they give rise to all the other major brain cell types , which include oligodendrocytes , astrocytes , and neurons . however , molecular pathways , which are complicated and control the proliferation and differentiation of nscs , have been characterized incompletely to date . there is multiple indirect evidence suggesting a critical role of mirnas in nscs [ 47 ] . post - transcriptional control mediated by mirnas was identified as a further important regulation level , especially in neural proliferation where the action of these negative modulators of gene expression is pervasive . mirnas are small single - stranded non - coding rnas molecules which participate in the transcriptional regulation of eukaryotic genes and produce biological effects through inhibiting translation or destabilizing target mrnas [ 1012 ] . mirnas act as vital adjusters in multiple biological processes , including cell metabolism , differentiation , proliferation , and apoptosis . mir-126 is highly enriched in endothelial cells and previous studies found that mir-126 plays critical roles in vascular integrity and can promote angiogenesis during embryonic development [ 1518 ] . reported that the expression level of mir-126 was downregulated after spinal cord injury and that it can promote angiogenesis and attenuate inflammation in rats . however , the effects of mir-126 in nscs is still unclear ; therefore , we intended to study its role in nscs proliferation and survival . the immortalized human nsc lines hb1.f3 and hb1.a4 were maintained with dulbecco s modified eagle s medium ( dmem , sigma ) containing 10% fetal bovine serum ( fbs ) . total rna was extracted from cell lines with a mirvanamirna isolation kit ( applied biosystems , foster city , ca , usa ) according to the manufacturer s instructions . the expression level of mir-126 was detected by qrt - pcr according to the taqman mirna assays protocol ( applied biosystems ) and normalized by u6 small nuclear rna ( rnu6b ; applied biosystems ) with the 2 method . lv3-pglv - h1-gfp+puro plasmids with mir-126 mimics and negative control ( lv - mir-126 and lv - mir - nc ) were purchased from genepharma ( shanghai , china ) . hb1.f3 and hb1.a4 stably expressing mir-126 were established by transfecting lentivirus according to the manufacturer s instructions . cells were plated in 96-well plates in triplicate at approximately 1000 cells per well and cultured in the growth medium . cells were then treated with the indicated reagent and the numbers of cells per well were measured by the absorbance ( 450 nm ) of reduced water - soluble tetrazolium salt ( wst ) at the indicated time points . cells were plated into 6-well plates at a density of 1000 cells / well in 2 ml medium . the colonies were observed using a phase - contrast microscope at a magnification of 4 ( we counted the colonies containing at least 50 cells ) . cells were transfected with mir-126 mimics or their respective controls and were harvested after transfection at 48 hours and then marked with the annexinv / pi double staining kit ( bd biosciences , usa ) according to the manufacturer s instructions . flow cytometry was used to assess the apoptotic cells in triplicates and all assays were repeated at least 3 times . student s t test or one - way anova were used for analysis when appropriate . the immortalized human nsc lines hb1.f3 and hb1.a4 were maintained with dulbecco s modified eagle s medium ( dmem , sigma ) containing 10% fetal bovine serum ( fbs ) . total rna was extracted from cell lines with a mirvanamirna isolation kit ( applied biosystems , foster city , ca , usa ) according to the manufacturer s instructions . the expression level of mir-126 was detected by qrt - pcr according to the taqman mirna assays protocol ( applied biosystems ) and normalized by u6 small nuclear rna ( rnu6b ; applied biosystems ) with the 2 method . lv3-pglv - h1-gfp+puro plasmids with mir-126 mimics and negative control ( lv - mir-126 and lv - mir - nc ) were purchased from genepharma ( shanghai , china ) . hb1.f3 and hb1.a4 stably expressing mir-126 were established by transfecting lentivirus according to the manufacturer s instructions . cells were plated in 96-well plates in triplicate at approximately 1000 cells per well and cultured in the growth medium . cells were then treated with the indicated reagent and the numbers of cells per well were measured by the absorbance ( 450 nm ) of reduced water - soluble tetrazolium salt ( wst ) at the indicated time points . cells were plated into 6-well plates at a density of 1000 cells / well in 2 ml medium . the colonies were observed using a phase - contrast microscope at a magnification of 4 ( we counted the colonies containing at least 50 cells ) . cells were transfected with mir-126 mimics