长链非编码RNA BMP2-1通过调控BMP2表达影响人牙周膜干细胞成骨分化的功能及机制研究

Using microarrays, our previous work have detected the changes of long non-coding RNA (lncRNA) and mRNA in human periodontal ligament stem cell (hPDLSCs) before and after osteogenic induction in the previous work. The results showed that lncRNA BMP2-1 and mRNA bone morphogenetic proteins 2 (BMP2) were both significantly up-regulated after the induction, and the coding-non-coding gene co-expression analysis also indicated their significant positive correlation. And bioinformatics analysis showed their close location. The mRNA and protein level of BMP2 decreased significantly after down-regulated lncRNA BMP2-1. Therefore, we hypothesized that lncRNA BMP2-1 might be involved in osteogenic differentiation of hPDLSCs by regulating mRNA BMP2. The present project will try to establish cell line of gene silencing of lncRNA BMP2-1, to study the impact of lncRNA BMP2-1 on the osteogenic differentiation of hPDLSCs in vivo and in vitro, to observe subcellular distribution of lncRNA BMP2-1, to investigate the interactions between mRNA BMP2 and lncRNA BMP2-1. The objective of this project is to elucidate the effects and mechanism of involvement of lncRNA BMP2-1 in the osteogenic differentiation of hPDLSCs by regulating mRNA BMP2. This study would be significant for revealing the important role of lncRNA in the reconstruction of periodontal tissue defects, and it also provides a new idea for periodontal tissue regeneration in the future.

前期研究采用基因芯片检测发现长链非编码RNA（lncRNA）BMP2-1和mRNA 骨形成发生蛋白2（BMP2）在成骨诱导的人牙周膜干细胞（hPDLSCs）中显著上调表达，PCR验证显示两者表达趋势一致；两者位置毗邻且共表达分析提示两者之间存在显著正相关；抑制lncRNA BMP2-1表达后BMP2在基因和蛋白水平均显著下降。据此我们推测，lncRNA BMP2-1可能通过靶向正调控BMP2促进hPDLSCs的成骨分化。本课题拟构建lncRNA BMP2-1过/抑制表达的稳定细胞系，分析lncRNA BMP2-1对成骨分化的影响；观察lncRNA BMP2-1的细胞内定位，明确其调控的靶基因及作用机制；进一步探索lncRNA BMP2-1通过调控BMP2可能参与的信号通路。研究旨在阐明lncRNA BMP2-1调控hPDLSCs成骨分化的功能及机制，为牙周再生提供新思路。

长链非编码RNA(lncRNA)在基因调控中起关键作用并与众多疾病相关，但其与牙周组织再生的研究才刚起步。我们首次用lncRNA和mRNA表达谱芯片进行人牙周膜干细胞成骨分化中lncRNA和mRNA差异筛选，发现成骨分化14天lncRNA BMP2-1和其临近基因骨形成蛋白2（BMP2）均显著上调，分别构建lncRNA BMP2-1的慢病毒过表达载体、BMP2的慢病毒过表达载体并将载体分别转染牙周膜干细胞，观察其对牙周膜干细胞成骨分化的影响，且抑制lncRNA BMP2-1后BMP2表达水平显著下降。进一步采用荧光原位杂交技术分析发现lncRNA BMP2-1主要位于细胞质内，我们推测该lncRNA可能以竞争性内源RNA角色发挥调控作用。在此基础上采用芯片分析牙周膜干细胞成骨分化过程中差异表达的微小RNA（miRNA），联合位点预测发现lncRNA BMP2-1与miR-361-5p结合，而BMP2是miR-361-5p一个重要的成骨分化相关靶基因，双荧光素酶报告均显示lncRNA BMP2-1与miR-361-5p、miR-361-5p与BMP2存在结合。功能试验发现lncRNA BMP2-1促进人牙周膜干细胞成骨分化，而miR-361-5p显著抑制。共转染实验证实miR-361-5p能够部分回复lncRNA BMP2-1对BMP2的促进作用。综上所述本研究发现lncRNA BMP2-1/miR-361-5p/BMP2通路调控人牙周膜干细胞成骨分化，为确立基于lncRNA BMP2-1/miR-361-5p/BMP2调控轴的牙周膜干细胞作为调控牙周再生靶点提供科学依据。