Preliminary Research On The Expression Of The REEP2 Gene On The Production Of Colony-stimulating Factor 1 Receptor In Glioma Cells

Objective:To preliminarily investigate the expression of receptor accessory protein 2(REEP2)in glioma and the effect of REEP2 expression level on the production of glioma colony-stimulating factor receptor(CSF-1R).Methods:Immunohistnchemistry(IHC)staining was performed on parafin-embedded tissue sections from 63 glioma patients to assess the expression of REEP2;real-time fluorescence quantitative PCR(qRT-PCR),as well as protein immunoblotting(Western Blotting,WB),was performed after U251 glioma cells were transfected with plasmid vector to analyze and compare the REEP2 mRNA transcript levels and REEP2 protein expression levels in U251 cells after sh-REEP2 and OE-REEP2 interventions.The results of transcriptome sequencing analysis of sh-REEP2 and NC-intervened U251 cells were compared to uncover the differential genes,and then GO enrichment was performed to analyze the Biological Process(BP),Cellular Component(CC),Molecular Function(MF)and KEGG annotation,followed by protein protein interaction network(PPI)analysis of the differential genes to explore the specific role of the differential molecules in the development of glioma.The expression levels of CSF-1R mRNA and CSF-1R protein in sh-REEP2 and OE-REEP2-intervened U251 cells were observed to assess whether CSF-1R mRNA and CSF-1R protein expression were regulated by the reversion of REEP2 gene.Finally,CSF-1R mRNA expression levels and survival of glioma patients were analyzed using the database to evaluate if there is a correlation between them.Results:The results of IHC staining of paraffin-embedded tissue sections from 63 patients with glioma suggested a positive correlation between REEP2 protein expression levels and the pathological grade of glioma.Subsequently,the results of qRT-PCR and WB of plasmid-transfected U251 glioma cells suggested that the expression levels of REEP2 mRNA transcript and REEP2 protein were decreased in the sh-REEP2 group,while the that of REEP2 mRNA and REEP2 protein were increased in the OE-REEP2 group,demonstrating that U251 glioma cells were successfully transfected.The samples from the successfully identified sh-REEP2 group and NC negative control group were performed with transcriptome sequencing.And the results indicated a total of 65 differential genes,of which 31 genes were up-regulated and 34 genes down-regulated.By analyzing the transcriptome results and reviewing the related literature,it was hypothesized that CSF-1R might be the core factor among the above differential genes involved in the related signaling pathway and the regulation of PPI network.The results of qRT-PCR and WB experiments on U251 glioma cells transfected with sh-REEP2 plasmid and U2 51 glioma cells further transfected with OE-REEP2 plasmid confirmed that the mRNA and protein expression levels of CSF-1R in U251 glioma cells were suppressed by REEP2 gene regulation.Finally,CSF-1R was found to be expressed in both high-and low-grade glioma by GEPIA2.0,and CSF-1R mRNA showed high expression in gliomas compared with normal tissues.Meanwhile,the expression level of CSF-1R mRNA was negatively correlated with the survival rate of patients.Conclusions:High expression of CSF-1R in glioma patients may be detrimental to patient prognosis.Targeting REEP2 to affect CSF-1R expression is expected to be an effective strategy for clinical treatment of glioma.