Regulation Mechanism And Function Of MiR-210 In Neural Stem Cells Under Physiological Hypoxia

Neural stem cells (NSCs) are self-renewing and can differentiate into neurons, astrocytes or oligodendrocytes, so they have wide prospects of application of mammalian central nervous system diseases. The technical development of culture NSCs in vitro provide a stable and safe source for cellular transplantation. It is important to maintain the proliferation state and the ability of differentiation of NSCs for clinical application.Traditionally, NSCs culture is usually performed at atmospheric O2 concentration (20%), which is considered as a standard culture system. Actually, O2 content is much low during development or in adult brain tissue, the mean oxygen level is about 1-5%, which is called "physiological hypoxia" by some researchers. It hasn’t been paid extensive attention to the effect of hypoxia microenvironment on the proliferation and differentiation of NSCs. Recently, it has been reported that hypoxia can promote the survival and growth of NSCs, and regulate its’ differentiation. Hypoxia inducible factor-1(HIF-1) plays an important role in the cellular response to hypoxia. However, there are still little known about the regulation mechanism of hypoxia on the proliferation of stem cells such as NSCs.MicroRNAs (miRNAs) are an abundant class of newly identified endogenous non-coding small RNAs (ncRNAs) with-22 nucleotide length. MiRNAs have been considered one of the most important regulatory molecules in cell growth and development, which regulate gene expression at the posttranscriptional levels by targeting mRNA for direct cleavage of mRNA or repression of mRNA translation. Some research report that a number of miRNAs high-express in nervous system with time series and tissue specificity, which imply that they may have important regulation effect on the development of nervous system. As the physiological environment of embryonic development and adult brain tissue, hypoxia might regulate the expression of these miRNAs and influence the proliferation of NSCs.In this study, we examined the expression profiles of ncRNAs in NSCs under hypoxia by using microarray analysis. Then, the differentially expressed ncRNAs were confirmed by quantitative RT-PCR, and miR-210 was highly expressed in NSCs under hypoxic conditions. Next, the regulation mechanism of miR-210 expression in NSCs under hypoxia was investigated. In addition, the role of miR-210 was explored. The results were as follows:1. Isolation, culture and identification of NSCs and role of HIF-1αstabilization in NSCs under hypoxia.NSCs were mechanically dissociated from SD rat mesencephalon at embryonic day 13 (E13). Cells were identified by immunofluorenscence with Nestin. The results showed that almost all of the cells are Nestin positive. Then, cells were differentiated by 1% fetal bovine serum and identified by immunohistochemistry with markers of neurons, astrocytes or oligodendrocytes (Tuj1, GFAP or CNPase). These cells could differentiate into the three types of cells, which have typical characteristic of stem cells, so they were used to perform the following experiments.Hypoxia promotes the proliferation of NSCs and that HIF-1 is critical in this process. The expression of HIF-la were examined under either normoxia or hypoxia for 1,3,6,12,24,48, and 72h, and we found that expression of HIF-la levels increased in NSCs after exposure to hypoxia at above different time points compared with control. Then, we found HSP90 could regulate HIF-la stabilization in NSCs under hypoxia. Geldanamycin (GA), an HSP90 inhibitor, decreased the expression of HIF-1αin NSCs during hypoxia-driven proliferation and reduced the expression level of HIF-la protein under hypoxia in a time-dependent and dose-dependent manner. The expression of HIF-1’s target genes, erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in NSCs under hypoxia was also suppressed by GA. CCK-8 spectrophotometric analysis showed the OD value of NSCs increased under hypoxia, indicating that hypoxia promoted the survival of NSCs, and the survival rate of NSCs induced by hypoxia was inhibited after GA treatment for 24h. We next examined the proliferation index (PI) of NSCs by performing a flow cytometric measurement, and found the PI of NSCs increased after exposure to hypoxia and decreased after treatment with GA. The above data indicated that HSP90 might be involved in regulation of hypoxia-driven proliferation of NSCs by influence the stabilization of HIF-1α.2. Analysis and identification of the ncRNAs expression profile in NSCs under hypoxia by microarray and Q-PCR.NSCs were cultured under normoxia and hypoxia (3% oxygen) for 3 days, and total RNA were extracted for ncRNA microarray analysis. The data showed 20 highly expressed ncRNAs in neural stem cells. Next, we identified differentially expressed ncRNAs using SAM analysis and found 15 up-regulated and 11 down-regulated small ncRNAs. Some up-regulated miRNAs