CPEB2 M6A Methylation Regulates Blood-Tumor Barrier Permeability By Increasing Splicing Factor SRSF5 Stability

Objective: Malignant gliomas are the most common primary tumors in the brain.Due to the existence of the blood-tumor barrier(BTB),it is difficult for macromolecular chemotherapeutic drugs to reach the tumor tissue and achieve the expected therapeutic effect.Therefore,selective BTB opening is an effective method to improve the chemical efficacy of glioma.The alteration of BTB permeability mainly includes two main ways,transcellular and paracellular pathways.The paracellular pathway is regulated by tight junction(TJ).Tight junction proteins(TJPs)include the cytoplasmic plaque protein ZO-1and the transmembrane proteins occludin and claudin-5.Several studies have shown that reduced expression of ZO-1,occludin,and claudin-5 is able to increase BTB permeability.N6-methyladenosine(m6A)RNA methylation modification is the most common post-transcriptional modification in eukaryotes,and plays an important physiological role in cell differentiation,embryonic development,and stemness maintenance of tumor stem cells effect.In the process of m6 A methylation modification,three types of molecules are involved: m6 A methyltransferase(Writer),m6 A demethylase(Eraser)and m6 A binding protein(Reader).METTL3(methyltransferase like 3),the gene encodes the 70 k Da MT-A subunit,METTL3 as a methyltransferase can regulate the m6 A modification of RNA in vivo.The high expression in many tumors such as glioma,acute myeloid leukemia,liver cancer,lung cancer,and breast cancer indicates that it may participate in tumor cell proliferation by promoting m RNA translation.Studies have shown that m6A-binding proteins can specifically recognize and bind to the m6 A modification site of RNA,thereby regulating the translation,splicing and stability of RNA.IGF2BP3(insulin like growth factor 2 m RNA binding protein 3,also known as IMP3),as a m6 A binding protein,enhances the CPEB2 m RNA stability by recognizing the CPEB2 m6 A methylation site.RBPs(RNA binding proteins)are a class of proteins that can bind to RNA,mediate m RNA splicing,RNA stability,maintain intracellular localization and translation control and other processes.CPEB2(Cytoplasmic poly(A)denylation element binding protein 2),which regulates the translation of its target m RNA by binding to the cytoplasmic polyadenylation element in the 3’ untranslated region.Alternative splicing is the process that produces different mature m RNA splicing isoforms through different splicing modes.SRSF5(serine and arginine rich splicing factor 5,also known as SRp40,HRS)is a member of the SR protein family and plays an important regulatory role in RNA splicing and translation.It has been reported that SRSF5 plays a tumor-promoting role in liver cancer and lung cancer by regulating alternative splicing.Transcription factor ETS1(E26 transformation-specific 1)is highly expressed in a variety of tumor tissues,and inhibiting ETS1 can block tumor proliferation,migration,and invasion in vivo,such as high levels of ETS1 was found in lung,gastric,colorectal,breast,ovarian and cervical cancers.In this study,the effect and mechanism of CPEB2 on the permeability of BTB were taken as a starting point to clarify the role of METTL3 in promoting the production of CPEB2 methylation sites,and to explain the binding of IGF2BP3 to CPEB2 m6 A methylation sites to enhance its m RNA The mechanism of stability,to clarify the role and mechanism of CPEB2 binding to SRSF5 and affecting the alternative splicing of ETS1 mediated by SRSF5,and then affecting the permeability of BTB.These results can not only provide a new theoretical basis for regulating the permeability of BTB,but also provide new targets and new strategies for increasing the entry of chemotherapy drugs into brain tumor tissues and for the comprehensive treatment of glioma.Method: 1.Astrocyte microvascular endothelial cells(AECs)and glioma microvascular endothelial cells(GECs)were prepared to construct in vitro blood-brain barrier(BBB)model and in vitro BTB model;2.q RT-PCR assays were used to detect the expression levels of METTL3,IGF2BP3,CPEB2,SRSF5 and ETS1;3.The expression levels of METTL3,IGF2BP3,CPEB2,SRSF5,ETS1,ZO-1,occludin and claudin-5 were detected by western blot assays;4.Stable transfection of METTL3,IGF2BP3,CPEB2,SRSF5 and ETS1 silent plasmids;5.Me RIP-q PCR assays were used to verify the m6 A methylation site of CPEB2;6.Nascent RNA assays were used to detect the nascent RNA of CPEB2 and SRSF5;7.Treatment with actinomycin D,combined with q RT-PCR method to detect the changes in the half-life of CPEB2 and SRSF5 m RNA;8.RIP and RNA pull-down assays were used to verify the binding of CPEB2 and SRSF5 m RNA;9.Transendothelial electrical resistance(TEER)assays were used to detect the