miR-495-3p靶向BUB1调控STAT3信号通路对食管癌细胞生物学行为的影响  

miR-495-3P targets BUB1 to regulate the effect of STAT3 signaling pathway on biological behavior of esophageal cancer cells

作　　者：杨晖[1,2,3] 石宁 陈晓伟[1,2,3] 宋雪杰 周茜[1,2,3] 司富春 Yang Hui;Shi Ning;Chen Xiaowei;Song Xuejie;Zhou Xi;Si Fuchun(Traditional Chinese Medicine School,Henan University of Chinese Medicine,Zhengzhou 450046;Henan Key Laboratory of Traditional Chinese Medicine Syndrome and Prescription in Signaling,Zhengzhou 450046;Henan International Joint Laboratory of TCM Syndrome and Prescription in Signaling,Zhengzhou 450046;Medicine College of Henan University of Chinese Medicine,Zhengzhou 450046)

机构地区：[1]河南中医药大学中医学院,郑州450046 [2]河南省中医方证信号传导重点实验室,郑州450046 [3]河南省中医方证信号传导国际联合实验室,郑州450046 [4]河南中医药大学医学院,郑州450046

出　　处：《安徽医科大学学报》2024年第8期1446-1454,共9页Acta Universitatis Medicinalis Anhui

基　　金：河南省科技发展计划项目(编号:222102310187、242102310582);河南省博士后科研项目(编号:202103092);河南中医药大学苗圃项目(编号:MP2022-6);河南省大学生创新创业训练项目(编号:202210471009)。

摘　　要：目的 探讨miR-495-3p靶向苯并咪唑出芽抑制解除同源物蛋白1(BUB1)调控信号转导及转录激活因子3(STAT3)信号通路对食管癌细胞生物学行为的影响。方法 使用cDNA芯片技术筛选出食管癌组织和正常组织差异表达基因,并用生物信息学方法进行分析。运用TargetScan数据库对miRNA的靶基因进行预测,并用双荧光素酶报告基因检测技术进行验证。将KYSE150细胞分为空白对照组、NC mimics组和miR-495-3p mimics组。通过细胞计数试剂盒8(CCK-8)检测细胞增殖活力。用流式细胞术测定细胞周期和凋亡。通过定量逆转录聚合酶链式反应(RT-qPCR)测定BUB1 mRNA的表达水平。通过蛋白质印迹法(Western blot)测量BUB1、STAT3、磷酸化(p)-STAT3、细胞周期蛋白B1(CCNB1)、细胞周期蛋白依赖性激酶1(CDK1)、B淋巴细胞瘤-2(Bcl-2)、半胱氨酸蛋白水解酶3(Caspase-3)和半胱氨酸蛋白水解酶9(Caspase-9)蛋白水平。用划痕和Transwell小室实验测定细胞的迁移和侵袭能力。结果 差异表达基因参与生物学过程、信号通路和网络构建主要与细胞周期相关,BUB1是关键的核心(Hub)基因,miR-495-3p靶向调控BUB1。体外实验表明,过表达miR-495-3p能显着抑制食管癌细胞的生长、迁移和侵袭,诱导细胞凋亡和G2/M期阻滞。过表达miR-495-3p处理后,食管癌细胞中Caspase-3、Caspase-9表达量升高(P<0.01),而Bcl-2、BUB1、CCNB1、CDK1、p-STAT3表达量降低(P<0.01)。STAT3信号通路也被发现在此过程中发挥着重要作用。结论 miR-495-3p可能通过下调BUB1介导STAT3信号通路影响食管癌细胞的生物学行为。Objective To investigate the effect of miR-495-3p targeting budding uninhibited by benzimidazoles(BUB1)on signal transducer and activator of transcription 3(STAT3)signaling pathway on biological behavior of esophageal cancer(EC)cells.Methods The differentially expressed genes in EC tissues and normal tissues were screened by the cDNA microarray technique.The differentially expressed genes were analyzed by bioinformatics methods.The target genes of miRNAs were predicted by the TargetScan database and verified by a dual luciferase gene reporter assay.KYSE150 cells were divided into blank control group,NC mimics group and miR-495-3p mimics group.The activity of KYSE150 cells were detected by the CCK-8 method.Cell cycle and apoptosis were measured by flow cytometry.The expression of BUB1 mRNA was measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR).The levels of BUB1,STAT3,phosphor(p)-STAT3,cyclin dependent kinase 1(CCNB1),cyclin dependent kinase 1(CDK1),B-cell lymphoma-2(Bcl-2),cysteinyl aspartate-specific proteinase-3(Caspase-3)and cysteinyl aspartate-specific proteinase-3(Caspase-9)were measured by Western blot.The migration and invasion abilities of the cells were measured by wound-healing and Transwell invasion assays.Results Differentially expressed genes were involved in biological processes,signaling pathways and network construction,which were mainly related to cell cycle.BUB1 is the key(Hub)gene,and BUB1 is the target gene of miR-495-3p.In vitro experiments showed that overexpression of miR-495-3p could significantly inhibit the migration and invasion of EC cells and induce apoptosis and G 2/M phase arrest.After treatment with overexpression of miR-495-3p,the expression of Caspase-3 and Caspase-9 in EC cells increased significantly(P<0.01),while the expression of Bcl-2,BUB1,CCNB1,CDK1 and p-STAT3 decreased significantly(P<0.01).Moreover,the STAT3 signaling pathway might play an important role in this process.Conclusion miR-495-3p may influence the biologic

关 键 词：食管癌 miR-495-3p BUB1 STAT3信号通路 生物学行为 

分 类 号：R735.1[医药卫生—肿瘤]