or their respective controls and were harvested after transfection at 48 hours and then marked with the annexinv / pi double staining kit ( bd biosciences , usa ) according to the manufacturer s instructions . flow cytometry was used to assess the apoptotic cells in triplicates and all assays were repeated at least 3 times . ibm spss 19.0 statistical software was used to analyze the data . student s t test or one - way anova were used for analysis when appropriate . to further study the potential function of mir-126 in nscs , hb1.f3 and hb1.a4 were stably transfected with mimics or negative control ( nc ) . as indicated in figure 1a , mir-126 mimics showed good overexpression effects both in hb1.f3 and hb1.a4 and both cell lines overexpressing mir-126 showed marked cell growth increase ( figure 1b , 1c ) . to further confirm the function of mir-126 on cell proliferation and growth , soft agar colony formation assay was performed . as shown in figure 2a , 2b , the colony formation was significantly increased by mir-126 compared with the negative control group . we next investigated the function of mir-126 in the regulation of cell apoptosis by flow cytometry . cells overexpressing mir-126 displayed a significant decrease in the apoptotic rate when compared with the control group ( figure 3a , 3b ) . these results suggest that mir-126 may inhibit apoptosis in nscs , which may contribute to the growth induction features of mir-126 . to further study the potential function of mir-126 in nscs , hb1.f3 and hb1.a4 were stably transfected with mimics or negative control ( nc ) . as indicated in figure 1a , mir-126 mimics showed good overexpression effects both in hb1.f3 and hb1.a4 and both cell lines overexpressing mir-126 showed marked cell growth increase ( figure 1b , 1c ) . to further confirm the function of mir-126 on cell proliferation and growth , soft agar colony formation assay was performed . as shown in figure 2a , 2b , the colony formation was significantly increased by mir-126 compared with the negative control group . we next investigated the function of mir-126 in the regulation of cell apoptosis by flow cytometry . cells overexpressing mir-126 displayed a significant decrease in the apoptotic rate when compared with the control group ( figure 3a , 3b ) . these results suggest that mir-126 may inhibit apoptosis in nscs , which may contribute to the growth induction features of mir-126 . they can profoundly regulate the expression of massive target genes that encode proteins , which may finally lead to the change of biological function . mir-126 , which is located in intron 7 of the egf - like domain 7 ( egfl7 ) gene , is a microrna that is highly enriched in endothelial cells   and regulated by the transcription factors ets-1 and ets-2 in endothelial cells . previous studies found that mir-126 promoted angiogenesis during embryonic development and after injury by targeting spred1 and pik3r2 . also , knockdown of mir-126 resulted in delayed angiogenic sprouting , collapsed blood vessels , widespread hemorrhages , and partial embryonic lethality during zebrafish and mouse embryogenesis . it is also involved in cell growth regulation in several organs , such as colorectal cancer , gastric cancer , and liver carcinoma , by regulating multiple target genes , including insulin receptor substrate , p85 , pi3k , akt , and crk [ 2629 ] . reported that mir-126 plays an important role in angiogenesis and inflammation after contusion spinal cord injury in rats . in the current study , we verified that when the expression level of mir-126 was overexpressed by mimics transfection , the proliferation and survival of nscs were both significantly promoted . cck8 assay and colony formation assay both demonstrated that mir-126 can induce the proliferation of nscs . although the mechanism is not yet completely understood , our study provides further evidence in this area .
backgroundthe aim of this study was to investigate the potential function of mir-126 in neural stem cells ( nscs).material / methodsexpression level of mir-126 was detected by quantitative real - time pcr ( qrt - pcr ) . 
 mir-126 overexpression was established by transfecting mir-126 mimics into human nsc lines ( hb1.f3 and hb1.a4 cells ) . 
 its effects on cell proliferation were studied using cell - counting kit-8 ( cck8 ) assay , colony formation assays . 
 flow cytometry was performed to evaluate the effect of mir-126 on cell survival.resultscck8 assay and colony formation assay showed that overexpression of mir-126 promoted cell proliferation and increased colony numbers in hb1.f3 and hb1.a4 cells . 
 the flow cytometry confirmed the results that mir-126 inhibited cell apoptosis.conclusionsmir-126 promoted the proliferation and survival of nscs .