integrity of BTB;10.Horseradish peroxidase(HRP)assays were used to detect the permeability of BTB;11.Immunofluorescence assays were used to detect the expression and distribution of ZO-1,occludin and claudin-5;12.Dual luciferase reporter gene assays were used to verify the binding site of P51-ETS1 with ZO-1,occludin and claudin-5;13.Ch IP assays were used to verify the binding effect of P51-ETS1 with ZO-1,occludin and claudin-5;14.BTB permeability was verified in vivo by GBM orthotopic xenograft mice.Results: 1.METTL3 was highly expressed in GECs.Knocking down METTL3 significantly decreased the TEER value in GECs,increased the HRP flux,significantly decreased the protein expression of ZO-1,occludin and claudin-5,and TJPs become discontinuously distributed on the cell membrane.2.IGF2BP3 was highly expressed in GECs.Knocking down IGF2BP3 significantly decreased the TEER value in GECs,increased the HRP flux,significantly decreased the protein expression of ZO-1,occludin,claudin-5,and TJPs become discontinuously distributed on the cell membrane.3.Knockdown of METTL3 in GECs significantly reduced the m6 A levels of CPEB2,reduced CPEB2 m RNA stability,and no significant change in CPEB2 nascent RNA levels.4 Knockdown of IGF2BP3 in GECs caused a significant reduction in CPEB2 m RNA stability and no significant change in CPEB2 nascent RNA levels.5.CPEB2 was highly expressed in GECs.Knocking down CPEB2 significantly decreased the TEER value in GECs,increased the HRP flux,significantly decreased the protein expression of ZO-1,occludin and claudin-5,and TJPs become discontinuously distributed on the cell membrane.6.After mutation of the m6 A methylation site of CPEB2 in GECs,the stability of CPEB2 m RNA was significantly reduced,the level of CPEB2 nascent RNA was not significantly changed,the level of CPEB2 protein expression was significantly reduced,the TEER value was significantly reduced,the HRP flux was significantly increased,the expression of ZO-1,occludin and claudin-5 protein was significantly reduced,and ZO-1,occludin,claudin-5 protein became discontinuous distribution on the cell membrane.7.Overexpression of CPEB2 wild-type plasmid at the same time of deletion of METTL3 significantly reversed the inhibition of METTL3 alone on TEER value,the promotion of HRP permeability,and the inhibition of ZO-1,inclusion,and claudin-5 protein expression;Knockdown of METTL3 while overexpressed the CPEB2m6 A site showed no significant change compared with knockdown of METTL3 alone.8SRSF5 was highly expressed in GECs,and knockdown of SRSF5 significantly reduced TEER values in GECs,increased HRP penetration,decreased ZO-1,occludin and claudin-5 protein expression,and ZO-1,occludin,claudin-5 became a discontinuous distribution at the cell membrane.9.CPEB2 was able to bind SRSF5 m RNA,knockdown of CPEB2 significantly reduced SRSF5 m RNA stability and reduced the protein expression level of SRSF5,and overexpression of CPEB2 significantly increased the protein expression level of SRSF5.10.Overexpression of SRSF5 in CPEB2 knockdown GECs significantly reversed the inhibitory effect of CPEB2 knockdown alone on TEER values,the promoting effect on HRP permeability,and the decreasing effect of the ZO-1,occludin and claudin-5 protein expression.11 SRSF5 is able to bind and promote the inclusion of ETS1 exon7,which in turn promotes the expression of the alternative spliceosome P51-ETS1 and suppresses P42-ETS1 expression.12 P51-ETS1 was highly expressed in GECs,and knockdown of P51-ETS1 significantly reduced TEER values in GECs,increased HRP penetration,significantly reduced ZO-1,occludin and claudin-5protein expression,and ZO-1,occludin,claudin-5 became a discontinuous distribution on the cell membrane.13.P51-ETS1 binds to the promoter regions of ZO-1,occludin and claudin-5,and promotes the transcription of ZO-1,occludin and claudin-5.14.sh-METTL3,sh-IGF2BP3,CPEB2-Mut,sh-SRSF5 and sh-P51-ETS1 combined with Dox treatment significantly inhibited the growth of GBM xenograft tumor in nude mice and made the nude mice have a longer survival time.Conclusion:1.METTL3,which is highly expressed in GECs,promotes the methylation of CPEB2 m6A,and IGF2BP3 specifically binds to CPEB2,thereby enhancing the stability of CPEB2 m RNA,upregulating its expression,and regulating BTB permeability.2.CPEB2,which is highly expressed in GECs,enhances the stability of SRSF5 m RNA,upregulates the expression of SRSF5,and then regulates BTB permeability.3.SRSF5 promotes the generation of P51-ETS1 and inhibits the generation of P42-ETS1 by mediating the alternative splicing of ETS1,thereby enhancing the transcriptional regulation of P51-ETS1 on ZO-1,occludin and claudin-5,and regulating BTB permeability.4.METTL3/IGF2BP3/CPEB2/SRSF5/P51-ETS1 signaling axis plays an important role in regulating BTB permeability.