The Effect Of PCBP2 On Oxidative Stress-induced Apoptosis Of Glioma Cells Through CGAS/STING Signaling Pathway And Its Mechanism

Background and objective:Glioma is an aggressive primary brain tumor that most commonly occurs in the central nervous system.At present,the effect of conventional treatment is not obvious,and most patients have poor prognosis.Therefore,it is particularly important to focus on the molecular biological mechanisms in the pathogenesis of glioma to find new therapeutic targets.PCBP2 is a member of the poly(C)-binding protein(PCBP)family.It has been reported that PCBP2 plays an important role in various human tumors(including glioma)by binding target m RNA,and PCBP2 reduces c GAS activation by inhibiting c GAS-DNA phase separation in vivo and in vitro,thereby maintaining the balance of c GAS-mediated innate immune response.Therefore,the role of PCBP2 in glioma may be related to the c GAS / STING signaling pathway.The c GAS / STING signaling pathway is an important component of the host ’s natural immune system and is related to the pathogenesis and progression of various diseases.After activation,it releases secretory factors and proteases such as oxidative stress and proinflammatory drugs,thereby accelerating cancer cell senescence and slowing tumor growth.Oxidative stress and inflammation play an important role in the pathogenesis of glioma.Studies have shown that high reactive oxygen species(ROS)are produced during the excessive proliferation of tumor cells,which optimizes ROS-driven proliferation by increasing its antioxidant status,while avoiding senescence,apoptosis or ferroptosis triggered by ROS thresholds.Evidence has shown that ROS is considered to be a key factor in tumor transformation,proliferation and metastasis,and the increase of ROS level is the cause of genetic and epigenetic mutations and proliferation signals.It can be seen that induction of oxidative stress is a common factor leading to the development of cancer,and many anti-cancer agents are now induced by oxidative stress.In addition,N6-methyladenosine(m6A)modification is the most common methylation modification in eukaryotes.It has attracted the attention of researchers at home and abroad because of its large enrichment in circ RNAs and key biological functions.The abnormal level of m6 A alternately affects the maturation,transcription,translation and metabolism of RNA,disrupts gene expression and important cellular processes,and ultimately leads to the occurrence and development of various tumors including glioma.Among them,methyltransferase-like 3(METTL3)is one of the ’writers ’ of m6 A methylation and participates in the regulation of various cellular biological processes.In recent years,many evidences have shown that METTL3 plays an important role in cancer as a m6 A methyltransferase.In summary,we speculate that :(1)PCBP2 may promote glioma progression;(2)PCBP2 regulates c GAS / STING signaling pathway to affect oxidative stress-induced apoptosis of glioma cells.(3)METTL3-mediated m6 A modification plays an important role in the regulation of c GAS / STING signaling pathway by PCBP2.Methods:(1)Collect glioma patient specimens and clinical data in our hospital to clarify the expression characteristics of PCBP2 in glioma and its influence on patient prognosis.(2)U251 glioma cell lines with PCBP2 overexpression and knockdown were constructed and divided into experimental groups.The effects of PCBP2 overexpression and si-PCBP2 on cell proliferation,migration and invasion were observed by q RT-PCR,CCK8,Transwell and EDU methods.The contents of SOD,GSH,Gsh-px,MDA and ROS in U251 cells were detected by ELISA and ROS detection kit,and the influence of PCBP2 on apoptosis rate was determined by flow cytometry.(3)Gene expression levels were detected by Microarray analysis,and KEGG term was used to confirm that c GAS and STING may be two important targets of PCBP2 in glioma.The protein and m RNA expressions of STING,c GAS and PCBP2 were detected by Western blot and q RT-PCR to study the effect of PCBP2 on c GAS/STING signaling pathway.The association between PCBP2 and c GAS was investigated by Co-IP,immunofluorescence and Western blot.