摘要
目的探究葡萄糖转运蛋白1(Glut 1)基因沉默对人结直肠癌HT-29细胞增殖、分化及凋亡的影响及机制。方法将Glut 1干扰序列(siRNA)转染至人结肠癌HT-29细胞作为shGlut 1组,非干扰序列转至HT-29细胞作为NC组,HT-29细胞作为Blank组,癌旁正常组织细胞作为Control组,应用qRT-PCR法和Western blot法检测各组细胞中Glut 1、TGF-β1、PI3K、AKT、PTEN、mTOR mRNA和相应蛋白的表达水平,并检测Bcl-2/Bax比值,p-PI3K、p-S 473-AKT、p-S 389-S6K1、p-T 70-4 EBP1、Cleaved Caspase-3、Cleaved-PARP蛋白的表达;MTT法检测各组细胞增殖能力变化,流式细胞术检测细胞周期和细胞凋亡情况,克隆形成实验检测肿瘤细胞体外增殖成瘤能力。结果免疫荧光结果表明shGlut 1组转染效率较高,符合实验标准;与Control组比较,结肠癌组织中Glut 1、TGF-β1、PI3K、AKT、mTOR、Bcl-2 mRNA和蛋白的表达上调,而PTEN、Bax的mRNA和蛋白的表达下调,p-PI3K、p-S 473-AKT、p-S 389-S6K1和p-T 70-4 EBP1蛋白表达上调,Cleaved Caspase-3、Cleaved-PARP蛋白表达下调(P均<0.05);与Blank组和NC组比较,shGlut 1组Glut 1、TGF-β1、PI3K、AKT、mTORC 1、Bcl-2 mRNA和蛋白的表达下调,PTEN、Bax mRNA和蛋白的表达上调,p-PI3K、p-S 473-AKT、p-S 389-S6K1和p-T 70-4 EBP1表达下调,Cleaved Caspase-3、Cleaved-PARP蛋白的表达上调,增殖率下调,G0/G1期较多而S期较少,凋亡率上调,克隆形成细细胞生长较慢(P均<0.05)。结论Glut 1基因沉默能抑制结直肠癌细胞的增殖与分化,促进其凋亡,考虑为抑制TGF-β/PI3K-AKT-mTOR信号通路。
Objective To investigate the effect and mechanism of glucose transporter 1(Glut 1) gene silencing on the proliferation, differentiation and apoptosis of human colorectal cancer HT-29 cells. Methods Glut 1 interference sequence(siRNA) was transfected into human colon cancer HT-29 cells as shGlut1 group, non-interfering sequence was transferred to HT-29 cells as NC group, HT-29 cells as Blank group, and normal tissue cells adjacent to cancer as Control Group. qRT-PCR and Western blot were used to detect the expression levels of Glut 1, TGF-β1, PI3 K, AKT, PTEN, mTORmRNA and corresponding proteins in each group of cells, and to detect the Bcl-2/Bax ratio, p-PI3 K, p-S 473-AKT, p-S 389-S6 K1, p-T 70-4 EBP1, CleavedCaspase-3, Cleaved-PARP protein expression;MTT method was used to detect changes in cell proliferation in each group, flow cytometry was used to detect cell cycle and apoptosis, clone formation experiments was used to detect tumor cell proliferation and tumor formation ability in vitro. Results The immunofluorescence results showed that the transfection efficiency of shGlut1 group was higher, which met the experimental standards. Compared with the Control group, the expression of Glut 1, TGF-β1, PI3 K, AKT, mTOR, Bcl-2 mRNA and protein were up-regulated in colon cancer tissues, while PTEN, Bax mRNA and protein expression were down-regulated, p-PI3 K, p-S 473-AKT, p-S 389-S6 K1 and p-T 70-4 EBP1 protein expression was up-regulated, Cleavedcaspase-3, Cleaved-PARP protein expression was down-regulated,(all P<0.05);compared with the Blank group and NC group, the expression of Glut 1, TGF-β1, PI3 K, AKT, mTOR, Bcl-2 mRNA and protein was down-regulated in the shGlut 1 group, and the expression of PTEN, Bax mRNA and protein was up-regulated, p-PI3 K, p-S 473-AKT, The expression of p-S 389-S6 K1 and p-T 70-4 EBP1 was down-regulated, the expression of Cleavedcaspase-3, Cleaved-PARP protein was up-regulated, the proliferation rate was down-regulated, the G0/G1 phase was more and the S phase was less, the apoptotic
作者
郭圣超
武雪亮
薛军
韩磊
孙光源
杨东东
屈明
Guo Shengchao;Wu XueLiang;Xue Jun(Graduate School of Hebei North University,Zhangjiakou 075000;Dept of General Surgery,First Affiliated Hospital of Hebei North University,Zhangjiakou 075000)
出处
《安徽医科大学学报》
CAS
北大核心
2021年第5期790-795,共6页
Acta Universitatis Medicinalis Anhui
基金
河北省财政厅临床医学优秀人才培养研究项目(编号:361009)
河北省卫计委医学科学研究重点课题计划(编号:20180834)
张家口市科技局指令性计划(编号:1621083D)。
关键词
Glut
1基因沉默
人结直肠癌HT-29细胞
增殖
分化
凋亡
机制
Glut 1 gene silencing
human colorectal cancer HT-29 cells
proliferation
differentiation
apoptosis
mechanism
