长链非编码RNA KCNQ1DN对尿源干细胞的干性调控研究  

Role of long non-coding RNA KCNQ1DN in the regulation of stemness of urine derived stem cells

作　　者：吴清剑[1] 陈伟[1] 杨帆[2] 方针强[1] 赵江[1] 王青青[1] 杨振兴[1] 张远宁[1] 李龙坤[1] 

机构地区：[1]第三军医大学附属新桥医院泌尿外科,重庆400037 [2]第三军医大学附属新桥医院中心实验室

出　　处：《临床泌尿外科杂志》2017年第4期290-293,297,共5页Journal of Clinical Urology

基　　金：国家自然科学基金(编号81600588)

摘　　要：目的:探讨长链非编码RNA KCNQ1DN在尿源干细胞干性调控中的作用。方法:收集健康成人(n=6)尿液,提取尿源干细胞并传代培养,分别收集P3、P5和P7代尿源干细胞,并应用实时荧光定量PCR(RTPCR)检测KCNQ1DN基因表达水平。构建靶向干扰KCNQ1DN的慢病毒载体LV3-shKCNQ1DN,转染P2代尿源干细胞并培养96小时后荧光显微镜观察感染效率,RT-PCR检测KCNQ1DN的表达,CCK8法检测细胞的增殖,RT-PCR和Western blotting检测干性相关转录因子c-Myc、Nanog、Rex1的mRNA及蛋白的表达。结果:随着尿源干细胞传代代数的增加,KCNQ1DN的表达量逐渐升高(P<0.05)。shKCNQ1DN转染尿源干细胞后KCNQ1DN的表达显著降低(P<0.01),敲低尿源干细胞中KCNQ1DN的表达能够促进尿源干细胞的增殖并上调干性相关转录因子c-Myc、Nanog和Rex1mRNA及蛋白的表达。结论:随着尿源干细胞的生长,长链非编码RNA KCNQ1DN的表达逐步被激活,并对尿源干细胞的干性起到负性调控作用。Objective：To investigate the role of long non-coding RNA KCNQ1 DN in the regulation of stemness in urine derived stem cells（USCs）.Method：The urine samples of healthy adults（n=6）were collected for the isolation and culture of USCs.Then the P3,P5 and P7USCs were collected to detect the expression of KCNQ1 DN by real-time PCR.KCNQ1 DN was down-regulated by lentivirus vector LV3-shKCNQ1 DN in the P2 USCs.After 96 h,the transfection efficiency was observed by fluorescence microscope.CCK8 assay was used to evaluate the proliferation of USCs.The expression of KCNQ1 DN and stemness-related transcriptional factors cMyc、Nanog and Rex1 were detected by real-time PCR,and the protein expression level of c-Myc、Nanog and Rex1 were assessed by western blotting.Result：With the continuous culture of USCs,the expression of KCNQ1 DN was increasing（P〈0.05）.The expression of KCNQ1 DN down-regulated by LV3-shKCNQ1DN（P〈0.01）,and the expression of c-Myc、Nanog and Rex1up-regulated both in mRNA and protein level with the down regulation of KCNQ1 DN.Conclusion：With the continuous culture of USCs,KCNQ1 DN was activated progressively,and KCNQ1 DN may play a negative role in the regulation of the stemness in USCs.

关 键 词：尿源干细胞 干性 长链非编码RNA KCNQ1DN 

分 类 号：R69[医药卫生—泌尿科学]