中国普外基础与临床杂志

中国普外基础与临床杂志

ADAM17-shRNA 通过 Akt/GSK3β 信号通路促进 HT29 结肠癌细胞的凋亡

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目的 探讨沉默解聚素-金属蛋白酶 17(ADAM17)基因的表达对 HT29 结肠癌细胞增殖和凋亡的抑制作用及其可能机制。 方法 将 HT29 结肠癌细胞分为干扰组、阴性对照组和空白对照组,干扰组细胞转染重组慢病毒载体以沉默 ADAM17 基因的表达,阴性对照组细胞转染阴性对照重组慢病毒载体,空白对照组细胞加入等量的 PBS 溶液。采用实时荧光定量 PCR 法(real-time PCR)检测 ADAM17 mRNA 的表达,采用 Western blot 法检测 ADAM17、半胱氨酸天冬氨酸蛋白酶-3(caspase3)、磷酸化蛋白激酶 B(P-Akt)、蛋白激酶 B(Akt)、磷酸化糖原合成酶激酶-3β(P-GSK3β)及糖原合成酶激酶-3β(GSK3β)蛋白的表达,采用 3-(4, 5-二甲基噻唑-2)-2, 5-二苯基四氮唑溴盐(MTT)法检测细胞增殖能力的变化,采用 Annexin-V-FITC/PI 试剂盒检测细胞凋亡情况。 结果 与阴性对照组和空白对照组比较,干扰组细胞中 ADAM17 mRNA 及其蛋白的表达水平均较低,同时点(培养 24、48 及 72 h)的吸光度值(A 值)也较低,细胞凋亡率较高,caspase3 蛋白的表达水平较高,P-Akt 和P-GSK3β 蛋白的表达水平均较低,差异均有统计学意义(P<0.05)。 结论 ADAM17 基因沉默可能通过抑制 Akt/GSK3β 通路的激活,发挥抑制细胞增殖和诱导细胞凋亡的作用。

Objective To investigate the inhibition effect of silence of a disintegrin and metalloproteinase 17 (ADAM17) gene on proliferation and apoptosis of HT29 colon cancer cells and its possible mechanism. Methods HT29cells were divided into 3 groups: cells of interference group were transfected with recombinant lentivirus vector, cells of negative control group were transfected with negative recombinant lentivirus vector, and cells of blank control group were treated with PBS. The expression of ADAM17 mRNA was detected by real-time PCR, the expressions of ADAM17 protein, caspase3, protein kinase B (Akt), glycogen synthase kinase-3β (GSK3β), phospho-protein kinase B (P-Akt), phospho-glycogen synthase kinase-3β (P-GSK3β) protein were detected by Western blot method, the cell proliferation was detected by MTT assay, and the apoptosis rate was detected by Annexin V-FITC/PI cell death detection kit. Results Compared with the control group and the negative control group, the interference group was related to low expressions of ADAM17 mRNA and its protein, low optical density value at the same time point (24, 48, and 72 h), high apoptosis rate, high expression level of caspase3 protein, but low expression levels of P-Akt and P-GSK3β protein (P<0.05). Conclusion Silent ADAM17 gene could significantly induces apoptosis and inhibits the proliferation of HT29 cells, which maybe via inhibiting Akt/GSK3β signaling pathway.

关键词: 解聚素-金属蛋白酶 17; 结肠癌; 蛋白激酶 B; 糖原合成酶激酶-3β; 凋亡

Key words: disintegrin and metalloproteinase 17; colon cancer; protein kinase B; glycogen synthase kinase-3β; apoptosis

引用本文: 张琪, 杨光华, 刘少鹏, 张国志, 王长友. ADAM17-shRNA 通过 Akt/GSK3β 信号通路促进 HT29 结肠癌细胞的凋亡. 中国普外基础与临床杂志, 2018, 25(5): 534-539. doi: 10.7507/1007-9424.201710044 复制

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