microRNA-24通过下调Cdk4和CyclinD3抑制胰岛β细胞增殖
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microRNA-24 inhibits islet β-cell proliferation by decreasing Cdk4 and CyclinD3 protein levels
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    目的:通过共同过表达Cdk4-CyclinD3与microRNA-24,观察miR-24所引起的胰岛β细胞增殖抑制是否得到逆转,从而判定Cdk4-CyclinD3是否介导了这种抑制作用-方法:在胰岛β细胞系MIN6细胞中过表达miR-24,运用WST-1法检测细胞活力,借助于流式细胞术检测细胞周期,Hoechst33342染色和BrdU标记检测细胞增殖凋亡情况-Western blot检测Cdk4-CyclinD3蛋白水平-构建cyclinD3-pCMV5-myc,cdk4-pCMV5-myc重组质粒,与pre-miR-24-pre-Neg miRNA precursors共转染MIN6细胞,BrdU标记检测细胞增殖-结果:过表达miR-24引起MIN6细胞活力降低,细胞周期G2期阻滞,细胞增殖受到抑制而并没有显著的凋亡;过表达miR-24降低了Cdk4-CyclinD3蛋白水平;而Cdk4-CyclinD3的过表达逆转了miR-24引起的胰岛β细胞增殖抑制-结论:miR-24通过降低Cdk4-CyclinD3蛋白水平抑制胰岛β细胞增殖,该作用可被Cdk4-CyclinD3蛋白的过表达逆转-

    Abstract:

    Objective:To explore whether Cdk4 and CyclinD3 are involved in the inhibition of β-cell proliferation caused by miRNA-24. Methods:miRNA-24(miR-24)was highly expressed in MIN6 cells. Cell viability was determined using WST-1 assays. Cells were collected for flow cytometry analysis to test cell cycle distribution. Protein levels of Cdk4 and CyclinD3 were analyzed by Western blot. CyclinD3-pCMV5-myc and cdk4-pCMV5-myc recombinant plasmid were constructed and transfected into MIN6 cells together with pre-miR-24 or pre-Neg miRNA precursors. Cell proliferation was measured by the BrdU labeling. Results:MIN6 cells proliferation was reduced because of transfected miR-24,mainly due to G2 phase arrest. No changes of the level of apoptotic cells were detected. Cdk4 and CyclinD3 were downregulated by overexpressed miR-24. Overexpressed cdk4 and cyclinD3 reversed the β-cell proliferation inhibition. Conclusion:miR-24 inhibits β-cell proliferation by decreasing Cdk4 and CyclinD3 protein levels. The inhibition may be reversed by overexpressed cdk4 and cyclinD3.

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常晓嫒,朱云霞,韩 晓,程 光. microRNA-24通过下调Cdk4和CyclinD3抑制胰岛β细胞增殖[J].南京医科大学学报(自然科学版),2016,(12):1413-1417

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  • 收稿日期:2016-07-19
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  • 在线发布日期: 2016-12-28
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