MicroRNA-26b Represses Pluripotency And Promotes Mesendodermal Differentiation Of Mouse Embryonic Stem Cells

Objective To investigate the regulatory role of miR-26 b in the pluripotency and differentiation of mouse embryonic stem cells(m ESCs).Methods1.Real-time quantitative PCR was used to detect the expression of miR-26 cluster precursors in m ESCs and MEF.2.Real-time quantitative PCR was used to detect the expression of miR-26 b during the formation of embryoid body(EB),and the expression of miR-26 b in m ESCs and MEF was compared.3.CCK-8 assay was used to detect the effect of miR-26 b on the proliferation ability of m ESCs.4.Flow cytometry was used to detect the regulatory effect of miR-26 b on cell cycle and apoptosis of m ESCs.5.Alkaline phosphatase(ALP)staining kit was used to detect the regulatory effect of miR-26 b on ALP activity in m ESCs.6.Real-time quantitative PCR was used to detect the effect of miR-26 b on the m RNA expression of pluripotent transcription factors(Oct4,Sox2 and Nanog)in m ESCs.7.Western blot(WB)was used to detect the effects of miR-26 b on the protein expression of three transcription factors in m ESCs.8.To observe the morphology of m ESCs after transfecting pc DNA-pre-miR-26 b in vitro.9.Real-time quantitative PCR was used to detect the effect of miR-26 b on the expression of germ layer markers of m ESCs that induced by retinoic acid(RA)and activin A(AA).10.Immunofluorescence was used to detect the effect of miR-26 b on mesoderm and endoderm markers(?-SMA and GATA4)during m ESCs differentiation.11.Real-time quantitative PCR was used to detect the expression of miR-26 b precursors in the typical tissues and organs that derived from the three germ layers.Results1.In m ESCs,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).2.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 m ESCs.3.Mi R-26 b overexpression repressed m ESCs proliferation and arrested cell cycle in G2 phase.Mi R-26 b did not affect m ESCs apoptosis.4.Alkaline phosphatase(ALP)staining showed that miR-26 b decreased the activity of ALP in m ESCs.5.Mi R-26 b overexpressing m ESCs reduced expression of pluripotency markers(Oct4,Sox2 and Nanog).6.The effect of miR-26 b on m ESCs differentiation was studied using an in vitro differentiation model.Differentiation was observed as early as day 4 of miR-26 b overexpression,where the m ESCs cluster started flattening out and detaching from the edges of the colonies.Subsequently,most colonies lost their typical monotype appearance and formed three-dimensional structures with different cell morphologies.7.RA treatment increased the expression of Nestin,an ectodermal marker in m ESCs.The induction was repressed in the presence of miR-26 b.In contrast,miR-26 b promoted the expression of AA-induced mesendoderm markers,including ?-SMA and Gata4.8.?-SMA-positive cells were present in miR-26 b overexpressing m ESCs colonies,which accounted for ~50% of cell population after 6 days.Gata4 was also greatly induced by miR-26 b induction.9.Mi R-26 b precursor was highly expressed in the organs mainly derived from mesoderm and endoderm,especially in intestine,stomach and liver,and relatively low expressed in ectoderm-derived tissues such as brain and spinal cord.Conclusion This study demonstrated that miR-26 b was a key miR-26 family member in m ESCs differentiation.Mi R-26 b inhibited the pluripotency and self-renewal.Mi R-26 b promoted the mesendodermal differentiation of m ESCs.