MiR-223-3p通过靶向SORBS1增加结直肠癌细胞对5-氟尿嘧啶的耐药性  

MiR-223-3p increases resistance of colorectal cancer cells to 5-fluorouracil via targeting SORBS1

作　　者：林依琳 王文波[1] 王雅[1] 付凯 LIN Yilin;WANG Wenbo;WANG Ya;FU Kai(Institute of Molecular Precision Medicine,Xiangya Hospital,Central South University,Changsha 410008;National Clinical Research Center for Geriatric Disorders,Xiangya Hospital,Changsha 410008,China)

机构地区：[1]中南大学湘雅医院精准分子医学研究所,长沙410008 [2]国家老年疾病临床医学研究中心(湘雅医院),长沙410008

出　　处：《中南大学学报（医学版）》2023年第3期356-368,共13页Journal of Central South University ：Medical Science

基　　金：湖南省自然科学基金(2021JJ20094)。

摘　　要：目的:5-氟尿嘧啶(5-fluorouracil,5-FU)是结直肠癌(colorectal cancer,CRC)的一线治疗药物,CRC细胞对5-FU的耐药是导致化学治疗(以下简称“化疗”)失败的主要原因,然而耐药机制仍不明确。本研究旨在探究参与CRC细胞对5-FU耐药的抑癌基因,并寻找对该基因存在调控作用的微RNA(microRNA,miRNA)。方法:在高通量基因表达(Gene Expression Omnibus,GEO)数据库中下载CRC数据集GSE28702和GSE69657,分别分析2个数据集中对FOLFOX化疗方案应答组和无应答组患者的差异表达基因并确定目标基因;采用在线生物信息学数据库TargetScan、miRwalk和miRDB预测靶向目标基因含山梨醇和SH3结构域蛋白1(sorbin and SH3 domain containing 1,SORBS1)的miRNA。采用瞬时转染技术在CRC细胞系HCT116、SW620中分别转染siSORBS1、HA-SORBS1、miR-223-3p mimic、anti-miR-223-3p及其相应的阴性对照(siNC、HA、miR-NC、anti-miR-NC),在HCT116细胞中共转染miR-NC和HA、miR-223-3p mimic和HA、miR-223-3p mimic和HA-SORBS1、anti-miR-NC和siNC、anti-miR-223-3p和siNC、anti-miR-223-3p和siSORBS1。采用实时反转录PCR(real-time reverse transcription PCR,real-time RT-PCR)和/或蛋白质印迹法检测细胞中SORBS1和miR-223-3p的表达水平。转染后用不同浓度的5-FU处理细胞。采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法检测细胞活力,双荧光素酶报告分析验证miR-223-3p与SORBS1的靶向关系。结果:GSE69657数据集和GSE28702数据集应答组分别有409和528个高表达基因,共有22个高表达交集基因。在22个高表达交集基因中,与CRC化疗敏感性有关的抑癌基因有3个,选择SORBS1作为目标基因进一步探索。3个在线生物信息学数据库预测的靶向SORBS1的miRNA为miR-223-3p。用5-FU(25μmol/L)处理HCT116、SW620细胞12~36 h后,2种细胞中miR-223-3p的表达水平均显著下调(均P<0.05)。转染siSORBS1或miR-223-3p mimic后,HCT116、SW620细胞中SORBS1表达水平均下调,细胞活力均增加(Objective:5-Fluorouracil(5-FU)is the first-line drug for treating colorectal cancer(CRC),and the resistance of tumor cells to 5-FU is the main cause of chemotherapeutic failure.However,the resistant mechanism is still unclear.This study aims to explore the tumor suppressor genes involved in 5-FU resistance in CRC,and to find the microRNA(miRNA)that regulates these genes.Methods:CRC data sets GSE28702 and GSE69657 were downloaded from Gene Expression Omnibus(GEO)database,and gene expression profiles of patients in the FOLFOX chemotherapeutic response group and the non-response group were analyzed,and differential expression genes were identified between the 2 groups.Target gene was then selected.Online bioinformatics databases TargetScan,miRwalk,and miRDB were used to predict miRNA targeting the interested gene sorbin and SH3 domain containing 1(SORBS1).siSORBS1,HA-SORBS1,miR-223-3p mimic,anti-miR-223-3p,and their corresponding negative controls(siNC,HA,miR-NC,and anti-miR-NC)were transfected into CRC cell lines of HCT116 and SW620 by transient transfection technique,respectively.Co-transfection was done with miRNA and plasmid(miR-NC+HA,miR-223-3p mimic+HA,or miR-223-3p mimic+HA-SORBS1)or anti-miRNA and siRNA(anti-miRNC+siNC,anti-miR-223-3p+siNC,or anti-miR-223-3p+siSORBS1)in HCT116 cells.Realtime reverse transcription PCR(real-time RT-PCR)and/or Western blotting were used to detect the expression levels of SORBS1 and miR-223-3p in cells.After transfection,the cells were treated with different concentrations of 5-FU,and the cell viability was detected by methyl thiazolyl tetrazolium(MTT)method.The targeting relationship between miR-223-3p and SORBS1 was comfirmed by dual luciferase reporter gene assay.Results:There were 409 and 528 highly expressed genes in the FOLFOX chemotherapeutic response group of GSE69657 and GSE28702,respectively.There were 22 overlapping genes in the response group,among which exist 3 tumor suppressor genes might be involved in chemosensitivity in CRC,and SORBS1 was selected as the target

关 键 词：结直肠癌 含山梨醇和SH3结构域蛋白1 微RNA-223-3p 5-氟尿嘧啶 化学治疗 耐药 

分 类 号：R735.34[医药卫生—肿瘤]