摘要
目的探讨潜在microRNA靶向下调p300表达水平对人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells,h UC-MSCs)增殖的影响。方法通过双荧光素酶报告基因(Firefly/Renilla Luciferase)系统筛选出作用于p300 3'UTR的目标microRNA,mimic转染UC-MSCs后RT-q PCR检测miRNA表达水平的变化,Western blot检测p300蛋白水平变化,RT-q PCR和Western blot检测多潜能转录因子表达水平的变化,MTT法检测UC-MSCs增殖能力的变化,并比较克隆形成能力的差异。结果证实mir 26b-5p通过不完全互补配对结合p300的3'UTR区,显著降低了荧光素酶的相对活性(P<0.05);mir 26b-5p mimic转染后抑制p300蛋白表达水平(P<0.05),并下调Nanog基因和蛋白表达水平(P<0.05);mir 26b-5p mimic转染后UC-MSCs的增殖受到抑制(P<0.05),克隆形成能力下降(P<0.05)。结论 UC-MSCs高表达mir 26b-5p能够显著抑制p300蛋白表达和干性相关基因Nanog的表达,并且抑制UC-MSCs的增殖能力和克隆形成能力。
Objective To investigate the target microRNA and its effect on the down-regulation of transcriptional coactivator p300, and then on the proliferation of human umbilical cord derived mesenchymal stem cells （hUC-MSCs）. Methods Dual luciferase gene reporter system （Firefly/Renilla Luciferase） was used to screen the effective microRNA targets which interacted with p300 3＇ UTR. Then the hUC-MSCs were transfected with microRNA mimics, and the expression of p300 3＇ UTR at miRNA and protein levels was detected by RT-qPCR and Western blotting respectively. The levels of pluripotency transcription factors including Nanog, Oct4 and Sex2 were measured by RT-qPCR and Western blotting too. MTF assay was employed to detect the proliferation and colony formation in hUC-MSCs before and after the transfection of microRNA mimics. Results It was found that mir 26b-5p binding to p300 3＇ UTR with imperfectly matched sequence. The Firefly/Renilla＇ s relative activity of mir 26b-5p/pmirGLO-p300 3＇ UTR co-transfected group was decreased significantly （P 〈 0.05 ）. The protein expression level of p300 was significantly reduced after mir 26b-5p transfection compared with NC-mimic transfection （P 〈 0. 05 ）, and the mRNA and protein expression levels of Nanog were significantly down-regulated after the transfection （ P 〈 0.05 ）. The mir 26b-5p transfection also reduced the proliferation and colony formation of UC-MSCs （P 〈 O. 05 ）. Conclusion mir 26b-5p suppresses the protein expression levels of p300 and Nanog, a pluripotency transcription factor in UC-MSCs, and also inhibits the cell proliferation and colony formation capability.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2015年第16期1614-1618,共5页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81371718)
重庆市教委科学技术研究项目(KJ110301)~~
