摘要
目的研究LIN28B对人乳腺癌细胞(MCF-7)肿瘤干细胞的干性状态维持的作用机制。方法在MCF-7细胞中瞬时过表达LIN28B,用q PCR和Western blot检测干性相关基因SOX2、NAONG、c-Myc和OCT4的表达;用流式细胞仪检测CD44high/CD24low细胞亚群的比例;利用体外成球培养检测肿瘤干细胞自我更新的能力,通过RNA第二代测序技术检测在LIN28B过表达后MCF-7细胞中miRNAs的表达差异,通过转染差异表达的miRNA,检测其对MCF-7细胞肿瘤干细胞比例和体外成球能力的影响。结果转染p-CMV6-LIN28B后能明显提高LIN28B的mRNA和蛋白水平(P<0.001);过表达LIN28B后MCF-7细胞中SOX2、NANOG和OCT4在RNA和蛋白水平表达都明显增高(P<0.05);CD44high/CD24low细胞比例由1.71%上升至11.60%(P<0.05);体外成球培养发现成球细胞数量明显增多(P<0.01);通过RNA测序发现有16个miRNAs在LIN28B过表达后差异表达倍数在2倍以上且P<0.05,LIN28B过表达后miR-92b-5p表达显著上调(P<0.01);转染miR-92b-5p mimic后MCF-7细胞的CD44high/CD24low细胞比例由1.14%上升至3.59%(P<0.05),体外成球能力明显增强(P<0.01)。结论 LIN28B能维持MCF-7肿瘤干细胞的干性状态,其作用机制可能是通过miRNAs的表达来实现的。
Objective To investigate the effect of LIN28B on maintaining the stemness of cancer stem cells in MCF-7 breast cancer cell line.Methods LIN28B was instantaneously overexpressed in MCF-7 cells （LIN28B-MCF-7）.The expression of stemness related genes in both mRNA and protein level was detected by either qPCR or Western blot;the proportion of CD44high/CD241ow cells was quantitated by flow cytometry;tumorsphere forming ability was analyzed by in vitro experiment;the differential expression of miRNAs either in LIN28B overexpression cells or wild type cells was detected by miRNA-sequencing or real-time PCR.The ability of tumorspheres forming was detected after transfecting miRNA mimics.Results The expression of LIN28B significantly increased in MCF-7 cells that transfected p-CMV6-LIN28B （P ＜ 0.001）.The expression of either mRNA and protein of SOX2,NANOG,c-Myc and OCT4 were up-regulated （P ＜ 0.05）;the proportion of CD44high/CD24low cells increased from 1.71％ to 11.60％ （P ＜ 0.05）;the number of tumorspheres was significantly improved （P ＜ 0.01） in LIN28B-MCF-7 cells;16 miRNAs of differential expression were found by miRNA-sequencing in LIN28B-MCF-7 cells comparing to wild type cells.The proportion of CD44high/CD24low cells was increased from 1.14％ to 3.59％ （P ＜ 0.05）,and the ability of tumorsphere forming was significantly increased in MCF-7 cells after transfecting miR-92b-5p mimic （P ＜ 0.01）.Conclusions LIN28B keeps the stemness of cancer stem cells by regulating miRNAs in MCF-7 cell line.
出处
《基础医学与临床》
CSCD
2015年第6期754-760,共7页
Basic and Clinical Medicine
基金
国家重点基础研究发展计划(2013CB967202)
