| Neural stem cells (Neural stem cells, NSCs) exist in embryonic nervous systemand adult brain tissue， that can self-renew to produce more stem cells anddifferentiate into neurons, astrocytes and oligodendrocytes. Neural stem cells is veryimportant for the embryonic development of the central nervous system, and can beused for the treatment of a variety of adult central nervous system injury and disease.The transplantation of neural stem cells can be used to treat Parkinson’s disease,Huntington’s disease, amyotrophic lateral sclerosis, Alzheimer’s disease, multiplesclerosis, stroke and spinal cord injury and other central nervous system diseases inanimals or humans. Currently, the factors of the oxygen concentration are oftenoverlooked in the process of culturing neural stem cells in vitro. Actually, the oxygenconcentration of stem cells in vivo is below5%, which is referred to as a"physiological hypoxia" environment. In recent years, more and more experimentalevidence proved that hypoxia has an important role in the regulation of proliferationand differentia-tion of neural stem cells. The findings in our laboratory have shownthat moderate hypoxia can promote the proliferation and differentiation of neural stemcells in vitro and in vivo. Therefore, the study of the regulation of hypoxia to thesurvival, proliferation and differentiation of neural stem cells, has importantimplications.As sensing molecule of hypoxia, Hypoxia-inducible factor (HIF) is a basicelement of hypoxia signal passway. Under hypoxic conditions, HIF-1is stabilizedand transfered to the nucleus, and combin with hypoxia response element (Hypoxiaresponsive element, HRE) to regulate the expression of multiple downstream targetgenes involved in cell proliferation, survival and differentiation. The latest findingsshow that microRNAs involved in the regulation of HIF signaling pathway upstreamand downstream. In hypoxia, HIF can induce the expression of miR-23, miR-24,miR-26, miR-107, miR-210and miR-373. And miR-199and miR-20b can induce theexpression of HIF. Among hypoxic regulated miRNAs, miR-210was the most studied.miR-210is consistently upregulated in hypoxia-treated cells, and has the greatest increase. miR-210is currently regarded as the “master miRNA” of thehypoxic response. Studies shown that HIF-1can combin with the HRE,40bpupstream of the transcription start site of miR-210, and regulate the expressiong ofmiR-210. Through the regulation of target genes, miR-210was involved in theregulation of cell survival, apoptosis, mitochondrial metabolism, DNA damage repairand angiogenesis. However, the expression and regulation of miR-210in neural stemcells has not been reported. Therefore, in this study, we investigate the regulation ofhypoxia on the expression of miR-210in neural stem cells and the mechanism.First: Regulation of hypoxia on the expression of miR-210in neural stem cells and thefuction of miR-210.1. NSCs were cultured in20%,3%and0.3%for1,2,3days,and the expression levelof miR-210in NSCs was analyzed by realtime-PCR.The results indicated that3%and0.3%hypoxia can significantly promote the expression of miR-210, and theexpression level of miR-210gradually increased as the hypoxia time prolongedand the concentration of oxygen reduced, which indicate that miR-210may haveimportant biological effect in neural stem cells.2. The inhibition of DNA methylation with a DNA methylation inhibitor AZA caninduce the expression of miR-210in normoxia and hypoxia, which indicate thatDNA methylation can regulate the expression of miR-210in HIF-1-independantmanner.3. By overexpression and inhibition of miR-210, we found miR-210cansignificantly inhibit apoptosis and promote cell survival of neural stem cells in0.3%oxygen concentration.But miR-210does not affect cell proliferation.4. By bioinformatics screening, we found that the coding area and3’UTR of BNIP3,a pro-apoptotic protein, contain two seeding sequences of BNIP3, which indicatethat BNIP3may be the target genes of miR-210.5. We detected the effect of overexpression and inhibition of miR-210on BNIP3protein expression, and found that miR-210could significantly inhibit theexpression of BNIP3protein. Sequence analysis found that the coding region andthe3’UTR of the BNIP3mRNA has two complementary sites which may bind tomiR-210. By the construction of these two loci to the luciferase reporter vector,we found that overexpression of miR-210could significantly inhibit the luciferaseactivity of the two plasmids, and point mutations can reverse the inhibition effectof miR-210. The above results indicate that BNIP3is a target gene of miR-210. 