长链非编码RNA-OPLLR在后纵韧带骨化发生过程中的功能及调控机制研究

Although researches showed that genes’ participation in the bone morphogenesis is critical for the development of ossification of the posterior longitudinal ligament (OPLL), but the underlying patho-mechanism of such heterotopic ossification is still unknown. Recently, studies showed that long non-coding RNAs (lncRNAs) are new participants in the regulatory network of many diseases. On the basis of previous fundings, we set out to find the differentially expressed lncRNAs by high-through-put sequencing. By functional analysis, we found that lncRNA-OPLLR (LINC00346) is highly expressed in OPLL, and can significantly affect the expression of bone morphogenesis related genes in normal ligament cells, thus influencing the efficacy of bone morphogenesis during induction. Based on our previous results, we hypothesize that lncRNA-OPLLR may play a vital role in OPLL genesis and development, especially in regulating bone morphogenesis related genes. Our current proposal tries to explain the underlying mechanism of lncRNA-OPLLR in regulating bone morphogenesis related genes during ossification of ligament cells by using technologies such as RNA-IP, ch-IP, mass spectrum, et al. Our study provides new insights into the mechanism of OPLL development through an lncRNA mediated manner, and may throw light on early diagnosis or conservative therapy of OPLL.

后纵韧带骨化症（OPLL）的分子机制研究表明骨化相关基因在其发生过程中起重要作用，但其作用具有普遍性，因此研究OPLL中骨化相关基因的特殊调控机制对于揭示OPLL的病理过程具有重要意义。近年来长链非编码 RNA（long non-coding RNA，lncRNA）被发现在疾病发生过程中起重要调控作用。在以往相关自然基金的基础上，我们通过高通量lncRNA测序发现OPLL中差异表达的lncRNA。经过功能筛选，发现lncRNA-OPLLR（LINC00346）能明显影响韧带细胞骨化诱导过程中骨化相关基因的表达，促使细胞产生成骨倾向。本课题拟通过RNA-IP、ch-IP、RNA-pulldown等相关技术手段研究lncRNA-OPLLR对骨化相关基因的影响与作用，及其在此过程中的具体调控机制，为揭示lncRNA在OPLL发病过程中的作用提供理论依据，同时为早期诊断治疗提供新的思路。

后纵韧带骨化症（OPLL）的相关致病分子机制研究表明骨化相关基因在OPLL发生过程中起到不可忽视的作用，但其作用具有普遍性，因此研究骨化相关基因于OPLL发生发展中的特殊调控机制对于全面揭示OPLL的病理过程具有重要意义。课题组针对OPLL疾病中长链非编码 RNA（lncRNA）进行高通量测序及差异表达筛选，并通过生物信息分析确认了LINC00346在OPLL疾病发生过程中可能存在重要功能。通过细胞实验明确了LINC00346具有显著促进韧带细胞骨化的作用。利用RNA-pull down技术，通过对LINC00346进行免疫共沉淀，将其结合蛋白整体拉下并进行蛋白质谱检测并发现delta-catenin与LINC00346结合密切。而后续功能实验也证实了LINC00346过表达确实能够提高delta-cadherin的生成量，进而明确LINC00346与delta-catenin的相互作用。项目首次提出了OPLL疾病中具有功能的lncRNA并阐明了其具体机制，为进一步认识OPLL疾病的发生以及疾病的相关分子诊疗提供重要理论基础。此外，课题组在项目实施过程中首次开发出了OPLL疾病特异性的小鼠模型，并利用该模型进行疾病研究，为今后的相关机制及临床研究提供有力保障。