胃癌干细胞差异表达基因筛选及其对肿瘤发生能力的调控机制  

Screening of differentially expressed genes from gastric cancer stem cells and analysis of their regulatory mechanism on tumorigenic ability

作　　者：徐桂华 董志会 谭雪怡 申杰 XU Guihua;DONG Zhuihui;TAN Xueyi;SHEN Jie(Key Laboratory of Precision Medicine,Dongguan Binhaiwan Central Hospital,Dongguan 523900,China;不详)

机构地区：[1]东莞市滨海湾中心医院精准医学研究重点实验室,广东东莞523900 [2]东莞市滨海湾中心医院中心实验室,广东东莞523900 [3]深圳市龙岗区第三人民医院神经内科,广东深圳518115

出　　处：《山东医药》2025年第3期58-63,共6页Shandong Medical Journal

基　　金：广东省医学科学技术研究基金项目(A2023232)。

摘　　要：目的筛选胃癌干细胞差异表达基因,分析其对胃癌干细胞肿瘤发生能力的调控机制。方法采用FACSCalibur流式细胞仪分选人胃癌细胞系MKN-45中的CD44^(+)、CD44^(-)细胞,并经CD44^(+)表达率、细胞球集落形成实验、裸鼠成瘤实验证实CD44^(+)胃癌细胞具有干细胞特性。取CD44^(+)、CD44^(-)胃癌细胞,HiSeq4000测序仪测序后筛选差异表达基因,经实时荧光定量PCR法和Westen blotting法证实差异具有统计学意义的仅有细胞周期蛋白依赖激酶抑制剂1A(CDKN1A)。设计CDKN1A siRNA1、siRNA2、siRNA3,其中CDKN1A siRNA2仅抑制CD44^(+)胃癌细胞CDKN1A mRNA表达但不会影响细胞存活率和细胞凋亡率。取CD44^(+)胃癌细胞,分为GAPDH siRNA组、NC siRNA组和CDKN1A siRNA2组,分别使用Lipofectamine2000转染GAPDH siRNA、NC siRNA、CDKN1A siRNA2,采用细胞球集落形成实验检测细胞球集落形成数量,BrdU标记法检测BrdU阳性细胞率,流式细胞术检测细胞周期比例。利用Webgestalt软件预测CD44^(+)胃癌细胞中调控CDKN1A表达的上游微小核糖核酸(miRNA),实时荧光定量PCR法证实CD44^(+)、CD44^(-)胃癌细胞中差异具有统计学意义的仅有miR-20a。取CD44^(+)胃癌细胞,随机分为mimic组、mimic NC组、inhibitor组、inhibitor NC组、空白对照组,分别使用Lipofectamine2000转染miR-20a mimic、miR-20a mimic阴性对照、miR-20a inhibitor、miR-20a inhibitor阴性对照和等量PBS,采用实时荧光定量PCR法检测miR-20a、CDKN1A mRNA表达,Westen blotting法检测CDKNIA蛋白表达,流式细胞术检测细胞周期比例,裸鼠成瘤实验检测其在裸鼠体内的成瘤能力(记录注射后0~24 d的肿瘤体积)。结果与NC siRNA组和GAPDH siRNA组比较,CDKN1A siRNA2组细胞集落形成数量、BrdU阳性细胞率、S期细胞比例均升高而G1期细胞比例降低(P均<0.05)。与Control组、mimic NC组、inhibitor NC组比较,mimic组miR-20a表达升高而CDKN1A mRNA及蛋白相对表达量降低,inhibitor组miR-20a表�Objective To screen the differentially expressed genes of gastric cancer stem cells(GCSCs)and to analyze the mechanism of their regulation on tumorigenesis of GCSCs.Methods CD44^(+)and CD44^(-)cells from human gastric cancer cell(GCC)line MKN-45 were separated by FACSCalibur flow cytometer.CD44^(+)cells were confirmed to have stem cell characteristics by CD44^(+)expression rate,cellular colony formation and tumor formation experiments in nude mice.CD44^(+)and CD44^(-)GCCs were selected and sequenced by HiSeq4000 and the differentially expressed genes were screened.The only gene with statistically significant differential expression was cyclin-dependent kinase inhibitor 1A(CDKN1A),which was confirmed by real-time fluorescence quantitative PCR and Western blotting.We designed CDKN1A siRNA1,siRNA2,and siRNA3.Among them,CDKN1A siRNA2 only inhibited the expression of CDKN1A mRNA in CD44^(+)gastric cancer cells but did not affect cell survival rate and apoptosis rate.We collected CD44^(+)GCCs and divide them into the GAPDH siRNA group,the NC siRNA group,and the CDKN1A siRNA2 group,which were transfected with GAPDH siRNA,NC siRNA,and CDKN1A siRNA2,respectively,using Lipofectamine 2000.Colony formation assay was used to detect the number of colony formation,BrdU labeling was used to detect the BrdU positive cell rate,and flow cytometry was used to detect the proportion of cell cycle.Webgestalt software was used to predict upstream miRNAs regulating CDKN1A expression in CD44^(+)GCCs,and real-time fluorescence quantitative PCR(RT-PCR)method confirmed that only miR-20a had a statistically significant difference between CD44^(+)and CD44^(-)GCCs.We selected the CD44^(+)GCCs and randomly divide then into the mimic group,the mimic NC group,the inhibitor group,the inhibitor NC group and the blank control group,which were transfected with miR-20a mimic,miR-20a mimic negative control,miR-20a inhibitor,miR-20a inhibitor negative control,and equal volume of PBS and transfection reagent,respectively,by Lipofectamine 2000.RT-PCR was u

关 键 词：微小核糖核酸20a 胃癌干细胞 细胞周期蛋白依赖激酶抑制剂1A 细胞周期 肿瘤发生能力 

分 类 号：R735.2[医药卫生—肿瘤]