安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
Acta Universitatis Medicinalis Anhui
2015年
11期
1639-1644
,共6页
蒋秀敏%刘雨生%许波
蔣秀敏%劉雨生%許波
장수민%류우생%허파
miR-483-5p%ERK1/2-MAPK 通路%颗粒细胞%增殖凋亡平衡
miR-483-5p%ERK1/2-MAPK 通路%顆粒細胞%增殖凋亡平衡
miR-483-5p%ERK1/2-MAPK 통로%과립세포%증식조망평형
miR-483-5p%ERK1 /2-MAPK pathway%granular cells%proliferation-apoptosis balance
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。
目的：證明 miR-483-5p 及其靶基因 ERK1參與調控人類顆粒細胞增殖凋亡平衡。方法①體外培養人正常顆粒細胞，調控其 miR-483-5p 超錶達，聚閤酶鏈反應（PCR）及Western blot 法檢測 ERK1的 mRNA 及蛋白錶達情況；將包含野生型或突變型的 ERK1 mRNA 3′UTR 的 DNA 片段剋隆在熒光素酶標記的質粒，共轉染 miR-483-5p mimics、control-miR mimics 和野生型、突變型重組質粒，檢測其顆粒細胞相對熒光素酶活性；②將 miR-483-5p mimics 及 ERK1 siRNAs單獨及共同轉染顆粒細胞，MTT 法及 TUNEL 法分彆檢測三種情況下卵巢顆粒細胞的增殖、凋亡情況。結果① miR-483-5p 超錶達後，ERK1基因及蛋白錶達均下降；與 control-Wt（野生型）轉染組相比，miR-483-Wt 組的熒光素酶活性顯著降低；miR-483-Mut（突變型）與 control-Mut 轉染組間熒光素酶活性的比較差異無統計學意義；② miR-483-5p mimics與 ERK1 siRNAs 單獨及共同轉染顆粒細胞，三種轉染條件下顆粒細胞增殖均顯著受抑製、凋亡率顯著升高，且三種作用效果差異無統計學意義。結論 miR-483-5p 通過直接與靶基因 ERK1結閤，參與調控人類顆粒細胞增殖凋亡平衡。
목적：증명 miR-483-5p 급기파기인 ERK1삼여조공인류과립세포증식조망평형。방법①체외배양인정상과립세포，조공기 miR-483-5p 초표체，취합매련반응（PCR）급Western blot 법검측 ERK1적 mRNA 급단백표체정황；장포함야생형혹돌변형적 ERK1 mRNA 3′UTR 적 DNA 편단극륭재형광소매표기적질립，공전염 miR-483-5p mimics、control-miR mimics 화야생형、돌변형중조질립，검측기과립세포상대형광소매활성；②장 miR-483-5p mimics 급 ERK1 siRNAs단독급공동전염과립세포，MTT 법급 TUNEL 법분별검측삼충정황하란소과립세포적증식、조망정황。결과① miR-483-5p 초표체후，ERK1기인급단백표체균하강；여 control-Wt（야생형）전염조상비，miR-483-Wt 조적형광소매활성현저강저；miR-483-Mut（돌변형）여 control-Mut 전염조간형광소매활성적비교차이무통계학의의；② miR-483-5p mimics여 ERK1 siRNAs 단독급공동전염과립세포，삼충전염조건하과립세포증식균현저수억제、조망솔현저승고，차삼충작용효과차이무통계학의의。결론 miR-483-5p 통과직접여파기인 ERK1결합，삼여조공인류과립세포증식조망평형。
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.
