Effect of miR-184 on proliferation of neural stem cells and its underlying mechanism
10.3760/cma.j.issn.1671-8925.2017.03.009
- VernacularTitle:miR-184对小鼠神经干细胞增殖的影响及其机制
- Author:
Fobao HUANG
1
;
Haoju ZHANG
;
Yugang LIU
;
Dan LUO
;
Ruxiang XU
;
Yiwu DAI
Author Information
1. 南方医科大学第二临床学院
- Keywords:
Micro-184;
Neural stem cell;
Numbl protein;
Notch signaling pathway;
Hesl protein;
Hes5 protein
- From:
Chinese Journal of Neuromedicine
2017;16(3):262-268
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect ofmiR-184 on proliferation of neural stem cells (NSCs) and its mechanisms in mice.Methods The pHBLV-U6-GFP-miR-184 over-expression plasmid and pHBLV-U6-GFP-miR-184 inhibitor plasmid were used to construct recombinant lentivirus.And the NSCs derived fiom subventricular zone of E14d CD1 mouse were confirmed by immunofluorescence assay.There were four groups that contain a miR-184 overexpression group,a miR-184 inhibitor group and two control groups.The NSCs which infected with lentiviral vectors were selected for puromycin resistance for 5-7 days,and then surviving cells were cultivated to three generations.The expression level ofmiR-184 was detected by real time-quantitative PCR (RT-qPCR).And the target genes ofmiR-184 were predicted through TargetScan,IRTarBase and MiRanda,and were confirmed by Western blotting and RT-qPCR.The cells in the four groups were culttared under proliferating conditions incorporated bromodeoxyuridine (BrdU) in cell proliferation analyses.The protein expressions of Hesl and Hes5,the target proteins of Notch signaling pathways,and their mRNA expressions were detected by Western blotting and RT-qPCR.Results There were 90％ of cells in each group expressing both Nestin and Sox2.The miR-184 level in the miR-184 overexpression group was 67.63±7.53 times of that of the control group,with significant difference (P＜0.05).The percent of BrdU+/DAPI+ of the miR-184 overexpression group was 1.47±0.05 times of that in the control group,with significant difference (P＜0.05);and the percent of BrdU+/DAPI+ of the miR-184 inhibitor group was 0.84±0.03 times of that in the inhibitor control group,with significant difference (P＜0.05).Numbl was a target gene ofmiR-184 indicated by IRTarBase and MiRanda.The miR-184 could inhibit Numbl protein expression;the Numbl protein expression level in the miR-184 overexpression group was 0.73±0.07times of that in the control group,and the Numbl protein expression level in the miR-184 inhibitor group was 1.30±0.05 times of that in the control group,with significant difference (P＜0.05);but miR-184 did not change the Numbl mRNA level.The miR-184 could activate Notch signaling pathway through inhibiting the Numbl protein expression,and increase the Hes1 and Hes5 protein and mRNA expression levels (P＜0.05).Conclusion The miR-184 promotes the NSCs proliferation through inhibiting the Numbl protein translation and further activating the Notch signaling pathway.
