Effects Of MiR-184 On Proliferation And Differentiation Of Neural Stem Cells And Its Underlying Mechanisms

BackgroundNeural stem cells(Neural Stem Cells,NSCs)is a class of cell that have the ability of proliferation and differentiation,and the NSCs main exists in the Subventricular zone of lateral(SVZ)and the subgranular zone of hippocampus(SGZ).Research show that NSCs have the ability of regenerating and repairing central nervous system injury.But we can not well-understand currently the mechanisms of the proliferation and differentiation of NSCs.So it is necessary to further inquiry the mechanism of proliferation and differentiation of NSCs.MicroRNA(miRNAs)constitute a big family of small,approximately～22 nucleotide-long,non-coding RNAs that mediating post-transcriptional gene suppression by binding to the 3’ untranslated regions of target mRNAs.And inhibiting the translation process of target mRNAs or contribute to the degradation of the mRNA.The single-strand miR 184-3P is the mature form of miR 184 in multicellular organism.But the role of miR 184 in embryonic nervous system has less been studied.Here,we provide testimony to state that how the miR-184 controls the proliferation and differentiation of NSCs by overexpressing or inhibiting miR-184 in NSCs through constructing the Lentiviral vectors with pHBLV-U6-miR-184/miR 184-shRNA-ZsGreen plasmid.Purposes1.To construct the lentiviral vector with miR-184 gene overexpression or interference.2.To predict and verify the target gene of miR-184.3.To investigate the effect of miR-184 on proliferation and differentiation of NSCs and its underlying mechanisms.4.To investigate the interaction mechanisms of miR-184 and Notch signaling pathway on proliferation and differentiation of NSCs.Methods1.To amplify miR-184 and miR-184-shRNA gene by RT-PCR,and clone it into the pHBLV-U6-Scramble-ZsGreen carrier,then to construct Lentiviral vector with the carrier.And to culture and identificate the NSCs that were collected at E14 from pregnant mice.2.Then the cells were divide into four groups contain miR-184 overexpression group,miR-184 inhibition group and control groups,then adding lentivirus in four groups.Neurospheres infected with lentivirus vectors were selected for Puromycin resistance by culturing in NSC culture media that contains 3μg/ml Puromycin.Then continue to culture the surviving cells for less than 2 times to use directly in experiments.And measure the expression levels of miR-184-3p of each group by RT-qPCR.3.5-bromo-2-deoxyuridine(BrdU)cell cycle analysis was performed on proliferating cells.And recording the Brdu+/DAPI+ ratio of each group by using confocal microscopy.Collecting 3th cells of each group,and inoculating cells in differentiation media for 7d,then detecting and recording the MAP2+/DAPI+ ratio through confocal microscopy.Measuring the expression level of NeuN protein、Hes1 protein and Hes5 protein and their mRNA by using Western blotting and RT-qPCR.4.Predicting that the Numbl gene is one of potential target gene of the miR-184 through targetScan,iRTarBase,miRanda and other software.Then to validate the target gene of miR-184 by Western blotting method and RT-qPCR,and further verifying it by luciferase reporter gene system assays.Results1.The study results demonstrated that lentivirus vector with miR-184/miR 184-shRNA gene was successfully constructed.And immunofluorescence results showed that there are 90%cells that express Nestin and Sox2 protein that NSCs markers,and that state that cells meet the requirements of following experiments.2.There are 80%Cells that express green fluorescent protein(GFP)after infecting with Lentiviral for 72h in each group.Then changing the fresh media with puromycin.Then continue to culture survival cells,and the cells have a good growth.The RT-qPCR results show that the expression level of miR-184 of overexpression group is 67.63 ± 7.53 times than that of the control group.And the difference was statistically significant.3.Brdu cell infiltration experiments showed that the Brdu+/DAPI+ ratio of miR-184 overexpression group be increase significantly and that of miR-184 interference group be decrease significantly.The Brdu+/DAPI+ ratio of miR-184 overexpression group is 1.47 ± 0.05 times than that of control group,and that of interference group is 0.84 ± 0.03 times than control group.The MAP2+/DAPI+ ration of miR-184 overexpression group is lower than that of the control group,on thecontrary,the MAP2+/DAPI+ ration of miR-184 interference group is bigger than that of the control group.Comparing with the control group,the NeuN protein and their mRNA expression level of miR-184 overexpression groups be reduce significantly,and NeuN protein and their mRNA expression level of the miR-184 interference group be increase significantly.The study show that the expression level of Hes1 and Hes5 protein that the target gene of Notch signaling pathway of miR-184 overexpression group be increase significantly,comparing with the control group,by using Western blotting method,on the contrary,those protein expression level of miR-184 interference group be reduce significantly.In addition,the paper show same results on mRNA expression level by using RT-qPCR.4.The research state that the Numbl gene is one potential target genes of miR-184 by using miRanda and iRTarBase software.Comparing with the control group,the Numbl protein expression level of miR-184 overexpression groups be reduce significantly,and Numbl protein expression level of the miR-184 interference group be increase significantly.In addition,comparing with the control group,the difference of Numbl mRNA expression level of miR-184 overexpression group is not statistically significant(t=0.593,P=0.613).So,the paper show that Numbl is a potential target gene of miR-184,and miR-184 can inhibit the Numbl protein translation,without changing the Numbl mRNA expression levels.And the conclusion is verified by luciferase reporter gene system assays.