lncRNA H19通过miR-141/SPAG9影响根尖牙乳头干细胞生物学特性的机制研究

Stem cells from apical papilla (SCAPs) are one of important seed cells for the tooth root development and regeneration. SCAPs combined with tissue engineering could be used to provide a new therapeutic approach for the periapical periodontitis in the immature permanent teeth. Our previous study found that the expression of lncRNA H19 showed an increased level in the odonto/osteogenic differentiation of SCAPs, accompanied by the reduced expression of miR-141 and raised expression of SPAG9, the MAPK signal pathway related gene. On the contrary, the expression of miR-141 was boosted and SPAG9 was decreased in the downregulated lncRNA H19 SCAPs. And the odonto/osteogenic differentiation potential of SCAPs was weaked. Bioinformatics analysis reminded that there are possible binding sites between miR-141 and SPAG9 3’UTR. We propose that lncRNA H19 might inhibit miR-141 to defend SPAG9 from binding to it, further activate the downstream pathway MAPK to promote he odonto/osteogenic differentiation of SCAPs. We will test this hypothesis in cell biological characteristics, molecular biological mechanism and cell transplantation in vivo. Results from this study will be helpful to clarify the molecular mechanism of regulation of lncRNA H19 in dental stem cells, thus will provide experimental and theoretical basis for the application of stem cells in the tooth root engineering regeneration.

根尖牙乳头干细胞（SCAPs）是牙根发育和再生过程中的重要种子细胞之一，运用组织工程技术将其应用于年轻恒牙根尖周炎的治疗，促进牙根再生有望成为新的治疗途径。我们的前期研究发现SCAPs牙/骨向分化过程中lncRNA H19的表达显著升高，并伴随miR-141的表达降低和MAPK通路相关SPAG9的表达升高；相反，低表达lncRNA H19的SCAPs，其miR-141表达升高、SPAG9表达降低，且牙/骨向分化能力减弱。生物信息学分析发现miR-141与SPAG9的3’UTR存在结合位点。由此我们提出lncRNA H19通过调控miR-141/SPAG9及其下游通路MAPK的激活影响SCAPs干细胞特性的假说，并拟从细胞生物学特性、分子生物学机制和动物体内移植三个层面进行验证。研究将有助于揭示lncRNA H19调控牙源性干细胞的分子机制，为基于干细胞的牙根组织工程再生提供实验和理论依据。

非编码RNA对干细胞的调控作用已成为近年转录后调控研究的热点，也影响着基于干细胞的组织工程再生的研究效果。本项目着眼于LncRNA-H19，通过细胞生物学、分子生物学和组织形态学三个层面论证了其对根尖牙乳头干细胞（SCAPs）生物学特性的影响，并就LncRNA-H19/miR-141/SPAG9调控轴对SCAPs牙/骨向分化的作用机制进行了阐明。首先，我们高/低表达SCAPs中的LncRNA-H19，通过CCK8 绘制生长曲线、流式细胞术和EdU多方面检测证实其对SCAPs的增殖能力没有明显影响。其次，通过对碱性磷酸酶、矿化结节、牙/骨向分化相关蛋白/基因的检测以及体内移植实验从体内外证实了LncRNA-H19对SCAPs牙/骨向分化的促进作用。再次，通过生物信息学分析，我们发现了LncRNA-H19与miR-141的结合关系，对高/低表达miR-141的SCAPs牙/骨向分化能力的体外实验也验证了miR-141对SCAPs的抑制作用。最后，通过对miR-141下游靶基因SPAG9的表达检测以及SPAG9激活下游JNK和P38通路促进SCAPs分化的验证，我们发现了miR-141与SPAG9的结合关系。另外，实验过程中我们发现虽然LncRNA-H19对SCAPs的增殖能力没有影响，但是miR-141却明显抑制了SCAPs的增殖能力。通过生物信息学分析和流式细胞术、EdU、Western Blot、免疫荧光等实验的验证，我们论证了miR-141通过靶基因YAP抑制SCAPs增殖能力、加速SCAPs衰老的作用机制。本项目通过探索总结，积累了一定的非编码RNA调控机制研究的经验和方法，得到了LncRNA-H19/miR-141/SPAG9调控SCAPs牙/骨向分化和miR-141/YAP调控SCAPs增殖和衰老两条作用通路，为以后组织工程再生的种子细胞调控提供了更多的研究依据和更广阔的应用前景。