Mechanism Of Mutant P53 Regulating The Sorting Of CircRNAs Into Exosomes From Glioblastoma Cell

Background:Glioblastoma(GBM)is the most malignant intracranial brain tumor with high heterogeneity,high invasiveness and high recurrence.Exosomes,as natural mediators of intercellular communication,can transport DNA,protein and non-coding RNA between cells.Circular RNAs(circRNAs)are a class of RNA molecules with high specificity,high conserved and high stability,which can be detected in peripheral blood.Tumor cells can secrete specific circRNAs to enter the extracellular microenvironment through exosomes and play an important role in biological processes such as GBM proliferation,invasion and immune invasion.In previous studies on circRNA differences between GBM cells and exosomes,we found that,compared with GBM containing wild-type p53,the secretion of circRNA in exosomes from GBM with mutant p53 was significantly reduced.However,the mechanism by which mutant p53 mediates circRNA sorting into exosomes of GBM cells is still unclear.Purpose:Screening and verifying the mechanism of mutant p53 inhibiting circRNA sorting into exosomes of GBM cells,and clarifying the characteristics of mutant p53 selectively secreting circRNA into exosomes and its role in cicr RNA sorting.Methods:(1)Analyze the expression and mutation status of p53 in GBM,and determine the mutation frequency of p53 in GBM;(2)The expression of circRNAs in U251 cells expressing mutant p53 and corresponding exosomes was compared by RNA sequencing analysis,and the number and expression of differentially expressed circRNAs were determined,which was verified by RT-q PCR.(3)By circRNA sequencing analysis,the characteristics of circRNAs profiles in U251 cells expressing mutant p53,U251 cells with mutant p53 konckdown and exosomes secreted by corresponding cells were analyzed,including expression,number,length,type,GC%,parental gene functional enrichment analysis and motif analysis.(4)The highly expressed RNA-binding proteins in U251 cells expressing mutant p53 and U251 cells with mutant p53 konckdown and corresponding cell secretion were analyzed,and the binding ability of these proteins with exosomal circRNAs and the relationship between the binding ability and the expression level were detected;The expression of high expression RBP in GBM cells was detected.(5)The correlation between RBP and p53 expression was analyzed through public database,and compared with the RBP in GBM in another study that was pulled down by exosomal circRNAs;(6)Starbase predicts circRNAs bound by RBP and detects the expression of circRNAs intersected.Result:(1)p53 is the most common mutation in GBM,up to 30%.The common mutation sites are R273 and R248,and it is often highly expressed in GBM.(2)In U251 cells expressing mutant p53,there was a significant selective distribution of intracellular circRNAs and exosomal circRNAs.The number of circRNAs in exosomes was significantly reduced,but the expression level of some circRNAs was significantly increased,even more than 4 times that of intracellular circRNAs.(3)Whether U251 cells with mutant p53 knockdown or U251 cells expressing the mutant p53,the exosomal circRNAs have the following characteristics compared with intracellular circRNAs: The overall expression level was lower,the number of circRNAs was fewer,the dominant type of circRNAs was intron-intron type,GC content was higher,and functional enrichment was mainly concentrated in clusters related to vesicle transportation or intercellular communication,prefer to containing EXOmotifs.Compared with U251 cells with mutant p53 knockdown,exosomal circRNAs of U251 cells expressing mutant p53 showed no significant difference in the characteristics of overall expression,number,type and GC content,while there were significant differences in functional enrichment and motif analysis.exosomal CircRNAs of U251 expressing mutant p53 were mainly enriched in clusters such as "cancer pathway" and EXOmotif such as "KARGRSRA".(3)GBM cells had abundant expression of RBP and high binding force with exosomal circRNAs,and some RBP were highly correlated with the expression of mutant p53.High correlation of RBP:HNRNPA2B1 predicts to bind hsa＿circ＿0001445,which was significantly overexpressed in exosomes from U251 expressing mutant p53 and contained unique mutant p53 associated EXOmotif such as "AGMGAYSA".Conclusions:.(1)Mutant p53 plays an important role in the selective distribution of GBM exosomal circRNAs.(2)Mutant p53 may regulate circRNAs sorting by influencing circRNAs’ parent gene transcription splicing and circRNAs’ EXOmotif.(3)Mutant p53 regulates the sorting of exosomal circRNAs in a manner that relies on RBP to recognize circRNAs’ mutp53-a EXOmotif.