miR-338-3p在成牙本质细胞和成骨细胞分化过程中的作用

Both precursors of odontoblast and osteoblast originate from neural crest,the mechanisms of odontoblast(OD)and osteoblast(OB)differentiation are significantly different. MicroRNAs are a kind of non-coding small RNAs, which play important roles during biological characteristics involving cell differentiation, proliferation and organogenesis. The object of this study is to investigate the roles of miR-338-3p during OD and OB differention. Firstly,the expression patterns of miR-338-3p during OD and OB differention were detected by real-time PCR.After overexpression and suppression of miR-338-3p in these two different kinds of cells, the expression levels of OD and OB differentiation markers were analysed by RT-PCR and western blot. Furthermore, bioinformatics methods were used to predict its downstream target gene.The binding interaction between miR-338-3p and its target genes will be further confirmed by dual luciferase assay. Last of all,the regulation effect of its upstream transcription factors on miR-338-3p will be detecteted by CHIP, and to explain the reason why miR-338-3p expresses differently in OD and OB differentiation.

尽管形成牙本质和骨质的前体细胞均来自神经嵴，但是成牙本质细胞和成骨细胞的分化过程和随后的矿化过程却显著不同。MicroRNA是一类长度仅为20～25nt的非编码的小分子RNA，在细胞的分化、增殖和器官发生等生物学行为中发挥了重要作用。本项目旨在探讨miR-338-3p在成牙本质细胞和成骨细胞分化过程中的作用。首先通过real-time PCR检测miR-338-3p在成牙本质细胞和成骨细胞分化过程的中的表达趋势；分别利用慢病毒过表达miR-338-3p和miR-338-3p拮抗剂抑制miR-338-3p后，检测细胞分化相关标志性基因的表达；进一步用生物信息学方法预测其下游靶基因，并用双荧光素酶实验检测miR-338-3p与靶基因的结合作用；最后使用染色体免疫共沉淀技术验证上游转录因子对miR-338-3p的调控作用,分析miR-338-3p在成牙本质细胞和成骨细胞中差异表达的原因。

形成牙本质和骨质的前体细胞均来自神经嵴细胞，但是成牙本质细胞和成骨细胞的分化过程和矿化过程却显著不同。MicroRNA是一类长度仅为20～25nt的非编码的小分子RNA，在细胞的分化、增殖和器官发生等生物学行为中发挥了重要作用。本项目旨在探讨miR-338-3p在成牙本质细胞和成骨细胞分化过程中的作用。首先我们发现miR-338-3p在成牙本质细胞分化过程中是显著上调的，而在成骨细胞分化过程的中的表达趋势却是显著下调；我们分别利用慢病毒过表达miR-338-3p和miR-338-3p拮抗剂抑制miR-338-3p后，发现多个细胞分化相关标志性基因的表达均受其调控；随后进一步用生物信息学方法预测miR-338-3p的下游靶基因，并用双荧光素酶实验验证了miR-338-3p可以与成牙本质细胞和成骨细胞分化都相关的关键基因Runx2结合；最后我们建立双侧卵巢摘除术的骨质疏松小鼠模型，发现在骨质疏松小鼠的BMSCs，miR-338-3p的表达显著高于对照组，抑制miR-338-3p的表达后，可以挽救由于骨质疏松造成BMSCs降低的矿化能力。