所有数据均进行方差齐性检验.结果 RT-PCR结果显示miR-106b在CIA小鼠膝关节中高表达.Micro-CT结果显示,降低miR-106b表达后,CIA小鼠骨关节破坏明显减轻;骨体积、骨体积分数、骨面积分数、骨小梁厚度分别为(6.85±0.24) mm3、(22.52±0.62)%、(9.46±2.02)/mm、(0.31±0.06) mm,与NC组[(5.61±0.35) mm3、(16.26±1.42)%、(13.32±1.85)/mm、(0.24±0.06)mm]比较,差异有统计学意义(P<0.01).TRAP染色结果显示CIA小鼠关节破坏局部有大片紫红色深染区域,Inhibitor组仅可见少量阳性染色区域,提示miR-106b inhibitor能够抑制破骨细胞活化.ELISA结果显示,miR-106b inhibitor能促进OPG的分泌,抑制RANKL的表达,使RANKL/OPG比值明显降低.结论抑制miR-106b能减轻CIA模型小鼠骨关节破坏.

Objective To explore the in vivo effect of microRNA (miRNA, miR)-106b on joint destruction in collagen-induced arthritic (CIA) mice. MethodsForty male DBA/1 mice (8-10 weeks of age) were randomly divided into fourgroups: sham control [no collagen Ⅱ injection/phosphate buffer (PBS)-only treatment], lentivirus-NC control (collagen Ⅱ injection/lentivirus treatment), mimic (collagen Ⅱ injection/miR-106b mimics) and inhibitor (collagen Ⅱ injection/miR-106b inhibitor). CIA mice are developed by injecting DAB/1 mice with bovine type Ⅱ collagen containing Freund’s Adjuvant. The level of miR-106b was evaluated via reverse transcriptase-polymerase chain reaction (RT-PCR). Morphological changes in the ankle joints were assessed via μCT. Mature osteoclasts were identified using tartrate-resistant acid phosphatase (TRAP) staining. Expression of receptor activator of nuclear factor-κB ligand (RANKL) and bone protection element (OPG) were identified using enzyme linked immunosorbent assay (ELISA). ResultsCIA mice were found to have increased miR-106b expression and CIA-associated bone loss. As assessed in the three-dimensional μCT reconstructions, severe bone destruction was observed in CIA mice. Treatment with miR-106b inhibitor significantly mitigated CIA-induced bone resorption compared with the NC groups. The miR-106b inhibitor treatment induced an obvious increase in bone volume [(6.85±0.24) mm3 vs.(5.61±0.35) mm3,P<0.01], bone volume fraction [(22.52±0.62)% vs.(16.26±1.42)%, P<0.01], trabecular thickness [(0.31±0.06) mm vs (0.24±0.06) mm, P<0.01], and a marked decrease in bone surface to volume ratio [(9.46±2.02)/mm vs.(13.32±1.85)/mm, P<0.01], when compared with that in NC groups. Histological results showed that NC-and miR-106b mimic-treated groups had larger numbers of TRAP-positive cells located on eroded bone surfaces than in the vehicle-treated group. However, few TRAP-positive cells presented in the miR-106b inhibitor treated group. Serum from CIA mice was then examined via ELISA analysis and showed serum RANKL levels to be significantly increased when compared to the control group, whereas OPG levels were not significantly altered relative to the control. In miR-106b inhibitor treated mice, RANKL levels were significantly down-regulated, while OPG levels were up-regulated. Consequently, the RANKL/OPG ratio increased in CIA mice and was reduced in miR-106b inhibitor treated mice. ConclusionIn summary, the results in the current study clearly demonstrated that miR-106b is involved in the development of CIA-induced bone destruction and blocking of miR-106b attenuated bone resorption in CIA mice.