摘要
目的明确miRNA-26b(miR-26b)对小鼠胚胎干细胞(mESCs)多能性的调控作用。方法实时荧光定量PCR检测miR-26簇前体在mESCs和小鼠胚胎成纤维细胞(MEF)中的表达。实时荧光定量PCR检测胚状体(EB)形成过程中成熟miR-26b的表达。CCK-8方法检测miR-26b对mESCs增殖能力的影响。流式细胞术测定miR-26b对mESCs细胞周期与凋亡的影响。使用碱性磷酸酶染色试剂盒检测miR-26b对mESCs中碱性磷酸酶活性的影响。实时荧光定量PCR检测miR-26b对mESCs中多能性转录因子(Oct4、Sox2和Nanog)mRNA表达的影响。使用蛋白质印迹法检测miR-26b对mESCs中3个关键转录因子的蛋白表达影响。用体外模型观察转染pcDNA-pre-miR-26b对mESCs形态的影响。结果 mESCs中,miR-26b的前体在miR-26簇群表达最高。MEF中所有miR-26前体均上调,其中pre-miR-26b上调幅度最大(上调约10倍)。成熟的miR-26b在EB形成过程中从第2天到第6天明显上调,并且成熟miR-26b在MEF中的表达水平明显高于其在mESCs中的表达水平。miR-26b的过表达抑制mESCs增殖,并显示miR-26b过表达诱导mESCs于细胞周期的G1期阻滞,miR-26b不影响mESCs的凋亡。ALP染色结果表明miR-26b导致mESCs中ALP活性降低。过表达miR-26b导致mESCs中多能性标志物(Oct4、Sox2和Nanog)的表达水平降低。体外模型研究miR-26b对mESCs分化的影响,在miR-26b诱导的第4天观察到了分化的细胞。结论此研究证实miR-26b对mESCs多能性的调控作用。
Objective To clarify the regulatory role of miRNA-26 b(miR-26 b) in the pluripotency of mouse embryonic stem cells(mESCs).Methods Real-time quantitative PCR was used to detect the expression of miR-26 cluster precursors in mESCs and MEF.Real-time quantitative PCR was used to detect the expression of miR-26 b during the formation of embryoid body(EB).CCK-8 assay was used to detect the effect of miR-26 b on the proliferation ability of mESCs.Flow cytometry was used to detect the regulatory effect of miR-26 b on cell cycle and apoptosis of mESCs.Alkaline phosphatase staining kit was used to detect the regulatory effect of miR-26 b on ALP activity in mESCs.Real-time quantitative PCR was used to detect the effect of miR-26 b on the mRNA expression of pluripotent transcription factors(Oct4, Sox2 and Nanog) in mESCs.Western blot was used to detect the effects of miR-26 b on the protein expression of three transcription factors in mESCs.The morphology of mESCs after transfecting pcDNA-pre-miR-26 b in vitro was observed. Results In mESCs, the expression of pre-miR-26 b was highest among all members in miR-26 clusters. All miR-26 precursors were up-regulated in differentiated mouse embryonic fibroblasts(MEF), pre-miR-26 b showed the most significant increase among the three members(about 10-fold increase).Mature miR-26 b was significantly up-regulated from day 2 to day 6 during embryonic body(EB) formation, and the expression level of mature miR-26 b in MEF was significantly higher than that in mESCs.MiR-26 b overexpression repressed mESCs proliferation and arrested cell cycle in G1 phase. MiR-26 b did not affect mESCs apoptosis.Alkaline phosphatase staining showed that miR-26 b decreased the activity of ALP in mESCs.MiR-26 b overexpressing mESCs reduced expression of pluripotency markers(Oct4, Sox2 and Nanog).The effect of miR-26 b on mESCs differentiation was studied using an in vitro differentiation model. Differentiation was observed as early as day 4 of miR-26 b overexpression.Conclusion This study confirms the regulatory effects of miR-26 b on mESCs pluripotency.
作者
曹靓
金瑞
孟祥云
王浩浩
李天立
刘小燕
王康林
吴繁荣
臧洪梅
Cao Liang;Jin Rui;Meng Xiangyun(School of Pharmacy,Anhui Medical University,Hefei 230032;Dept of Pharmacy,The Second Peoplers Hospital of Hefei 9 Hefei 230011)
出处
《安徽医科大学学报》
CAS
北大核心
2020年第7期997-1001,共5页
Acta Universitatis Medicinalis Anhui
基金
安徽省自然科学基金项目(编号:1708085QH96)
合肥市科技重大专项(编号:ZR201711290008)。
