山东医药2025，Vol.65Issue(3)：58-63,6.DOI:10.3969/j.issn.1002-266X.2025.03.012
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
摘要
Abstract
Objective To screen the differentially expressed genes of gastric cancer stem cells(GCSCs)and to ana-lyze 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 siR-NA,NC siRNA,and CDKN1A siRNA2,respectively,using Lipofectamine 2000.Colony formation assay was used to de-tect 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 di-vide 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 inhibi-tor negative control,and equal volume of PBS and transfection reagent,respectively,by Lipofectamine 2000.RT-PCR was used to detect the relative expression levels of miR-20a and CDKN1A mRNA,Westen blotting was used to detect the relative expression levels of CDKNIA protein,and flow cytometry was used to detect the cell cycle ratio.Tumor formation experiment was used to detect tumor formation ability in nude mice(tumor volume was recorded at 0-24 days after injec-tion).Results Compared with the NC siRNA group and GAPDH siRNA group,the number of cell colonies,the percent-age of BrdU-positive cells and the proportion of cells in the S-phase significantly increased in the CDKN1A siRNA2 group,while the proportion of cells in the G1 phase significantly decreased(all P<0.05).Compared with the mimic NC group,Control group,mimic NC group and inhibitor NC group,the expression of miR-20a increased while the relative expression of CDKN1A mRNA and protein decreased in the mimic group;the expression of miR-20a decreased and the relative ex-pression of CDKN1A mRNA and protein increased in the inhibitor group(all P<0.05).Compared with the control group,mimic NC group and inhibitor NC group,the proportion of cells in the G1 phase decreased while the proportion of cells in the S phase and the tumor volume from 15 to 24 days increased in the mimic group;the proportion of cells in the G1 phase significantly increased,but the proportion of cells in the S phase and the tumor volume from 15 to 24 days significantly de-creased in the inhibitor group(all P<0.05).There was no significant difference in tumor volume from 0 to 12 days among all groups(all P>0.05).Conclusions The differentially expressed gene miR-20a can regulate the cell cycle of GCSCs by negatively targeting CDKN1A expression,thus enhancing their tumorigenic ability.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Key words
microRNA-20a/gastric cancer stem cells/cyclin-dependent kinase inhibitor 1A/cell cycle/tumori-genic ability分类
临床医学引用本文复制引用
徐桂华,董志会,谭雪怡,申杰..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[J].山东医药,2025,65(3):58-63,6.基金项目
广东省医学科学技术研究基金项目(A2023232). （A2023232）
