DANCR竞争性结合miR-1305在滑膜间充质干细胞成软骨分化中的作用及机制

Articular cartilage defect (ACD) repair is currently a research hotspot in Joint Surgery. Our previous study has demonstrated that over-expression of differentiation antagonizing non-protein coding RNA (DANCR) promoted chondrogenic differentiation and proliferation of synovium-derived mesenchymal stem cells (SMSCs), but the precise mechanism remains to be elucidated. Bioinformatics predicted that there were direct binding sites between DANCR and miR-1305, and between miR-1305 and Smad4 mRNA. Since our preliminary experiment showed that over-expression of DANCR significantly down-regulated the expression of miR-1305 while up-regulated Smad4, knockout of miR-1305 leading to higher Smad4 protein level, we speculated DANCR may competitively bind with miR-1305 to up-regulate Smad4 expression, and then enhance chondrogenic differentiation and proliferation of SMSCs for the repair of ACD. This project will be accomplished to investigate the relationship between DANCR and miR-1305, miR-1305 and Smad4 in an animal model of ACD by a combination of many techniques, and to provide new mechanism, target, and experimental evidence for repair of ACD.

关节软骨缺损（ACD）修复是目前关节外科研究的热点。我们前期研究证实：DANCR可促进滑膜间充质干细胞（SMSCs）向软骨细胞增殖和分化，从而在ACD修复中起重要作用，但机制有待进一步阐明。相关生物信息学预测DANCR与miR-1305、miR-1305与Smad4之间均有直接结合区域，基于预实验结果：SMSCs中过表达DANCR可显著引起miR-1305表达降低及Smad4表达增加；miR-1305敲除组Smad4蛋白水平显著增高，推测DANCR可作为一种竞争性内源RNA，下调miR-1305，进而上调Smad4，从而促进SMSCs向软骨细胞增殖和分化。本项目拟在ACD动物模型上，运用多种技术明确DANCR在ACD修复中的作用，探讨DANCR与miR-1305、miR-1305与Smad4的关系，有望揭示DANCR调节软骨代谢的新机制，并为DANCR作为ACD修复的新靶点提供实验依据。

由于关节软骨中无血管、神经和淋巴分布的自身特点，故一旦损伤关节软骨，其自身修复的能力往往十分有限。目前发现lncRNA在软骨代谢的表观转录水平、翻译水平、蛋白修饰过程和遗传水平中均发挥着重要的调控作用。通过前期预实验，课题申请人提出DANCR是通过下调miR-1305从而作用于Smad4从而完成其促SMSCs向软骨细胞增殖和分化的生理过程这一科学问题。发现：①从兔膝关节滑膜中培养获取的SMSCs，其细胞形态大致均一，细胞呈梭形分布；表面分布间充质干细胞标志性表面抗原；免疫荧光显示表达α-SMA、Oct4、Sox2等；在一定诱导培养条件下，SMSCs可以向成软骨、成骨、成脂等不同方向分化；②细胞周期显示DANCR+SMSCs为正常细胞周期，荷瘤实验结果阴性；③CCK-8显示DANCR-SMSCs的细胞增殖速度显著快于Vector-SMSCs对照组；细胞培养显示DANCR+SMSCs细胞分化更显著；qRT-PCR显示DANCR+SMSCs的Ⅱ型胶原、蛋白多糖、SOX9基因的表达显著增加；④基因芯片结果显示miR-1305明显下调，靶基因预测Smad4为miR-1305的靶基因；双荧光素酶报告基因试验显示miR-1305组中，Smad4组荧光较Smad4突变组明显增加；Western blot结果显示miR-1305过表达时，Smad4蛋白含量下降，而转染了miR-1305特异性拮抗剂时，Smad4蛋白水平上升；⑤在DANCR+SMSCs组中miR-1305的表达明显较对照组降低，DANCR被阻断后miR-1305的表达量较对照组明显上升；RIP实验结果显示在DANCR共轭生物素组的免疫沉淀中发现miR-1305的表达；双荧光素酶报告基因实验显示miR-1305组中DANCR组荧光较DANCR突变组明显减少，miR-1305敲除组中DANCR组荧光较DANCR突变组明显增加。以上结果证实了科学问题，既揭示DANCR的功能，而且为DANCR作为ACD修复的新靶点提供了基础数据。