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

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(均P<0.05);转染HA-SORBS1或antimiR-223-3p后,HCT116、SW620细胞中SORBS1表达水平均上调,细胞活力均下降(均P<0.05)。双荧光素酶报告分析结果显示:共转染SORBS13'-非翻译区(untranslated region,UTR)野生型质粒和miR-223-3p mimic的细胞荧光素酶活性显著低于共转染SORBS13'-UTR野生型质粒和miR-NC的细胞(P<0.05)。与共转染miR-223-3p mimic和HA的细胞比较,共转染miR-223-3p mimic和HA-SORBS1的细胞活力明显降低(P<0.01);与共转染anti-miR-223-3p和siNC组比较,共转染anti-miR-223-3p和siSORBS1组细胞活力明显增加(P<0.05)。结论:MiR-223-3p通过靶向SORBS1基因增加CRC细胞对5-FU的耐药性,miR-223-3p有望成为临床治疗CRC的新靶点。

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 gene for further study.Three online bioinformatics databases predicted miRNAs targeting SORBS1 and obtained an intersection molecule miR-223-3p.After treatment with 5-FU(25μmol/L)for 12−36 h,the levels of miR-223-3p in HCT116 and SW620 cells were significantly down-regulated(all P<0.05).After transfection with siSORBS1 or miR-223-3p mimic,the expression levels of SORBS1 in HCT116 and SW620 cells were downregulated,and the cell viability was increased(all P<0.05).After transfection with HASORBS1 or anti-miR-223-3p,the expression levels of SORBS1 in HCT116 and SW620 cells were up-regulated,and the cell viability was decreased(all P<0.05).The result of dual luciferase reporter gene assay showed that the luciferase activity of cells co-transfected with SORBS13'-UTR wild plasmid and miR-223-3p mimic was significantly lower than that of the 3'-UTR wild plasmid and miR-NC cells(P<0.05).Compared with cotransfection with miR-223-3p mimic and HA,the cell viability of cells co-transfected with miR-223-3p mimic and HA-SORBS1 was decreased significantly(P<0.01).Compared with the co-transfected anti-miR-223-3p and siNC,the cell viability of the co-transfected anti-miR-223-3p and siSORBS1 was significantly increased(P<0.05).Conclusion:MiR-223-3p increases 5-FU resistance in CRC cells by targeting SORBS1,and miR-223-3p is expected to become a new target for clinical treatment of CRC.