ALCAM基因1沉默对胃癌细胞增殖和凋亡的影响及分子机制

目的 探讨活化白细胞黏附分子(ALCAM)基因沉默对胃癌细胞增殖和凋亡的影响,并对其分子机制进行初步研究.方法 将ALCAM 和对照shRNA转染至胃癌细胞SGC-7901中,建立稳定细胞系,分别采用实时荧光定量(RT-PCR)技术和蛋白免疫印迹法(Western blot)检测转染后SGC-7901细胞中ALCAM mRNA和蛋白水平;采用CCK-8法检测转染后SGC-7901细胞的增殖情况;采用流式细胞术检测转染后SGC-7901细胞凋亡情况.采用胃癌肿瘤模型分析ALCAM基因沉默对肿瘤生长的影响.结果 ALCAM shRNA稳定细胞系ALCAM mRNA(1.01 ±0.26)和蛋白质的表达水平(1.66 ±0.23)低于对照 shRNA组细胞ALCAM mRNA(0.12 ±0.06)和蛋白质水平(0.23 ±0.09),差异有统计学意义(P<0.05).与对照shRNA稳定细胞系比较,ALCAM shRNA 稳定细胞系细胞增殖活性下降(P<0.05)、凋亡水平升高(P<0.05)、荷瘤小鼠肿瘤生长速度减缓(P<0.05).ALCAM下调并不影响总蛋白激酶B(AKT)和哺乳动物雷帕霉素靶蛋白(mTOR)的表达,而降低了AKT和mTOR磷酸化水平,抑制了mTOR活性,此外,ALCAM蛋白敲低提高了Caspase-3蛋白的表达水平,激活了凋亡信号通路.结论 ALCAM基因沉默能够降低胃癌细胞中ALCAM的表达水平,抑制胃癌细胞的增殖,增加细胞凋亡.

Effects of ALCAM gene silencing on proliferation and apoptosis of gastric cancer cells and its molecular mechanism

Objective To investigate the effects of gene silencing of activated leukocyte adhesion molecule(ALCAM)on the prolif-eration and apoptosis of gastric cancer cells and its molecular mechanism.Methods ALCAM shRNA was transfected into gastric cancer cells SGC-7901.ALCAMmRNA and protein levels of SGC -7901 cells were detected after transfected respectively by real -time fluorescence quantitative(RT-PCR)and Western blotting.SGC-7901 cell proliferation level was detected by CCK -8 after transfection.SGC-7901 cell apoptosis level was detected by flow cytometry after transfection.The effect of ALCAM gene silencing on tumor growth was analyzed using gastric cancer model.Results Compared with control shRNA groupmRNA(0.12 ±0.06), protein(0.23 ±0.09)], ALCAM mRNA (1.01 ±0.26)and protein levels(1.66 ±0.23)in ALCAM shRNA SGC -7901 cells were significantly reduced(P<0.05).Compared with control shRNA group,the proliferation level of ALCAM shRNA SGC -7901 cells significantly decreased(P<0.05).Compared with control shRNA group,the apoptosis level of ALCAM shRNA SGC -7901 cells significantly increased(P<0.05).Compared with control shRNA group,tumor growth of ALCAM shRNA SGC -7901 cells was significantly reduced(P<0.05).Down-regulation of ALCAM did not affect the expression of AKT and mTOR protein,but it decreased AKT and mTOR phosphorylation level,and inhibited mTOR activity.Knock-down of ALCAM protein significantly increased the expression level of Caspase -3 protein.Conclusion ALCAM gene silencing significantly reduces the expression of ALCAM in gastric cancer cells,inhibits the proliferation level and increases the apoptosis level of gastric cancer cells.