结肠癌耐药细胞株LoVo/5-Fu中microRNA表达的初步研究  

Preliminary study of microRNA expression in colon cancer drug-resistant cell line LoVo/5-Fu

作　　者：何冬雷[1] 谢小明[1] 刘玉[1] 夏立平[1] 许荣华[1] 

机构地区：[1]海南医学院附属医院胃肠肿瘤外科,海南海口570102

出　　处：《中国现代医学杂志》2013年第18期31-35,共5页China Journal of Modern Medicine

基　　金：海南省卫生厅基金课题(No:琼卫2011-32)

摘　　要：目的建立结肠癌细胞耐药模型LoVo/5-FU并初步筛选可能的耐药相关的microRNA。方法采用5-FU浓度递增法建立人结肠癌细胞耐药模型LoVo/5-FU,观察其生长规律并绘制细胞生长曲线;用MTT法鉴定耐药细胞株耐药性并计算耐药指数(RI);用microRNA芯片技术检测耐药细胞株LoVo/5-FU与其亲本细胞株LoVo中mi-croRNA的表达谱,筛选差异表达的microRNA;用实时荧光定量PCR方法对筛选出的部分差异microRNA在耐药细胞及其亲本细胞中的表达情况进行验证。结果 LoVo/5-FU细胞与LoVo细胞相比,生长缓慢,细胞体积增大,耐药株LoVo/5-FU细胞相对于其亲本LoVo细胞的耐药倍数为8.08。microRNA芯片的结果显示,耐药株LoVo/5-FU细胞与亲本LoVo细胞比较,有10个microRNA表达上调,13个microRNA表达下调;实时荧光定量pcr的结果进一步证实mir-210、mir196a、mir-1281在LoVo/5-FU中显著上调,而let-7f、mir-1228显著下调,其中mir-210在LoVo/5-FU与LoVo中的表达有明显差异。结论 LoVo/5-FU细胞株耐药性稳定,筛选获得的差异miRNA可能通过调控其靶基因而参与人结肠癌细胞对5-Fu的耐药,为进一步研究microRNA在结肠癌耐药机制中的作用奠定基础。To construct the LoVo/5-FU drug-resistance cell model of human colon cancer and to screen potential drug-resistance related microRNA.【Methods】LoVo/5-FU drug-resistance cell model has been established by exposing the LoVo cells in gradually increased concentration of 5-FU,observed the cell growth and portrayed the growth curve.The drug resistance of LoVo/5-FU was measured by MTT assay and calculated the drug resistance index.The microRNA expression difference between LoVo/5-FU cell line and the parent cell line LoVo was tested by microarray and selected several drug resistance related microRNA candidates,and further confirm them by Q-PCR.【Results】By contrast with parent cell line,the LoVo/5-FU cell grows slowly,shows larger cell body morphology and has 8.08 fold drug resistance.Microarray suggest,by contrast with parent LoVo cell line,10 microRNA are up-regulated and 13 microRNA are down-regulated.Q-PCR further confirmed mir-210,mir196a,mir-1281 are up-regulated dramatically,but let-7f,mir-1228 are down-regulated markedly,and mir-210 expression is markedly different between LoVo/5-FU and LoVo.【Conclusions】The drug resistance of LoVo/5-FU cell line is stable.The selected candidate miRNA of human colon cancer cell probably regulate their target gene to involve in the resistance to 5-FU.Taking together,this work provides a good foundation for basic research of the function of microRNA in the drug resistance mechanism of human colon cancer cell.

关 键 词：结肠癌 耐药相关microRNA 5-氟尿嘧啶 LOVO 5-FU细胞株 microRNA芯片 

分 类 号：R735.35[医药卫生—肿瘤]