稳定过表达环状RNA circ-Fam114a2膀胱癌T24细胞株的建立  

Construction of lentiviral vector-induced circ-Fam114a2 overexpression in bladder cancer T24 cells

作　　者：王敬梓 韩杰 于浩[1] 周锐 卢泓成 杨潇[1] 李鹏超[1] 吕强[1] WANG Jing-zi;HAN Jie;YU Hao;ZHOU Rui;LU Hong-cheng;YANG Xiao;LI Peng-chao;LV Qiang(Department of Urology,The First Affiliated Hospital of Nanjing Medical University,Nanjing 210029, China)

机构地区：[1]南京医科大学第一附属医院泌尿外科

出　　处：《现代泌尿外科杂志》2019年第6期474-479,共6页Journal of Modern Urology

基　　金：国家自然科学基金(No.81772711,81602235);江苏省“333”工程(No.LGY201600);江苏省重点人才计划(No.ZDRCA201600);江苏省高等学校重点学科建设(No.JX10231801);江苏省优秀学科省级启动计划(No.BE2016791)

摘　　要：目的利用慢病毒载体建立稳定过表达环状RNAcirc-Fam114a2的膀胱癌T24细胞株。方法利用pLVX-CMV-minicrRNA-EF1-GFP-Puro质粒,制备慢转录病毒pLVX-CMV-minicrRNA-EF1-GFP-Puro-circ-Fam114a2(circ-Fam114a2)和pLVX-CMV-minicrRNA-EF1-GFP-Puro-Vector(Vector),并转染膀胱癌T24细胞系。用嘌呤霉素筛选阳性表达细胞,并用荧光显微镜,实时荧光定量PCR(qRT-PCR)和Northern印记等实验验证circ-Fam114a2基因成环及过表达情况。最后,通过CCK-8实验和克隆形成实验初步探究circ-Fam114a2对T24细胞增殖的影响。结果转染并用嘌呤霉素药筛后的T24细胞在荧光显微镜下呈现出绿色荧光。qRT-PCR显示过表达组细胞circ-Fam114a2水平是对照组的28倍,而Fam114a2mRNA没有明显变化。Northern印迹显示circ-Fam114a2在T24细胞中成功成环并过表达。CCK-8实验和克隆形成实验表明过表达circ-Fam114a2显著抑制T24细胞增殖。结论该实验成功建立了稳定高表达circ-Fam114a2的膀胱癌T24细胞模型,并初步探究circ-Fam114a2对膀胱癌细胞的增殖抑制作用。为进一步研究circ-Fam114a2在膀胱癌中的作用和机制奠定了基础。Objective To establish bladder cancer T24 cells with stable overexpression of circ-Fam114a2. Methods The pLVX-CMV-minicrRNA-EF1-GFP-Puro plasmid was used to construct lentiviruses with pLVX-CMV-minicrRNA-EF1-GFP-Puro-circ-Fam114a2 (circ-Fam114a2) or pLVX-CMV-minicrRNA-EF1-GFP-Puro-Vector (Vector).Lentiviruses were transfected into bladder cancer T24 cells.The fluorescence intensity of GFP and expression of circ-Fam114a2 were measured with fluorescence microscope,qRT-PCR assays and Northern blot.The effects of circ-Fam114a2 overexpression on T24 cell proliferation were determined with CCK-8 assay and colony formation assay. Results After puromycin treatment,the transfected T24 cells showed green fluorescence under a fluorescence microscopy.The qRT-PCR results showed that the expression level of circ-Fam114a2 in experimental group was 28 folds of that in control group,whereas Fam114a2 mRNA was not changed significantly.Northern blot showed that circ-Fam114a2 was successfully overexpressed in T24 cells.CCK-8 assay and colony formation assay showed that circ-Fam114a2 overexpression significantly inhibited the proliferation of T24 cells. Conclusion This study successfully established stable overexpression of circ-Fam114a2 bladder cancer T24 cells and preliminarily explored the inhibitory effects of circ-Fam114a2 on the proliferation of bladder cancer cells,which paved the way for further study on the role and mechanism of circ-Fam114a2 in bladder cancer.

关 键 词：circ-Fam114a2 环状RNA 转染 膀胱癌 

分 类 号：R737.14[医药卫生—肿瘤]