miR-483-5p 通过靶基因 ERK1调控人类颗粒细胞增殖凋亡平衡

目的：证明 miR-483-5p 及其靶基因 ERK1参与调控人类颗粒细胞增殖凋亡平衡。方法①体外培养人正常颗粒细胞，调控其 miR-483-5p 超表达，聚合酶链反应（PCR）及Western blot 法检测 ERK1的 mRNA 及蛋白表达情况；将包含野生型或突变型的 ERK1 mRNA 3′UTR 的 DNA 片段克隆在荧光素酶标记的质粒，共转染 miR-483-5p mimics、control-miR mimics 和野生型、突变型重组质粒，检测其颗粒细胞相对荧光素酶活性；②将 miR-483-5p mimics 及 ERK1 siRNAs单独及共同转染颗粒细胞，MTT 法及 TUNEL 法分别检测三种情况下卵巢颗粒细胞的增殖、凋亡情况。结果① miR-483-5p 超表达后，ERK1基因及蛋白表达均下降；与 control-Wt（野生型）转染组相比，miR-483-Wt 组的荧光素酶活性显著降低；miR-483-Mut（突变型）与 control-Mut 转染组间荧光素酶活性的比较差异无统计学意义；② miR-483-5p mimics与 ERK1 siRNAs 单独及共同转染颗粒细胞，三种转染条件下颗粒细胞增殖均显著受抑制、凋亡率显著升高，且三种作用效果差异无统计学意义。结论 miR-483-5p 通过直接与靶基因 ERK1结合，参与调控人类颗粒细胞增殖凋亡平衡。

Characterization of miRNA-483-5p and targeted gene ERK1 in the regulation of proliferation-apoptosis balance of human granulosa cells

Objective To demonstrate that miR-483-5p and its targeted gene ERK1 involved in the regulation of proliferation and apoptosis of human granulosa cells.Methods ① The ERK1 mRNA and protein level expression were detected by PCR and Western blot after miR-483-5p overexpression in vitro normal human granular cells.De-tected the relative luciferase density after cotransfecting miR-483-5p mimics and its control respectively with wild or mutant ERK1 mRNA 3`UTR cloned luciferase report vector in granular cells.② Granular cells proliferation and ap-optosis were detected by MTT and TUNEL after transfecting miR-483-5p mimics or ERK1 siRNAs alone or simulta-neously.Results ① It showed that both ERK1 mRNA and protein in granular cells were markedly downregulated after the transfection of miR-483-5p mimics.A significant relative luciferase activity decrease were detected in the granular cells co-transfected with ERK1 wild and miR-483-5p mimics comparison with the control mimics,but not in the cells co-transfected with the ERK1 mutant and miR-483-5p mimics.② When miR-483-5p mimics and ERK1 siRNAs were alone or co-transfected into granular cells,the proliferation was inhibited while apoptosis trend was monitored to be promoted in all cases.No obvious statistic difference was shown between each other.Conclusion MiR-483-5p is involved in the regulation of the proliferation and apoptosis of human granulosa cells by directly binding to the target gene ERK1.