6. BNIP3siRNA can inhibit the apoptosis of NSCs induced by miR-210inhibitor,which indicate that miR-210—BNIP3play an important role in the inhibition ofapoptosis under hypxia.7. By Western Blot and immunofluorescence experiments, we prove that miR-210inhibit BNIP3expression, which inhibit the translocation of AIF into the nucleus,and ultimately inhibit apoptosis of neural stem cells.Second: Regulation of hypoxia on the secretion of circulating miR-210and thefuction of circulating miR-210.The latest study found miRNAs can be secreted into the cell culture medium andthe fluid circulation system. The secreted miRNAs, which is wrapped by protein andmembrane, is named circulating miRNAs. As circulating miRNAs are stable and easyto obtain and detect, they are considered as ideal disease diagnostic molecules. Butthe functions of circulating miRNAs are rarely reported.1. We analyzed the level of miR-210in NSCs and cell culture medium underdifferent concentration of oxygen by real-time PCR and revealed that theexpression of miR-210was upregulated not only in the NSCs, but also in cellculture medium in a hypoxia time and concentration dependent pattern.2. Exosomes and microvesicles containing circulating miR-210were isolated byultracentrifugation and their micro-structure were observed. Fluorescent dyespecific staining experiments showed that exosomes and microvesiclescould beabsorbed by cells, and the circulating miR-210fromexosomes andmicrovesicleshad an access into the receptor cells to upregulate the level ofmiR-210in the cells.3. We collected the medium from NSCs cultured in different concentration ofoxygen and analyzed their regulation on the survival of NSCs under normoxia.The results displayed that medium from NSCs which were cultured in3%O2could promote NSCs survival, while medium from0.3%O2-treated NSCs inhibitcell survival. However, the effect of the medium on NSCs survival could bereversed by an addition of miR-210inhibitors into the medium, indicating aregulatory role of circulating miR-210on NSCs survival.4. Furthermore, NSCs were treated in normoxia condition by serial dilutedcirculating miR-210-containing medium to analyze the potential association ofcirculating miR-210concentration and NSCs survival. We found that NSCssurvival could be inhibited by circulating miR-210at a high concentration, whereas whether circulating miR-210at a low concentration could promote cellsurvival needs further investigation.5. SD rats were treated with5000m altitude hypobaric hypoxia for0.5-2hours, andthe expression level of miR-210was detected by Realtime-PCR. The results showthat5000m hypobaric hypoxia can dramatically increase the level of circulatingmiR-210in rat plasma and cerebrospinal fluid circulation, however theunderlying mechanism still remains unclear.In summary, our results revealed that:(1)Hypoxia significantly up-regulated theexpression of miR-210in neural stem cells;(2) DNA methylation, independent ofHIF-1pathway, was involved in the regulation of miR-200expression;(3)In hypoxia,miR-210could promote the survival of neural stem cells,and inhibitapoptosis;(4)BNIP3is a target gene of miR-210, through the inhibition of which,miR-210suppressedthe translocation of AIF into the nucleus, leading to the inhibitionof NSCs apoptosis.(5)Hypoxiy can promote the secretion of miR-210from neuralstem cells.(6)Circulating miR-210could regulate NSCs survival, which wasassociatedwith its concentration.(7) Hypoxia could increase the content of circulating miR-210in rat blood and cerebrospinal fluid. The above results show that miR-210has animportant role in the regulation of the balance between neural stem cell survival andapoptosis in hypoxia. Circulating miR-210is not only a marker of hypoxia and isinvolved in the regulation of survival of neural stem cells. The study also provides anew theoretical foundation and ideas for the regulation of neural development andneural stem cells transplantation in the treatment of nerve injury andneurodegenerative diseases. |
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