SLC2A6基因敲减诱导A549肺癌细胞G2/M期阻滞研究  

Knockdown of SLC2A6 gene inhibits the proliferation of lung adenocarcinoma A549 cells by inducing G2/M arrest

作　　者：田迪[1] 陈春发 刘佳[1] 黄钊 赵平[1] TIAN Di;CHEN Chun-fa;LIU Jia;HUANG Zhao;ZHAO Ping(Institute for Medical Biology&Hubei Provincial Key Laboratory for Protection and Application of Special Plants in the Wuling Area of China,College of Life Sciences,South-Central University for Nationalities,Hubei Medical Biology International Science and Technology Cooperation Base,Wuhan 430074,China)

机构地区：[1]中南民族大学,生命科学学院,医学生物学研究所,湖北省武陵山区特种植物保护与应用重点实验室,湖北省医学生物国际科技合作基地,武汉430074

出　　处：《生物学杂志》2020年第5期24-29,34,共7页Journal of Biology

基　　金：国家自然科学基金(31070744,81573561,81774000);中南民族大学基础研究基金(CZR18003,CZP17060,CZP17048);武汉科技应用基础计划(2017060201010217);湖北省重点实验室建设基金(No.2018BFC360)。

摘　　要：SLC2A6是促进性糖转运蛋白家族中的一员,它编码GLUT6蛋白。SLC2A6基因的表达在许多癌症中都有上调,但在正常组织中没有得到广泛表达。为了阐明SLC2A6控制癌细胞增殖和凋亡的作用及其相关机制,利用CRISPR/Cas9技术筛选了两株敲减SLC2A6基因的肺腺癌A549细胞系,以阐明SLC2A6在生物学过程中的影响。与野生型A549细胞相比,KD1和KD2细胞中SLC2A6的蛋白水平的表达分别下降74%和83%,RNA水平的表达分别下降70.6%±2.0%和76.9%±4.1%。MTT实验的结果表明KD细胞增殖速度较慢,第5天的细胞数目的改变倍数由WT细胞的22.1±1.0下降到KD1细胞的13.5±0.9和KD2细胞的12.2±1.0(P<0.001)。流式细胞术细胞周期分析表明,SLC2A6基因敲减(KD)细胞发生了G2/M期阻滞。G2期细胞占比由A549野生型(WT)细胞的16.0%±1.0%升高到KD1细胞的23.0%±1.1%或KD2细胞的27.4%±0.4%(P<0.05)。细胞周期调节因子的mRNA表达的结果表明,G2/M阻滞与APC、Rad9a、CDK1、cdc25和p21 Waf1/Cip1的表达调控有关,揭示了SLC2A6在调控癌细胞生长方面的新作用,这表明SLC2A6可能是一个可行的抗癌药物靶点。SLC2A6 is a member of the facilitative glucose transporter family,it codes GLUT6 protein.SLC2A6 is up-regulated in several numerous cancers,but is not widely expressed in normal tissues.In order to elucidate the functions and underlying mechanisms for SLC2A6 controlling cancer cell proliferation and apoptosis,we screened two SLC2A6 knockdown lung adenocarcinoma A549 cell lines by CRISPR/Cas9 to illustrate the effects of SLC2A6 on biological process.SLC2A6 expression in mRNA and protein levels has been reduced in SLC2A6-knockdown A549 cells.Western Blot analysis confirmed that SLC2A6 protein expression decreased by 74%in KD1 and 83%in KD2 cells,respectively,compared with the control vehicle.In SLC2A6 mRNA expression level,there are 70.6%±2.0%and 76.9%±4.1%reduction in KD1 and KD2 cells,respectively,compared with wild-type A549 cells.The results of MTT assay indicated that the proliferation rate of KD cells was slower.The fold change of cell number on the fifth day of growth decreased from 22.1±1.0 in WT cells to 13.5±0.9 or 12.2±1.0 than in KD1 and KD2 cells(P<0.001).The cell cycle analysis via flow cytometry demonstrateed that SLC2A6 KD cells arrested at G2/M phase,as the percentage of cells in the G2 phase increased from 16.0%±1.0%of wild type(WT)cells to 23.0%±1.1%of KD1 cells or 27.4%±0.4%of KD2 cells(P<0.05).Moreover,the results of cell cycle regulators RNA expression showed that G2/M arrest was associated with the regulation of expression APC,Rad9a,CDK1,cdc25 and p21 Waf1/Cip1.In conclusion,our findings revealed a novel role for SLC2A6 in regulating cancer cell growth,which suggested that SLC2A6 may represent a viable anti-cancer drug target.

关 键 词：SLC2A6 A549细胞 CRISPR/Cas9 细胞增殖 细胞周期 

分 类 号：Q789[生物学—分子生物学]