| Since the TGF-(3 family was discovered more than thirty years ago,its biological function has been the focus of research in the field of cell biology.TGF-?plays an important role in the development of organisms and homeostasis by controlling cell behavior.In recent years,the regulatory mechanism of TGF-?signaling for neural development and its role in neurological diseases have been gradually revealed.Long non-coding RNA(LncRNA)regulates gene expression in a variety of ways,thereby regulating a series of important life activities.Abnormal expression of LncRNA will change the expression level of target genes,leading to the occurrence of neurodegenerative diseases.A large number of studies have revealed the key role of LncRNA in regulating the nervous system.In the previous work,our laboratory discovered a new long non-coding RNA Lnc422 in mouse lateral ventricle neural stem cells,which has a reverse overlapping position relationship with transforming growth factor beta receptor 2(TGF?R?).In this study,mouse neural stem cells were used as research materials.By overexpressing and down-regulating Lnc422 in neural stem cells,study the regulatory relationship between Lnc422 and its target gene TGF?R?.By overexpressing Lnc422 to study its effect on neural stem cell proliferation,it was found that Lnc422 can regulate the proliferation of neural stem cells by targeting TGF?R?.To further study the mechanism by which Lnc422 regulates neural stem cell proliferation,we performed transcriptome sequencing on the total RNA of neural stem cells overexpressing Lnc422.Key genes were extracted by bioinformatics and their effects on cell proliferation were analyzed.The results are as follows:1.Using real-time fluorescence quantitative PCR technology to study the expression of Lnc422 and TGF?R? in different neuroanatomical regions of mice.The results show that they are generally expressed in various parts of the mouse brain region,but there are regional expression differences,and the differential expression of TGF?R? in the mouse brain regions is consistent with the differential expression of Lnc422 in the mouse brain regions.2.Real-time fluorescence quantitative PCR and Western Blot were used to study the changes of target gene TGF?R? at transcription and translation levels after overexpression and down-regulation of Lnc422 in neural stem cells.The results showed that overexpression of lnc422 resulted in up-regulation of TGF ? R?expression at both transcription and translation levels,and down regulation of lnc422 resulted in down regulation of TGF ? R? expression at both transcription and translation levels.3.The effect of overexpression of Lnc422 on the proliferation of neural stem cells was studied through the EDU cell proliferation experiment.The results showed that overexpression of Lnc422 caused a significant decrease in the proliferation rate of neural stem cells.4.Bioinformatics was used to analyze RNA transcriptome sequencing data.The results showed that the expression of 1367 genes changed significantly after overexpression of lnc422,among which 708 genes were up-regulated and 659 genes were down regulated.Through KEGG enrichment analysis,12 key differential genes in the cell cycle and apoptosis were concerned:Chekl,Atm,Cdc25a,Gadd45a,Sfn,Ddit3,Tradd,Bbc3,Ccnd2,Ccndl,Hdacl and Skpla.The results show that Lnc422 regulates the proliferation of neural stem cells by targeting TGF?R?.After overexpression of Lnc422 in neural stem cells,the expressions of related genes that promote apoptosis and hinder cell cycle progression are up-regulated,while the expression of genes that are crucial to cell cycle progression is down regulated,which leads to the inhibition of neural stem cell proliferation. |
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