退变性腰椎侧凸发病中Hsa-let-7f调控白细胞介素10/STAT3信号通路的作用

Downregulated Hsa-let-7f contributes to the loss of type Ⅱ collagen by targeting interleukin-10/STAT3 signaling pathway in degenerative lumbar scoliosis

背景:microRNAs(miRNAs)在多种疾病中扮演重要的角色,对退变性腰椎侧凸患者椎间盘中miRNAs表达谱的分析,有助于揭示其发病机制,为其治疗提供新的靶点。假设miRNA通过调控白细胞介素10/STAT3(椎间盘退变的一个潜在炎症通路)信号通路促使椎间盘退变。目的:比较退变性腰椎侧凸患者与正常对照组椎间盘组织中miRNAs表达谱的差异,确定退变性腰椎侧凸特异性相关的miRNAs,并对其进行功能验证。方法:对获取的退变性腰椎侧凸患者手术髓核组织和腰椎骨折患者正常髓核组织依次进行总RNA提取,其中,各取10例进行miRNA Solexa测序筛查,挑选表达有差异的microRNA。随后,采用q RT-PCR技术对其进行验证。对表达显著差异的miRNA进行探究。进一步对其进行功能验证,确定其与Ⅱ型胶原表达的关系。运用Western与荧光素酶报告系统进一步确定靶基因。结果与结论:与正常对照相比,30条miRNA表达存在显著差异(16条表达上调和14条表达下调)。随后进行q RT-PCR验证,与正常对照相比,Hsa-let-7f在退变性腰椎侧凸患者椎间盘组织中,表达显著下调。此外,Hsa-let-7f表达水平与椎间盘退变评分密切相关。高表达Hsa-let-7f促进Ⅱ型胶原表达。敲除白细胞介素10后,对Ⅱ型胶原表达表达类似与过表达Hsa-let-7f。生物信息学软件证实,白细胞介素10为Hsa-let-7f理论上的靶基因。荧光素酶报告系统及western blot进一步证实Hsa-let-7f的靶基因为白细胞介素10。此外,Hsa-let-7f影响其下游基因STAT3及基质金属蛋白酶2基因表达。研究表明,下调的Hsa-let-7f通过调控白细胞介素10/STAT3信号通路,导致椎间盘组织中Ⅱ型胶原的丢失,进而引起椎间盘退变及退变性腰椎侧凸;Hsa-let-7f可成为治疗退变性腰椎侧凸的一个新的生物治疗靶点。

BACKGROUND： MicroRNAs（miRNAs） play an important role in a variety of diseases. Investigation of miRNA expression profile in degenerative lumbar scoliosis is beneficial for understanding its pathogenesis, providing a novel therapeutic target. Therefore, we tested the hypothesis that miRNAs promote intervertebral disc degeneration through the interleukin-10/STAT3 signaling pathway, a potential regulator of intervertebral disc degeneration. OBJECTIVE： To compare the differentially expressed miRNAs in the intervertebral disc tissues from patients with degenerative lumbar scoliosis and normal controls and to identify specific miRNAs in degenerative lumbar scoliosis followed by functional validation. METHODS： An initial screening of miRNA expression in nucleus pulposus tissues by miRNA Solexa Sequencing was performed in samples from 10 patients with degenerative lumbar scoliosis and 10 controls, respectively. Subsequently, differentially expressed miRNAs were validated using q RT-PCR. The level of differentially expressed miRNAs in degenerative nucleus pulposus tissues was investigated. Then, functional analysis of the miRNAs in regulating type II collagen expression was carried out. Western blot and luciferase reporter assay were used to further confirm the target gene. RESULTS AND CONCLUSION： We identified 30 miRNAs that were differentially expressed（16 upregulated and 14 downregulated） in patients with degenerative lumbar scoliosis compared with controls. Following q RT-PCR confirmation, Has-let-7f was significantly down-regulated in degenerative nucleus pulposus tissues as compared with controls. Moreover, its level was correlated with the severity of disc degeneration. Overexpression of Has-let-7f promoted type II collagen expression in nucleus pulposus cells. Knockout of interleukin-10 induced effects on nucleus pulposus cells similar to Has-let-7f. Bioinformatics target prediction identified interleukin-10 as a putative target of Has-let-7f. Furthermore, luciferase reporter assays demonstrated that Has-let-7f altered the expression of STAT3 and matrix metalloproteinase-2. These findings indicate that the downregulation of Has-let-7f induces type II collagen loss by directly targeting inleukin-10, thereby resulting in intervertebral disc degeneration and degenerative lumbar scoliosis. Has-let-7f is likely to be a novel therapeutic target for degenerative lumbar scoliosis.