MicroRNA-450a-3p通过抑制Bub1基因的表达调控小鼠细胞增殖和胚胎发育

MicroRNA-450a-3p regulates cell proliferation and embryonic development by repressing Bub1gene in mouse

目的:探讨microRNA-450a-3p(miR-450a-3p)对小鼠细胞增殖和胚胎发育的调控是否通过抑制Bub1基因的表达实现。方法:用萤光素酶报告基因实验检测miR-450a-3p能否特异性结合于Bub1基因的3’-非翻译区(untranslated region,UTR);用Western blotting和实时荧光定量RT-PCR检测miR-450a-3p对Bub1蛋白和mRNA表达;分别通过MTT、Hoechst染色、流式细胞术等分析miR-450a-3p对小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)增殖、凋亡和细胞周期等生物学功能的调控;利用染色体核型分析技术检测miR-450a-3p对MEFs染色体数目的影响。结果:miR-450a-3p能与靶基因Bub1的3’-UTR结合,在翻译水平抑制MEFs中Bub1的表达,然而其转录水平的表达却不受影响。miR-450a-3p通过下调靶基因Bub1的表达,抑制MEFs的增殖,促进细胞的凋亡;而且miR-450a-3p还能使大多数细胞停滞在G1/G0期,使细胞分裂受阻,导致细胞染色体数目异常。结论:miR-450a-3p能够调控靶基因Bub1的表达,抑制MEFs增殖,并最终影响小鼠胚胎的发育。

AIM：To investigate the regulatory role of microRNA-450a-3p（miR-450a-3p） in cell proliferation and embryonic development in mouse. METHODS：The luciferase reporter gene assay was used to detect the specific binding ability of miR-450a-3p to Bub1 3’-untranslated region（UTR）. Western blotting and quantitative real-time PCR were used to evaluate the effect of miR-450a-3p on the expression of Bub1 at protein and mRNA levels. The biological functions of mouse embryonic fibroblasts（MEFs） regulated by miR-450a-3p, such as proliferation, apoptosis and cell cycle, were detected by MTT assay, Hoechst staining and flow cytometry analysis. The effect of miR-450a-3p on the chromosome numbers was observed by karyotype analysis. RESULTS：miR-450a-3p could bind to 3＇-UTR of the target Bub1 gene, and inhibited the expression of Bub1 at translational level instead of transcription. After interference with Bub1, miR-450a-3p inhibited the proliferation of MEFs, while the apoptosis ability was promoted. At the same time, miR-450a-3p arrested most of the cells in the G1/G0 phase, blocked the cell division, and resulted in abnormal number of chromosomes. CONCLUSION：miR-450a-3p regulates the expression of target Bub1 gene, then inhibits the proliferation of MEFs, and ultimately affects the development of mouse embryos.