The Role And Mechanism Of Filamin A In The Proliferation And Differentiation Of Neural Stem Cells

The cytoskeleton protein Filamin A(FLNA)is a member of the Filamin family.FLNA is widely expressed in the human body and can interact with more than 90 factors to participate in a variety of cell activities.Our previous studies showed that FLNA was up-regulated during the differentiation of embryonic stem cells into neural cells,indicating that FLNA may be involved in the developmental process of neural cells.In addition,in some tumor cells,FLNA has been reported to regulate gene transcription and closely related to cell proliferation.However,it is still unclear whether FLNA is involved in regulating the proliferation and differentiation of neural stem cells(NSCs).In this study,we explored the regulatory effect of FLNA in the proliferation and differentiation of NSCs and their underlying mechanisms.In order to explore the role of FLNA in the proliferation of NSCs,FLNA expression was knocked-down or overexpressed transiently in NE-4C and C17.2,and cell proliferation was assessed using cell counting and CCK-8 experiments.Results showed that transient silence of FLNA could increase the proliferation of NE-4C and C17.2,while its overexpression inhibited their proliferation.Immunofluorescence analysis demonstrated that FLNA could be co-localized with nucleoprotein fibrillarin,suggestting that FLNA is also a nucleolar protein.Further investigation showed that FLNA knockdown significantly enhanced the transcription of genes directed by Pol ? and Pol ?,while FLNA overexpression inhibited their expression.These results indicate that FLNA,a nucleolar protein,regulates Pol ? and Pol ? gene transcription,and the subsequent proliferation of NSCs.To further verify such findings,we established the NE-4C and C17.2 stable cell lines with FLNA knockdown or overexpression,and detected the effects of FLNA on cell proliferation and Pol ?/Pol ?-directed gene transcription.Data showed that FLNA did negatively regulate Pol ?/Pol ?-directed gene transcription and cell proliferation for both NE-4C and C17.2 cells.The detection of signalling factorsregulating cell proliferation showed that FLNA knockdown up-regulated the expression of the proto-oncogene MDM2 and inhibited the expression of the tumor suppressor genes PTEN and p53.Taken together,these results suggest that FLNA may regulate NSCs proliferation through modulating the PTEN/MDM2/p53 signaling pathways and Pol ?/Pol ?-directed gene transcription.In this study,we also preliminarily explored the effect of FLNA knockdown on NSCs differentiation.Data demonstrated that FLNA knockdown inhibited the differentiation of NE-4C cells into neurons.RT-qPCR was used to analyze the expression of transcription factors and the signal proteins related to NSCs differentiation.The results showed that FLNA knockdown decreased the expression of Ascl1 and Ngn2 driving neuronal differentiation and promoted the expression of Notch1 maintaining NSCs identity.Collectively,we found that FLNA inhibited the differentiation of NE-4C into neurons mainly by regulating Notch and bHLH signaling pathways.These findings not only broaden our understanding of FLNA function in NSCs,but also provide a new way for the diagnosis and treatment of hereditary neurological diseases caused by FLNA mutations.