| Background : Glioma is the most common adult central nervous system tumor,although its incidence is not as high as that of other tumors,but because of its high invasiveness and high recurrence rate,even the maximum safe surgical resection and supplemented by molecular guidance,the prognosis of patients is still poor.Therefore,it is urgent to conduct in-depth research on the occurrence and development mechanisms of gliomas,especially glioblastoma(GBM),to provide theoretical basis and experimental evidence for improving the prognosis and survival of glioma patients.Objectives:ELFN2 is epigenetically regulated by long non-coding RNA LINC00470,which was found to be hypomethylated and highly expressed in gliomas,and its encoded protein can bind to AURKA to promote autophagy in GBM cells.Meanwhile,ELFN2 has also been suggested to be a possible regulatory subunit of protein phosphatase 1(PP1)for it contains the classical RVSF binding motif,but little has been reported on whether ELFN2 can function as a regulatory subunit of PP1.In this study,we will further explore the function of ELFN2 as a regulatory subunit of PP1 to promote autophagy in GBM cells by regulating the activity of PP1 and investigate the transcriptional regulation of ELFN2 by LRRC4,a glioma tumor suppressor gene cloned by our group,and its role in ELFN2-induced autophagy,in order to provide an important reference target for the diagnosis and treatment of GBM.Methods:1.Co-immunoprecipitation,immunofluorescence and GST pull down were performed to confirm the interaction of PP1 regulatory subunit ELFN2 and catalytic subunit PPP1 CA and the structural basis of binding,Western blot and RT-q PCR to detect their regulatory relationship,and phosphorus colorimetric assay to detect PP1 activity.2.Single-cell transcriptome sequencing was used to analyze the distribution of ELFN2 and PPP1 CA in tumor cell subsets of GBM tissue,and the correlation between the two and tumor cell autophagy was analyzed by pseudo-chronological analysis.3.Phosphorylation mass spectrometry analysis was performed to screened the substrate protein distribution characteristics,phosphorylation level and the affected core molecular network of PP1 containing ELFN2 dephosphorylation modification,and western blot was performed to verify the regulatory mechanism and target of ELFN2-PP1 dephosphorylation modification on autophagy in GBM cells.4.Ch IP-q PCR,RNA-pull down and luciferase assays were performed to detect the transcriptional regulation mechanism of LRRC4 as a transcription factor on ELFN2,to clarify the regulatory relationship among LRRC4,LINC00470 and ELFN2,and the role of LRRC4 in ELFN2-induced autophagy in GBM cells.5.Nude mouse model of intracranial orthotopic GBM tumorigenesis was constructed to detect the effects of inhibiting ELFN2-regulated PP1 on intracranial tumor growth,prognosis and tumor suppressive function of LRRC4 in glioma.Results:1.It was confirmed that PPP1 CA is the catalytic subunit bound by the PP1 regulatory subunit ELFN2,and the LRR and FN3 domains of ELFN2 and the PP2 AC domain of PPP1 CA are the structural basis for their binding.There is no regulatory relationship between them at the protein and RNA levels,and ELFN2 inhibits PP1 activity by binding to the catalytic subunit PPP1 CA.2.Single-cell transcriptome sequencing analysis showed that PP1 regulatory subunit ELFN2 and catalytic subunit PPP1 CA were expressed in GBM tumor subset 1 which enriched in autophagy signaling pathway,and pseudo-chronological analysis showed that the expression of both in the tumor subpopulation was positively correlated with autophagy in GBM cells.3.It was clarified that the substrate proteins of containing ELFN2-PP1 dephosphorylation modification were mainly localized in the nucleus.Among them,the splicing factor SRRM1 is the substrate protein molecule with the most significant changes in the level of dephosphorylation modification,and the dephosphorylation modification of SRRM1 mainly promotes alternative splicing of the autophagy-related molecule TMIBM6 to inhibit autophagy in GBM cells.4.The regulatory relationship among LRRC4,LINC00470 and ELFN2 was clarified.(1)LRRC4 mainly functions as a transcription factor and binds upstream of the promoter of ELFN2,and inhibits the expression of ELFN2 by increasing the methylation content of H3K9me3 and H3K27me3 in its promoter region.The transcription of ELFN2 by LRRC4 does not depend on the disordered region of LRRC4.(2)Although LRRC4 can bind to LINC00470,it cannot act as a transcription factor to regulate the transcription of LINC00470;LINC00470 can regulate the transcriptional repression of ELFN2 by LRRC4 through blocking the nuclear entry of LRRC4.5.It was demonstrated that LRRC4 can promote the activity of PP1 by inhibiting the transcription of ELFN2,and induce the dephosphorylation of SRRM1 to inhibit the autophagy of GBM cells and exert tumor suppressive function.6.We screened and confirmed that vorinostat,an small molecule compound inhibitor of the PP1 regulatory subunit ELFN2,inhibited the growth and autophagy of intracranial orthotopic GBM xenografts and effectively enhanced the tumor suppressive function of LRRC4 in glioma.Conclusion:1.It was firstly demonstrated that ELFN2 inhibit PP1 activity as a PP1 regulatory subunit,and ELFN2-containing PP1 promoted autophagy-related molecule TMBIM6 alternative splicing by targeting the phosphorylation level of the splicing factor SRRM1 to promote autophagy in GBM cells.2.It was firstly confirmed that LRRC4,as a transcription factor,promotes the activity of PP1 by transcriptionally inhibiting the expression of ELFN2,and promotes the dephosphorylation of SRRM1 to inhibit autophagy and exert tumor suppressive function in GBM cells.3.It is the first time to identify and demonstrate that vorinostat,an inhibitory small molecule compound of the PP1 regulatory subunit ELFN2,which can promote the tumor suppressor function of the glioma tumor suppressor molecule LRRC4 by selectively inhibiting the activity of PP1 containing ELFN2.38 figures,130 references... |
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