MALAT1/miR-22-5p调控β-catenin信号通路影响颗粒诱导骨溶解中成骨细胞分化的机制研究

In particle induced osteolysis, the differentiation of the osteoblast is in obstacle and the bone formation is decreased obviously. It is reported that many lncRNAs and miRNAs could affect the differentiation of the osteoblast. Our previous studies have demonstrated that in osteolysis synoviums the expression of lncRNA MALAT1 was higher and miR-22-5p was lower. The preliminary experiments demonstrated that them could bind specifically and regulate the differentiation of the osteoblast. On the base of these, we put forward the hypothesis that in osteoblast, miR-22-5p could target APC (Adenomatous polyposis coli, APC), which could inhibit the β-catenin, activate β-catenin signal pathway and promote the differentiation and bone formation of the osteoblast. While MALAT1 acts as a ceRNA of miR-22-5p and inhibits its function. Injection of MALAT1 Interference adenovirus may inhibit the progression of osteolysis. We intend to prove the hypothesis from clinical samples, cell experiments and animal models with the methods of FISH, siRNA, over expression plasmid transfection and so on, aim to enrich the molecular mechanism and regulatory network of osteolysis induced by particles, and provide theoretical strategies for treatment.

颗粒诱导的假体周围骨溶解中成骨细胞分化受阻，骨形成明显减少。文献表明多种长链非编码RNA（LncRNA）与微小RNA（miRNA）可以影响成骨细胞的分化，课题组前期研究表明骨溶解界膜中LncRNA MALAT1高表达，miR-22-5p低表达，且预实验证实二者可以特异性结合，并影响成骨细胞的分化。基于此，我们提出假设：成骨细胞中，miR-22-5p可能直接作用于APC（结肠腺瘤样息肉蛋白），上调β-catenin通路，促进成骨细胞的分化和骨形成，MALAT1作为miR-22-5p的ceRNA，抑制其发挥作用，给予其干扰腺病毒可能抑制骨溶解的进展。拟从临床样本、细胞实验、小鼠模型层面，采用siRNA、荧光原位杂交、过表达质粒转染等技术，证实上述假设，旨在丰富骨溶解发病的分子机制和调控网络，为其临床防治拓展新思路。

目的：探讨骨髓间充质干细胞(BMSCs)分泌外泌体传递MALAT1调控骨质疏松症的机制。.方法：分离原代人骨髓间充质干细胞提取外泌体或对其转染后提取外泌体。采用透射电镜（Transmission Electron Microscope，TEM）、TRPS（Tunable resistive pulse sensing）分析和WB方法对分离的外泌体进行鉴定。将提取的外泌体与人成骨细胞hFOB1.19共培养，通过CCK-8（Cell Counting Kit-8）、ALP（alkaline phosphatase）染色和茜素红染色检测hFOB1.19细胞增殖、ALP活性和矿化结节情况。此外，通过双荧光素酶报告实验、Pull down和RIP（RNA Immunoprecipitation）实验验证MALAT1、miRNA-34c和SATB2之间的关系。QRT-PCR检测MALAT1、miRNA-34c和SATB2的表达变化。WB检测SATB2、Runx2、ATF4和Hoxa2蛋白表达情况。最后，通过建立骨质疏松模型（ovariectomized，OVX模型）验证骨髓间充质干细胞(BMSCs)分泌外泌体传递MALAT1靶向miRNA-34c/SATB2对成骨生成和骨质疏松症的调控作用。.结果：人成骨细胞hFOB1.19能够内吞骨髓间充质干细胞(BMSCs)分泌的外泌体及外泌体中的MALAT1。沉默SATB2会抑制人成骨细胞hFOB1.19ALP活性及矿化结节，抑制Runx2、ATF4蛋白表达，促进Hoxa2蛋白表达。双荧光素酶报告实验、Pull down和RIP实验表明MALAT1能与miRNA-34c竞争性结合，miRNA-34c靶向抑制SATB2，MALAT1能够间接促进SATB2表达。过表达外泌体中的MALAT1能够促进人成骨细胞hFOB1.19ALP活性及矿化结节，促进Runx2、ATF4蛋白表达，抑制Hoxa2蛋白表达。体内实验表明，miRNA-34c能够逆转MALAT1对骨质疏松症小鼠的改善作用，而SATB2能够逆转miRNA-34c加重小鼠骨质疏松的作用。.结论：骨髓间充质干细胞(BMSCs)分泌外泌体传递MALAT1靶向miRNA-34c/SATB2促进成骨生成进而改善骨质疏松症。