miR-1292-5p对肝癌SNU-449细胞自噬与增殖及细胞周期影响

EFFECT OF MIR-1292-5P ON AUTOPHAGY, PROLIFERATION, AND CELL CYCLE OF SNU-449 HEPATOMA CELLS

目的研究miR-1292-5p对肝癌SNU-449细胞自噬、增殖及细胞周期影响。方法实时荧光定量PCR(qRT-PCR)检测肝癌HuH7、HCC9204、SNU-449细胞和正常肝L-02细胞中miR-1292-5p表达水平。肝癌SNU-449细胞中转染miR-1292-5p mimics,细胞计数试剂盒8(CCK-8)检测细胞增殖,碘化丙啶(PI)单染法检测细胞周期,膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)双染法检测细胞凋亡,蛋白质印迹法检测相关蛋白表达。以蛋白激酶B/磷脂酰肌醇3激酶(Akt/PI3K)信号激活剂处理转染miR-1292-5p mimics以后的肝癌SNU-449细胞,以同样方法检测上述指标。结果肝癌HuH7、HCC9204、SNU-449细胞中miR-1292-5p表达水平低于正常肝L-02细胞(F=260.440,q=13.159~20.505,P<0.05)。转染miR-1292-5p mimics后的肝癌SNU-449细胞存活率降低,G 0/G 1期比例升高,凋亡率升高,C-Caspase-3、P21蛋白表达水平升高,Cyclin D1、Beclin1、ATG5蛋白表达水平降低,差异有显著性(F=27.553~611.793,q=7.116~24.042,P<0.05);p-Akt、p-PI3K蛋白表达水平降低,差异有显著性(F=42.043、60.545,q=10.307、7.603,P<0.05)。Akt/PI3K信号激活剂可逆转miR-1292-5p mimics对肝癌SNU-449细胞增殖、周期、凋亡和相关蛋白表达影响(t=5.154~15.949,P<0.05)。结论上调miR-1292-5p可以抑制肝癌SNU-449细胞自噬和增殖,阻滞细胞周期,诱导细胞凋亡,机制可能与抑制Akt/PI3K信号激活有关。

Objective To investigate the effect of miR-1292-5p on the autophagy,proliferation,and cell cycle of SNU-449 hepatoma cells.Methods Quantitative real-time PCR was used to measure the expression level of miR-1292-5p in HuH7,HCC9204,and SNU-449 hepatoma cells and normal L-02 liver cells.SNU-449 hepatoma cells were transfected with miR-1292-5p mimics;CCK-8 assay was used to measure cell proliferation,propidium iodide single staining was used to observe cell cycle,Annexin V-fluorescein isothiocyanate/propidium iodide double staining was used to measure apoptosis,and Western blotting was used to measure the expression of related proteins.The SNU-449 hepatoma cells transfected with miR-1292-5p mimics were treated with protein kinase B/phosphatidylinositol 3-kinase signal activator,and the same methods were used to measure the above indicators.Results The expression level of miR-1292-5p in HuH7,HCC9204,and SNU-449 hepatoma cells was lower than that in normal L-02 liver cells(F=260.440,q=13.159-20.505,P<0.05).The SNU-449 hepatoma cells transfected with miR-1292-5p mimics showed a significant reduction in cell viability,a significant increase in the proportion of cells in G 0/G 1 phase,and a significant increase in apoptosis rate,as well as significant increases in the protein expression levels of C-caspase-3 and P21 and significant reductions in the protein expression levels of Cyclin D1,Beclin1,and ATG5(F=27.553-611.793,q=7.116-24.042,P<0.05),with significant reductions in the protein expression levels of p-Akt and p-PI3K(F=42.043,60.545;q=10.307,7.603;P<0.05).The Akt/PI3K signal activator reversed the influence of miR-1292-5p mimics on proliferation,cell cycle,apoptosis,and expression of related proteins in SNU-449 hepatoma cells(t=5.154-15.949,P<0.05).Conclusion Upregulation of miR-1292-5p can inhibit the autophagy and proliferation of SNU-449 hepatoma cells,block the cell cycle,and induce cell apoptosis,possibly by inhibiting the activation of Akt/PI3K signaling.