miR-338-3p/155-5p在根尖周炎破骨细胞分化中的作用

MicroRNAs are a kind of single-stranded non-coding small RNAs, which play important roles during every aspect of physiological and pathological processes. The object of this study is to investigate the roles of miR-338-3p/155-5p during periapical osteoclast differentiation. Firstly, we detect the expression pattern of miR-338-3p/155-5p during osteoclast differentiation by real-time PCR and northern blot. After over-expression and inhibition of miR-338-3p/155-5p,the osteoclast differentiation markers were determined by real-time PCR. Then, we use bioinformatics method and dual luciferase activity to confirm the binding interaction between miR-338-3p/155-5p and target gene MafB. And the roles of miR-338-3p/155-5p during osteoclast differentiation will be further detected by in vivo experiments. Lastly, we will co-transfected miR-338-3p/155-5p inhibitors and siRNAs of MafB to explain why miR-338-3p/155-5p could regulate osteoclast differentiation.

MicroRNA是一类长度约为20-24nt的单链非编码小分子RNA，在生物体生理病理过程的各个方面发挥了重要作用。本项目旨在探讨miR-338-3p/155-5p在根尖周炎破骨细胞分化过程中的作用。首先，利用real-time PCR和northern blot技术检测miR-338-3p/155-5p在破骨细胞分化中的表达趋势；利用mimics及inhibitors过表达和抑制microRNAs后检测破骨细胞分化相关基因的表达；再利用生物信息学方法和双萤光素酶报告实验确认miR-338-3p/155-5p与靶基因MafB之间的结合关系；进一步利用体内实验证实microRNAs在破骨细胞分化中的作用；最后，共转染microRNAs及靶基因抑制剂，探讨miR-338-3p/155-5p调控破骨细胞分化的机制。

破骨细胞分化受到细胞因子、信号通路、表观遗传等多方面因素的调控。microRNA是一类长度约为20-24nt的非编码小分子RNA。前期研究表明，破骨细胞分化受到作为表观遗传调控因子之一的micro RNA的调控。本项目中，首先利用RAW264.7破骨细胞系建立细胞分化体外模型，利用TRAP染色和real-time PCR技术检测相关基因的表达；再通过过表达和抑制microRNA的方法验证microRNA在破骨细胞分化中的功能；利用生物信息学方法预测靶基因；最后将microRNA和靶基因共表达确认其对破骨细胞分化的影响。研究结果发现miR-338-3p在破骨细胞分化过程中逐步上调，过表达/抑制miR-338-3p分别促进和抑制破骨细胞分化，生物信息学研究提示MAFB基因是miR-338-3p的靶基因之一，过表达MAFB抑制破骨细胞分化，而抑制MAFB促进破骨细胞分化，同时抑制MAFB和过表达miR-338-3p对破骨细胞分化无影响。此外，miR-155-5p在破骨细胞分化过程中逐步下调，抑制miR-155-5p促进破骨细胞分化，而敲除miR-155-5p促进破骨细胞分化，BACH1为miR-155-5p的靶基因。以此证明miR-338-3p/155-5p分别通过作用于MAFB/BACH1基因调控破骨细胞分化，从而为骨质疏松症等与骨代谢相关疾病的治疗提供一定的理论依据。