(4)The methylation modification sites near PCBP2 were detected by m6 A methylation sequence analysis;In U251 cells,PCBP2 m RNA and protein expression levels were detected by Western blot and q RT-PCR,respectively,after m6 A modification and METTL3 knockout.Sequence analysis of PCBP2 transcriptome was performed to find m6 A enrichment sites,and then q RT-PCR was used to detect the effects of m6 A methylation at different sites and si-METTL3 on PCBP2 m RNA levels.The effect of METTL3 knockout at different mutation sites on its activity was examined by luciferase reports compared with the WT group.Results:(1)The m RNA and protein levels of PCBP2 were significantly higher in glioma patients than in adjacent tissues;The OS and DFS of glioma patients in PCBP2 high expression group were lower than those in PCBP2 low expression group.(2)Overexpression of PCBP2 promoted the proliferation,invasion and metastasis of U251 cells,while silence of PCBP2 inhibited the proliferation,invasion and metastasis of U251 cells.Overexpression of PCBP2 increased the expressions of SOD,GSH and Gsh-Px,decreased the expressions of MDA and ROS,and significantly reduced the number of apoptotic cells.Silence of PCBP2 reduced the expressions of SOD,GSH and Gsh-Px,increased the expressions of MDA and ROS,and significantly increased the number of apoptotic cells.(3)Microarray analysis was used to detect gene expression levels,and it was found that c GAS and STING may be two important targets of PCBP2 in glioma.In U251 cells,m RNA and protein expression levels of c GAS and STING were decreased due to overexpression of PCBP2,but increased due to downregulation of PCBP2.CO-IP confirmed the association between PCBP2 and c GAS proteins.Immunofluorescence showed that PCBP2 inhibited the fluorescence expression of c GAS in vitro.c GAS agonist(SR-717,1μM)induced the expression of c GAS and STING in U251 cells by upregulation of PCBP2.c GAS inhibitors(PF-06928215,2μ-MPA)can inhibit the expression of c GAS and STING proteins in U251 cells by down-regulating PCBP2.(4)PCBP2 was found to have many suspected methylation modification sites near the stop codon.The results of q RT-PCR showed that compared with Ig G group,the expression level of PCBP2 m RNA in group m6 A was significantly increased.However,PCBP2 m RNA expression was significantly decreased after METTL3 was silenced.The results of WB showed that the expression level of PCBP2 protein in si-METTL3 group was significantly decreased compared with Ig G group.Sequence analysis of the PCBP2 transcriptome revealed that m6 A was located in the 3 ’-end untranslated region(UTR).RT-PCR results showed that compared with Ig G group,PCBP2 m RNA expression was significantly increased in the m6 A enriched group at sites 1 and 2.Compared with Negative group,the m6 A level of PCBP2 in si-METTL3 group was significantly decreased.In addition,results reported by luciferase showed that the transcription level of WT PCBP2 was reduced in the si-METTL3 group,but not in the mutant PCBP2.These results suggest that Mett L3-induced m6 A changes can increase PCBP2 expression.Conclusion:(1)PCBP2 was highly expressed in glioma tissues compared with paracancer tissues;High expression of PCBP2 is positively correlated with low prognosis in glioma patients.(2)In U251 cells,upregulation of PCBP2 overexpression promoted cell proliferation,migration and invasion,and reduced oxidative stress-induced apoptosis;Silencing PCBP2 inhibits cell proliferation,migration,and invasion,and increases oxidative stress-induced apoptosis.(3)PCBP2 inhibits glioma progression by targeting c GAS/STING pathway;PCBP2 protein is correlated with c GAS,and c GAS is one of the downstream targets of PCBP2.(4)METTL3-mediated m6 A alteration specifically boosts PCBP2 stability(5)PCBP2 reduces oxidative stress-induced glioma apoptosis by targeting METTL3-mediated m6A-modified c GAS / STING signaling pathway.Blocking PCBP2 is a potential method for the treatment of glioma,and revealed a biochemical pathway in this study,which may be a possible target for preventing glioma oxidative stress-induced death.