were confirmed by using quantitative RT-PCR including miR-210,376a,221,497,338,301,148a,34a,146b,181d,350,29b,344c. In addition we found that the level of miR-210 expression increased by 15 times or 80 times in NSCs exposed to 3% or 0.3% oxygen. The results strongly suggest that these miRNAs are hypoxia-regulated. By bioinformatic software, we found 6 miRNAs or clusters (miR-210, miR-338, miR-497, miR-29b/29c, miR-148b) among differentially expressed ncRNAs had 24 potential HIF-la binding sites. We inferred the expression of these miRNAs might be regulated by HIF-1.3. Regulation mechanism of miR-210 in NSCs under hypoxia.To investigate whether HIF-1 regulate the expression of miRNAs, we cloned the potential promoter sequence of miR-210, miR-338 and miR-497 to pGL3-basic vector, and constructed three miRNAs promoter-driven luciferase reporter plasmids and pEGFP-HIF-1 plasmids which could over express the HIF-la. The relative luciferase activities of miR-210,338 and 497 promoter reporter constructs were analyzed in HeLa cells after treated with hypoxia for 48h or co-transfection with pEGFP-HIF or empty vector under normoxia for 48h. Our results showed that the relative luciferase activities after transfection with the reporter constructs increased significantly under hypoxia, and HIF-la induced a robust activation of the miR-210 promoter-luciferase constructs but not miR-338 or miR-497, supporting a direct role of HIF-la in miR-210 up-regulation. Additionally, we confirmed the dynamic recruitment of HIF-la to the promoter of miR-210 using a ChIP assay. The HIF-la antibody, but not the control IgG antibody, immunoprecipitated the miR-210 promoter fragments in NSCs under hypoxia. This result suggests that HIF-1αbound to the putative promoter of miR-210 directly and regulated the expression of miR-210.Next, we investigated the DNA methylation control of miR-210 expression. Software analysis showed that miR-210 gene contained a conferted CpG islands. 5-aza-2’-deoxycytidine (Aza), an inhibitor of DNA methyltransferase, was added to the cell medium and NSCs were cultured under normoxia for 24h, Q-PCR analysis showed the express level of miR-210 increased in a dose-dependent manner. After treated with 2μM Aza for 48h, the level of miR-210 in NSCs had 3.75 times increased. As control, the expression of miR-338, which had no CpG islands in the sequence, was not influenced by Aza. Then, the DNA methylation status of miR-210 gene in NSCs under 20%,3% or 0.3% oxygen were analyzed using bisulfite genomic sequencing (BGS). The result showed the methylation level of miR-210 gene decreased by 22% or 49% in NSCs after exposed to 3% oxygen or 0.3% oxygen compared with 20% oxygen. The 58th CG site in miR-210 gene was changed from hemimethylation status under normoxia to nearly demethylation status under hypoxia. These results suggested hypoxia might reduce the methylation level of miR-210 gene and induced the expression of miR-210.The activity of DNA methyltransferase (Dnmts) and histone acetyltransferase (HAT) in NSCs was assessed under normoxia and hypoxia using enzyme activity assays. The activity of Dnmts was decreased and the activity of HAT was increased in NSCs exposed to hypoxia in a degree-independent manner. In addition, the level of Dnmt3b expression was significant decreased in NSCs under 0.3% oxygen. These indicated that hypoxia could influence the activity of Dnmts and HAT in NSCs. As a result, the DNA methylation level was decreased while the histone acetylation level was increased, which was benefited for transcription factor to activate the genes transcription.4. Role of miR-210 in NSCs and during development.In this part, we used the miR-210 lentivirus expression vector to transfect NSCs, CCK-8 spectrophotometric analysis showed the OD value of NSCs increased while over expression of miR-210 under 0.3% oxygen compared with the control. In addition, Q-PCR detection showed the expression of miR-210 in the E13,E16 and E21 rat brains increased day after day. Specific oligonucleotide for inhibition of miR-210 was synthesized and electroporated into the rat brain at embryo 16 days in vivo. We found that development of the embryos arrested and died after inhibition of miR-210. As control, most of the rat’s embryos transfered with NC-RNA were alive. These results suggested miR-210 might have important effect on nervous system development.In summary, hypoxia promotes the proliferation of NSCs and that HIF-1 is critical in this process, destruction of HIF-1 stabilization decreases the proliferation of NSCs induced by hypoxia. Hypoxia induces a number of miRNAs expressions. MiR-210 was up-regulated in NSCs under hypoxia and directly by HIF-1 especially. Furthermore, the DNA methylation level of miR-210 gene decreased in NSCs under hypoxia. Thus, the expression of miR-210 was regulated by the HIF-1 pathway and the DNA methylation pathway. The high-expression of miR-210 in NSCs is beneficial for cell growth and embryo development.