长链非编码RNA PVT1调控TGF-β1信号通路在糖尿病性骨关节炎中的作用及其机制研究

It has been proved that diabetes is an independent risk factor for osteoarthritis. And diabetic osteoarthritis has been individualised based on recent research. Meantime China is now among the countries with the highest diabetes prevalence. So we need to pay more attention to diabetic osteoarthritis. Diabetic osteoarthritis is responsible for pain, functional decline, and affects health and quality of life of the older population. Recent evidence suggests that lncRNA may play a major role in regulating multiple diseases. Our study found that the highly expressed PVT1 promoted the development and progression of diabetic osteoarthritis through mechanisms involving inflammation, chondrocyte apoptosis, inhibition of type Ⅱ collagen secretion and destruction of the extracellular matrix. We also found that PVT1 bound to TGF-β1. And it is generally known that TGF-β1 signal pathway is crucial for the pathogenesis of OA. As a result, we infer that TGF-β1 signal pathway mediated by PVT1 induce diabetic osteoarthritis. To demonstrate this, we will mediate the PVT1 expression level in vitro and vivo study respectively. Then we will evaluate the function of PVT1 on the biological characteristics. To understand the potential mechanisms, RNA pulldown, RIP and FISH are used to validate the interaction protein and potential target. Overall, there has an urgent need to thoroughly understand the function of PVT1 on the biological characteristics. This will enhance the understanding of disease mechanisms responsible for diabetic osteoarthritis and lead to novel and improved therapeutic strategies and targets for this chronic condition.

研究表明糖尿病是骨关节炎的独立危险因素，我国是糖尿病第一大国，糖尿病性骨关节炎严重威胁中老年人的健康与生活质量，而长链非编码RNA可能参与调控该疾病的发生发展。我们的前期工作表明长链非编码RNA PVT1在糖尿病性骨关节炎软骨组织中高表达，其可促进炎症反应和软骨细胞凋亡，并可抑制Ⅱ型胶原分泌、破坏细胞外基质；进一步研究发现其与TGF-β1特异性结合，鉴于TGF-β1信号通路是骨关节炎发病最重要的细胞机制之一，据此我们推测PVT1可能通过调控TGF-β1信号通路促进糖尿病性骨关节炎的发生。本项目拟通过体内外实验，探讨PVT1对软骨细胞增殖、凋亡及细胞外基质分泌的影响；同时采用RNA pulldown，RIP及FISH等技术，阐明PVT1通过靶向调控TGF-β1信号通路促进糖尿病性骨关节炎发生的作用机制。研究结果将丰富糖尿病性骨关节炎的病因学研究，并为糖尿病性骨关节炎的治疗提供新的思路和靶点。

研究表明糖尿病是骨关节炎的独立危险因素，我国是糖尿病第一大国，糖尿病性骨关节炎严重威胁中老年人的健康与生活质量。目前糖尿病性骨关节炎的发病机制还不清楚。在本研究中，我们首先收集对比了糖尿病骨关节炎患者和骨关节炎（OA）患者，两组患者一般资料无差异，但糖尿病骨关节炎患者的功能、软骨损伤和软骨下骨的破坏更严重，证明了糖尿病促进骨关节炎的发生和发展。在体外研究中，采用qRT-PCR对比分析PVT1和miR-26b在正常软骨组织，OA软骨组织和糖尿病性OA软骨组织中的表达，进一步验证PVT1在糖尿病性OA软骨细胞中的高表达，miR-26b的低表达。同时发现PVT1与miR-26b和COL2AI的表达呈负相关。再通过吡格列酮调控细胞外葡萄糖浓度，证明了高糖可以促进IL-6、MMP-13、CGTF和TGF-β1的表达，吡格列酮可以抑制IL-6、MMP-13、CGTF和TGF-β1的表达。采用生物信息学分析、荧光素酶分析、RNA pulldown、RIP-qPCR和FISH检测验证PVT1通过miR-26b靶向调控CTGF/TGF-β1信号通路。通过慢病毒感染调控PVT1和miR-26b的表达，检测软骨细胞生物特性关键蛋白和CTGF/TGF-β1信号通路关键蛋白的表达。在体内研究中，首先将小鼠分为假手术组、OA组、糖尿病骨关节组和吡格列酮+糖尿病骨关节组，在体证明了糖尿病对小鼠OA的促进作用，验证了在糖尿病骨关节组PVT1的高表达，miR-26b的低表达，PVT1和miR-26b及COL2A1表达呈负相关，与Mankin Score 评分呈正相关。再通过siRNA调控PVT1和miR-26b的表达，检测软骨细胞生物特性关键蛋白和CTGF/TGF-β1信号通路关键蛋白的表达。研究中我们还发现糖尿病可以通过对小鼠软骨下骨结构和力学性能的破坏进一步加重OA的进展。本项目阐明了PVT1通过miR-26b靶向调控CTGF/TGF-β1信号通路促进糖尿病性骨关节炎发病的机制，研究结果丰富了糖尿病性骨关节炎的病因学研究，为糖尿病性骨关节炎的治疗提供了新的思路和靶点。