Ji Ming-liang, Lu Jun, Shi Pei-liang, Zhang Xue-jun, Wang Shan-zheng, Chang Qing, Chen Hui, Wang Chen
The Department of Orthopaedic Surgery, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, China.
Key Laboratory of Model Animal for Disease Study of Ministry of Education, Model Animal Research Center, Collaborative Innovation Center of Genetics and Development, Nanjing University, Nanjing, China.
J Bone Miner Res. 2016 Apr;31(4):900-9. doi: 10.1002/jbmr.2753. Epub 2015 Dec 23.
Intervertebral disc degeneration (IDD) is associated with dysregulated expression of microRNAs (miRNAs). However, the precise molecular mechanisms underlying this disorder remain unclear. Therefore, we tested the hypothesis that miRNAs modulate IDD through effects on the IL-6/STAT3 signaling pathway, a potential regulator of IDD. The miRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing miRNA microarray and quantitative real-time PCR (RT-qPCR). Biological functions of differential expression miRNAs were further investigated using immunofluorescent staining. Luciferase reporter assays and Western blotting were performed to determine miRNA targets. We identified 41 miRNAs that were differentially expressed in patients compared with controls. Following RT-qPCR confirmation, miR-98 was significantly downregulated in degenerative NP tissues. Moreover, its level was inversely correlated with grade of disc degeneration. Through gain-of-function and loss-of-function studies, miR-98 was shown to significantly promote type II collagen expression in NP cells. Interleukin-6 (IL-6) was identified as a target of miR-98. Knockdown of IL-6 induced effects on NP cells similar to those induced by miR-98. In contrast, IL-6 treatment abrogated the effects induced by miR-98 upregulation. Moreover, miR-98 dramatically suppressed expression of STAT3 target gene, MMP2. IL-6 treatment antagonized this effect, whereas knockdown of IL-6 by IL-6 short hairpin RNA (shIL-6) induced inhibitory effects on the expression of p-STAT3 and its main target genes, similar to miR-98. The mRNA level of IL-6 was inversely correlated with that of miR-98 in degenerative NP tissues. These results suggest the downregulation of miR-98 could promote IDD through the IL-6/STAT3 signaling pathway. Our findings also highlight miR-98 as a novel hopeful therapeutic target for IDD.
椎间盘退变（IDD）与微小RNA（miRNA）表达失调有关。然而，这种疾病背后的确切分子机制仍不清楚。因此，我们检验了这样一个假设，即miRNA通过影响白细胞介素-6（IL-6）/信号转导和转录激活因子3（STAT3）信号通路来调节IDD，该信号通路是IDD的一个潜在调节因子。采用miRNA微阵列和定量实时聚合酶链反应（RT-qPCR）技术，测定了IDD患者和对照组髓核（NP）组织中的miRNA表达谱。使用免疫荧光染色进一步研究差异表达miRNA的生物学功能。进行荧光素酶报告基因检测和蛋白质免疫印迹法以确定miRNA的靶标。我们鉴定出41种在患者中与对照组相比差异表达的miRNA。经RT-qPCR确认后，miR-98在退变的NP组织中显著下调。此外，其水平与椎间盘退变程度呈负相关。通过功能获得和功能缺失研究表明，miR-98能显著促进NP细胞中II型胶原蛋白的表达。白细胞介素-6（IL-6）被确定为miR-98的一个靶标。敲低IL-6对NP细胞产生的影响与miR-98诱导的影响相似。相反，IL-6处理消除了miR-98上调所诱导的影响。此外，miR-98显著抑制STAT3靶基因基质金属蛋白酶2（MMP2）的表达。IL-6处理拮抗了这种作用，而用IL-6短发夹RNA（shIL-6）敲低IL-6则诱导了对磷酸化STAT3及其主要靶基因表达的抑制作用，类似于miR-98。在退变的NP组织中，IL-6的mRNA水平与miR-98的mRNA水平呈负相关。这些结果表明，miR-98的下调可能通过IL-6/STAT3信号通路促进IDD。我们的研究结果还突出了miR-98作为IDD一种新的有希望的治疗靶点。
