老年黄韧带骨化症软骨细胞miR24成骨相关的分子网络及机制探究

MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression that control osteoblast mediated bone formation. Deregulation of miRNA mediated mechanisms is emerging as an important pathological factor in bone degeneration (eg, osteoporosis) and other bone-related diseases. MiRNAs are intriguing regulatory molecules that are networked with cell signaling pathways and intricate transcriptional programs through ingenuous circuits with remarkably simple logic. Ossification of the ligamentum flavum (OLF) is one of the important causes of the stenosis of the spinal canal. OLF can result in slowly progressive paraphrases or acute paraplegia. OLF usually exists with myelopathy in the setting of thoracic spinal, but its etiology is not well defined. Our previous study first showed that the miR-24 was expression as half level of the normal ligament flavum, and the miR-24 down regulation could promote the bone formation with the Tcf1 level increase. But how or which molecular networks are involved the miR-24 regulating the post-transcription bone formation have not definited yet. The present study is aimed at illustration which nolecular netword was engaged in the bone formation under the miR-24 down-regulation. 1) the miR-24 is constructed expression and knocked in the MCSc，OLF chondrocytes and the ligamtum flavum fibroblast, the target genes are analysis by the slice. Bone formation is also evaluated. 2) the above step is repeated under the 3D matrix cultured 3) in vivo function of miR-4 in bone formation is exam by the cre-loxp mice, the miR-24 potential usage in the OLF and osteoporosis treatment is evaluated.

老年黄韧带骨化（ossification ligamentum flavum, OLF）症是非瘤性老年成骨现象，探索其成骨机制有利于防治老年OLF和骨质疏松（osteoporosis,OP）症。前期工作发现，老年OLF软骨细胞miR24是对照组表达量的1/2，并证实miR24抑制成骨伴有Tcf1降低。但目前尚不明确miR24成骨功效的确切转录后调控网络，尤其是αv整合素黏附（3D黏附）状态下的成骨调控网路，缺乏miR24抑制OLF和预防OP的证据。本次申报：1、检测αv整合素黏附状态和常规培养状态下，miR24对全转录组测序影响,分析与β-catenin/Tcf1相互作用的细胞内通路； 2、在体外筛选和验证miR24/β-catenin/Tcf1相关分子网络；3、分别建立OLF和OP小鼠的miR24转染动物模型，在体内验证miR24升高对OLF和miR24降低对OP的防治作用。

黄韧带骨化是一种常见的脊柱疾病，可引起脊柱脊髓病和神经根病。非编码RNA (ncRNA)可参与参与许多病理过程;然而，很少有ncRNA被证实与OLF相关。在这里，我们通过mRNA、lncRNA、circRNA芯片和microRNA测序，比较了OLF患者和健康志愿者的OLF组织中lncRNA、mRNA、circRNA、microRNA的表达情况。OLF发生的过程中共改变2054个mRNA, 2567个lncRNA, 627个circRNA, 28个microRNA (miRNA)。我们通过qPCR证实了所选mRNA和ncRNA的差异表达，并且通过对差异表达RNA转录本的相关性分析，构建lncRNA-mRNA - mrna共表达网络、miRNA-mRNA靶点预测网络和竞争性内源性RNA (ceRNA)网络。随后，分别对差异表达的mRNA和预测的miRNA靶基因进行gene ontology（GO）和Kyoto Encyclopedia of genesand genome（KEGG)通路分析。此外，通过功能实验，证实了一个以反常表达的miRNA-19b-3p 为基础的miRNA-circRNA-lncRNA-mRNA网络以及miR-342-3p在骨化过程中的作用。本研究不仅对ncRNA和mRNA在OLF的发病过程中的潜在功能给出了系统的认识，也阐明了OLF潜在的发病机制，为OLF的诊治和研究提供了新的靶点。