| Neural stem cells (neural stem cells, NSC) are a kind of cells that has differentiation potential and can differentiate into neurons, oligodendrocytes and astrocytes. It can provide cells for brain by self-renewal and differentiation. Precisely because it has such characteristics, NSC becomes an important research direction of neurological disease treatment. It not only can be used as an alternative means of treating brain tumors, degenerative diseases and diseases of central nervous system injury, but also can be used to explore the development of molecular mechanisms of the nervous system. Therefore, establishing a stable culture system of neural stem cells in vitro is the basis of various research studies to be carried out in the nervous system.MiRNAs are a new class of small RNAs encoded by endogenous genes and highly conserved in a variety of species, and they also can regulate gene expression. MiRNAs are a kind of small endogenous RNA molecules in vivo and they are expressd specifically and timingly, generally containing20to24oligonucleotides. MiRNAs do not have an open reading frame and do not encode a protein expression, In recent years, a large number of miRNAs have been extracted from neural stem cells in the brain, suggesting that miRNAs may play a certain important role in malignant transformation and development of nervous system. But up to now, most of the studies of miRNA focus on their regulation on development and differentiation of nervous system, instead of on their regulation on miRNA malignant transformation of neural stem cell.This work intends to compare miRNA expression of neural stem cells with that of Neuro-2a cells. Then, we try to detect whether there is a relationship between malignant performance of Neuro-2a and these different miRNA expression. Finally, we select a group of miRNA which meet our expectations, and perform a preliminary study of its mechanism of regulating malignant transformation.Aims:1. To establish a stable culture system of neural stem cells in vitro;2. To select a group of miRNA which differentially express between neural stem cells and Neuro-2a, then find the corresponding target gene;3. To explore the relationship between the selected miRNA and its function of malignant transformation on Neuro-2a cells by phenotyping methods;4. To prove the direct interaction between the miRNA we selected and their target genes by using Luciferase report;5. To validate the expression levels of of protein targeted by our miRNA between Neuro-2a cells and neural stem cells.Results:1. We successfully established a technology platform of neural stem cell culture. The technique of NSC isolation from mouse, cell proliferation and culture in vitro are stable and well repeated;2. Real-time quantitative PCR showed that, compared with NSC, miR-193a-3p, miR-30b, miR-335were increased significantly, but mir-124, mir-34a and mir-455-5p were reduced significantly in Neuro-2a cell line. miR-193a-3p is increased nearly tenfold.3. After transfection with miR-193a-3p inhibitor, the Neuro-2a cells’ proliferation has been significantly inhibited, and its apoptosis was significantly increased, compared to control group;4. Luciferase reporter system results showed that the activity of luciferase containing3’UTR region of PTEN can be obviously inhibited after introduction of miR-193a-3p mimics into HEK293cells;5. Expression of PTEN in Neuro-2a cells was significantly lower than that in neural stem cells.Conclusion:1. The expression of miR-193a-3p in Neuro-2a cells is up-regulated. miR-193a-3p inhibitor can promote apoptosis of Neuro-2a cells;2. MiR-193a-3p can directly interact with3’UTR region of PTEN. In Neuro-2a cells, miR-193a-3p promote the occurrence of neuroblastoma by directly reducing the expression of PTEN. |
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