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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Brd U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-p CMV5-myc,cdk4-p CMV5-myc重组质粒,与pre-miR-24、pre-Neg miRNA precursors共转染MIN6细胞,Brd U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。
OBJECTIVE: To investigate whether Cdk4 and CyclinD3 mediate this inhibitory effect by co-overexpression of Cdk4, CyclinD3 and microRNA-24 in order to observe whether the inhibition of pancreatic β-cell proliferation induced by miR-24 is reversed. Methods: miR-24 was overexpressed in pancreatic β-cell line MIN6 and the cell viability was measured by WST-1. The cell cycle was detected by flow cytometry. Hoechst33342 staining and BrdU labeling were used to detect cell proliferation and apoptosis. Western blot detection Cdk4, CyclinD3 protein levels. The CMV5-myc and cdk4-p CMV5-myc recombinant plasmids were constructed and co-transfected into MIN6 cells with pre-miR-24 and pre-neg miRNA precursors. BrdU labeling was used to detect cell proliferation. Results: Overexpression of miR-24 caused a decrease in the viability of MIN6 cells and G2 arrest in cell cycle, with no significant apoptosis in cell cycle. Overexpression of miR-24 reduced the protein levels of Cdk4 and CyclinD3. However, the expression of Cdk4, CyclinD3 Overexpression reversed miR-24-induced inhibition of pancreatic beta cell proliferation. Conclusion: miR-24 can inhibit the proliferation of pancreatic β-cells by decreasing the protein levels of Cdk4 and CyclinD3, which may be reversed by overexpression of Cdk4 and CyclinD3.