摘要
目的探究长链非编码RNA(lncRNA)SRY相关高迁移率族盒蛋白2(SOX2)重叠转录本(OT)对食管癌细胞上皮-间充质转化(EMT)及干细胞特性的影响。方法采用实时荧光定量PCR法检测正常人食管上皮细胞(HET-1A)及食管癌细胞系(KYSE30、KYSE70和KYSE270)中lncRNA SOX2OT和miR-200b表达水平,并选择其中lncRNA SOX2OT表达水平最高的食管癌细胞系(KYSE30)进行后续实验。取处于对数生长期的KYSE30细胞,随机分为空白对照组、阴性对照(NC)-小干扰RNA(siRNA)组和SOX2OT-siRNA组,比较各组细胞中lncRNA SOX2OT、miR-200b、SOX2、EMT标志物、干细胞特性相关蛋白表达水平及干细胞成球能力。采用双荧光素酶报告基因实验验证lncRNA SOX2OT、miR-200b及SOX2的靶向调控关系。结果与HET-1A细胞相比,KYSE30、KYSE70、KYSE270细胞中lncRNA SOX2OT表达水平均显著升高(P均<0.05),且KYSE30细胞中lncRNA SOX2OT表达水平最高。与空白对照组和NC-siRNA组相比,SOX2OT-siRNA组细胞中lncRNA SOX2OT、SOX2、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、ATP结合盒亚家族G成员2(ABCG2)、醛脱氢酶1家族成员A1(ALDH1A1)、八聚体结合转录因子4(OCT4)表达水平均显著降低,而miR-200b、E-钙黏蛋白(E-cadherin)表达水平均显著升高,且细胞球形成效率显著降低,差异均有统计学意义(P均<0.05)。双荧光素酶报告基因实验检测结果显示,与SOX2OT/SOX2野生型+miR-200b NC组相比,SOX2OT/SOX2野生型+miR-200b mimic组细胞的荧光素酶活性显著减弱(P<0.05);SOX2OT/SOX2突变型+miR-200b NC组与SOX2OT/SOX2突变型+miR-200b mimic组细胞的荧光素酶活性差异无统计学意义(P>0.05)。结论食管癌细胞系中lncRNA SOX2OT表达水平显著升高,下调其表达可抑制食管癌细胞EMT,并减弱食管癌细胞的干细胞特性,其机制可能与miR-200b/SOX2轴有关。
Objective This paper is to investigate the effects of long non-coding RNA(lncRNA)SRY-related high mobility group box protein 2(SOX2)overlapping transcripts(OT)on epithelial-mesenchymal transition(EMT)and stem cell characteristics of esophageal cancer cells.Methods Real time fluorescence quantitative PCR was used to detect the expression levels of lncRNA SOX2OT and miR-200b in normal human esophageal epithelial cells(HET-1A)and esophageal cancer cell lines(KYSE30,KYSE70,and KYSE270),and the esophageal cancer cell line with the highest expression level of lncRNA SOX2OT(KYSE30)was selected for subsequent experiments.KYSE30 cells in logarithmic growth phase were selected and randomly divided into the blank control group,the NC-siRNA group,and the SOX2OT-siRNA group.The expression levels of lncRNA SOX2OT,miR-200b,SOX2,EMT markers,stem cell characteristic related proteins,and stem cell spheroid formation ability in each group of cells were compared.Dual luciferase reporter gene experiments were conducted to verify the targeted regulatory relationship of lncRNA SOX2OT,miR-200b,and SOX2.Results Compared with HET-1A cells,the expression levels of lncRNA SOX2OT in KYSE30,KYSE70 and KYSE270 cells are significantly increased(P<0.05),and the expression level of lncRNA SOX2OT is the highest in KYSE30 cells.Compared with the blank control and the NC-siRNA groups,the expression levels of lncRNA SOX2OT,SOX2,N-cadherin,Vimentin,ATP-binding box subfamily G member 2(ABCG2),aldehyde dehydrogenase 1 family member A1(ALDH1A1),and octamer-binding transcription factor 4(OCT4)in SOX2OT-siRNA group cells are significantly decreased.The expression levels of miR-200b and E-cadherin are significantly increased,and the efficiency of cell sphere formation is significantly decreased,with statistically significant differences(P<0.05).The dual-luciferase reporter gene assay shows that compared with the SOX2OT/SOX2 wild-type+miR-200b NC group,the luciferase activity in the SOX2OT/SOX2 wild-type+miR-200b mimic group is significantly decreased(P<0.05).There is no statistically significant difference in luciferase activity between the SOX2OT/SOX2 mutant+miR-200b NC group and the SOX2OT/SOX2 mutant+miR-200b mimic group(P>0.05).Conclusions The expression level of lncRNA SOX2OT in esophageal cancer cell lines is significantly increased.Down-regulation of its expression can inhibit EMT in esophageal cancer cells and weaken the characteristics of esophageal cancer stem cells.The mechanism may be related to the miR-200b/SOX2 axis.
作者
王立平
田笑
WANG Liping;TIAN Xiao(Department of Health Medicine,General Hospital of Northern Theater Command,Shenyang 110000,China;Department of Clinical Laboratory,General Hospital of Northern Theater Command,Shenyang 110000,China)
出处
《国际消化病杂志》
2025年第4期225-230,236,共7页
International Journal of Digestive Diseases
