Effects Of Hypoxia On The Proliferation And Metabolism Of Nscs And MiR-21 Expression And Function

Neural stem cells (NSCs) are self-renewing and can differentiate into neurons, astrocytes or oligodendrocytes, which has been hot point of research in central nervous field. Recently, it has been reported that hypoxia can promote the survival and proliferation of NSCs, and hypoxia inducible factor-1(HIF-1) plays an important role in this process, but there are still little known about the regulation mechanism of hypoxia on the proliferation and metabolism of NSCs. Thus, we will first investigate the effect of hypoxia on proliferation and metabolism of NSCs.MicroRNAs(miRNAs) are endogenous noncoding small RNAs(ncRNAs) with 21～23 nucleotide length. MiRNAs are important regulatory molecules in cell growth and development, which regulate many kinds of genes expression. Recently, many reports focus on the expression regulation and function of miRNAs in tumour cell; as the physiological environment of tumor, hypoxia might regulate the expression of these miRNAs and influence the proliferation of NSCs. In this study, we next examined the expression profiles of ncRNAs in NSCs under hypoxia by using microarray analysis. Then, the function of miR-21, which highly expressed in NSCs, was investigated. The results were as follow:1. Effect of hypoxia on proliferation and metabolism of NSCs derived from rat embryonic mesencephalon in vitro.a. NSCs were mechanically dissociated from rat mesencephalon (E13.5), and almost all of the cells are Nestin positive. These cells could differentiate into neurons, astrocytes or oligodendendrocytes after induced with 1 % FBS, which showed typical characteristic of stem cells, so they were used to perform the following experiments.b. After cultured for three or five days under hypoxia, the number of NSCs was obviously increased, which means hypoxia could promote the proliferation of NSCs. After cultured under hypoxia, the metabolism of NSCs changed obviously. Following the culture time, the utilization of glucose was increasing, the generation of pyroracemic acid and lactic acid were also increasing, and the pH value in the culture medium decreased, which might promote the proliferation of NSCs exposed to hypoxia.c. Hypoxia promotes the proliferation of NSCs, and the generation of ATP in cells culture for three days or five days decreased under hypoxia compared with normoxia. Hypoxia regulated the metabolism of NSCs under hypoxic condition by increasing the transport of glucose and reinforcing the expression of pyruvate kinase through the glycolytic pathway.2. Effect of miR-21 overexpression on proliferation of NSCs in vitro.In this part we examined the expression profiles of non-coding small RNAs in NSCs under hypoxia by using microarray analysis. Then we investigated the expression and function of high expressed miR-21 in NSCs exposed to hypoxia.a. 12 up-regulated and 8 down-regulated small ncRNAs were found in NSCs exposed to hypoxia by using microarray analysis, these ncRNAs may play an important role in the proliferation of NSCs.b. miR-21, which high-expressed in NSCs, decreased its express level in cells under 3 % oxygen compared with 20 % oxygen. But after treated with 0.3 % oxygen, the expression of miR-21 increased obviously compared with normoxia.c. Overexpression of miR-21 could promote the survival of NSCs culture in vitro, and increased the number of Brdu positive cells compared with the control group. Furthermore, the number of survival cells after overexpression of miR-21, increased under anoxia condition (0.3 % oxygen). The regulatory mechanism of these process need more investigation in future.In summary, moderate hypoxia can promote the proliferation of NSCs in vitro. Under hypoxic condition, the metabolism of NSCs changes conspicuous, the utilization of glucose obviously increases and increases the generation of energy by glycolysis metabolic pathway. The expression level of a number of miRNAs change in NSCs exposed to hypoxia. MiR-21, which is high-expression in NSCs, can promote the survival and proliferation of NSCs under anoxia condition. These results provide new clues for proliferation regulation and cellular transplant of NSCs.