软骨相关lncRNAs作为ceRNA参与miR-381调控HDAC4促ADSCs成软骨分化的机制研究

To date, we have not been able to differentiate the mesenchymal stem cells in chondrocytes to form a bioengineered cartilage without early degeneration. LncRNAs, acting as a ceRNA, have been established to participate in the crucial processes of MSCs chondrogenesis and cartilage degeneration. Supported by the National Natural Science Foudation of China, we have demonstrated that miR-381 can alter chondrogenesis of ADSCs as well as cartilage degeneration at the same time. One possible mechanism is the interactions of lncRNA-miRNA-mRNA as a ceRNA network. Our analysis has led to three candidates of chondrocyte-related lncRNAs, which may competitively combine to miR-381 and suppress its regulation to HDAC4, causing the early degeneration of chondrocytes. Our preliminary data also confirmed this possibility. In this study, we will provide novel insight for better understanding of ceRNA-mediated gene regulation of lncRNAs-miR-381-HDAC4 in chondrogenesis and early chondrocyte degeneration. With the understanding of the ceRNA network, we will facilitate the regulation of ADSCs differentiation and degeneration and provide possible new strategies for the construction of cartilage tissure engineering.

组织工程软骨的种子细胞成软骨分化后存在稳定性较差且早期退变的问题，与细胞调控密切相关，而lncRNAs与miRNAs组成的ceRNA调控网络在间充质干细胞成软骨分化和软骨退变中发挥重要作用。申请人在国自然青年基金资助下除证实miR-381在ADSCs成软骨分化中特异性高表达外，还发现它既可促进成软骨分化又可促进软骨退变，具双向调控能力。可用表观遗传及ceRNA调控网络协同作用来解释。经实验及信息学分析发现3个软骨相关lncRNAs可作为ceRNA竞争结合miR-381而拮抗其对HDAC4的调控，致软骨早期退变，进一步预实验亦证实这一可能性。本研究拟通过体内外实验从细胞-组织-整体层面阐明软骨相关lncRNAs、miR-381和HDAC4组成的ceRNA调控网络，进而干预这一网络使ADSCs成软骨分化早期更好地分化，中晚期退变得以抑制，为解决组织工程软骨早期退变的难题提供新思路。

组织工程软骨的种子细胞成软骨分化后存在稳定性较差且早期退变的问题，与细胞调控密切相关，而lncRNAs在间充质干细胞成软骨分化和软骨退变中发挥重要作用。我们通过hADSCs成软骨分化的lncRNA表达谱基因芯片分析lncRNA在软骨分化过程中的调控网络图谱，并挑选出其中的lncNA H19进行功能验证，沉默H19可增强成骨相关基因ALPL和RUNX2的表达，过表达H19则显示相反的作用，这一研究初步描绘了hADSCs成骨分化过程中的lncRNAs表达谱，为进一步研lncRNAs的功能提供了一个初步的数据库。我们发现linc000473在NA细胞中低表达，且在IL-1β刺激模型中随着浓度及时间增高。这些结果表明linc000473在软骨发育及退变中具有重要作用。我们在linc000473第一个外显子后面插入3*flag核酸序列。通过lipo3000转染HEK293T细胞，western blot 检测发现linc000473可以表达一个15KD左右的多肽。我们在软骨中转染ORF及linc000473，发现是编码的蛋白而非lncRNA本身可以诱导软骨退变。同时我们分别转染ADSC并成软骨诱导21天，显示ORF、linc000473编码的蛋白而非lncRNA本身可以干扰ADSC成软骨过程。我们还通过研究hADSC外泌体，探讨lncRNA在成软分化过程中的调控网络，进一步揭示lncRNAs促进ADSCs成软骨分化、调控软骨退变的具体作用机制。这些成果为解决组织工程软骨中种子细胞成软骨分化后早期退变这一难题提供新的理论基础。