Mechanism Of CELF2 Inhibiting Malignant Progression In Glioblastoma By Regulating Proneural-Mesenchymal Transition Through Alternative Splicing

Glioblastoma(GBM),as one of the most malignant tumors,has typical characteristics of tumor heterogeneity.GBM cells with different molecular phenotypes have the ability to transform each other,and the strong transformation ability enables GBM intratumoral cells to reshape the tumor microenvironment by regulating the chemotaxis and recruitment of tumor-associated macrophages(TAM),and the immune escape ability obtained under the action of TAM finally induces proneuralmesenchymal transition(PMT).This process can promote the malignant progression of GBM to proliferation,invasion,and migration,which in turn leads to poor prognosis of patients in clinical practice.Research on the molecular mechanism of GBM molecular phenotype transformation is an important link in the field of glioma research.Previous studies have shown that alternative splicing factors can promote the mesenchymal phenotype transformation of GBM cells by forming a splicing complex and participating in the activation of the NF-kB pathway.However,the specific control mechanism still needs to be further clarified.In this study,we will focus on exploring the deep mechanism of the splicing factor CLEF2(CUGBP Eva-like family number 2)that regulates the PMT of GBM and the remodeling process of the tumor microenvironment through alternative splicing,and promotes the malignant progression and treatment resistance of tumors.According to existing research results,CELF2 plays a tumor suppressor role in many tumors,and the expression of CELF2 in GBM has obvious differences among different molecular phenotypes.Therefore,we speculate that CELF2 has the potential to regulate PMT.We have confirmed the clinical significance of CELF2 as a cancer-suppression splicing factor through clinical data analysis,and the regulatory effects of CELF2 on the proliferation,migration and dedifferentiation of GBM cells were confirmed by functional experiment.Then,according to the results of transcriptome sequencing,the alternative splicing events regulated by CELF2 were screened out,and the biological process involved in the downstream splicing events of CELF2 in GBM was explored through functional enrichment analysis combined with immune cell co-culture experiments,so as to clarify the regulatory mechanism of CELF2 as a splicing factor on PMT and tumor microenvironment.After bioinformatics analysis and a series of experimental verification,we found that as a molecular marker of PN GBM,CLEF2 is closely related to the good prognosis of patients,and plays a role in the process of inhibiting the proliferation and migration of GBM cells.Transcriptome sequencing results showed that in MES GBM,downregulation of CELF2 could promote exon 6 skipping of fibroblast growth factor receptor 1(FGFR1)pre-mRNA to form more truncated FGFR1 splicing subtype(FGFR1-s).As the signature splicing isoform of MES GBM,the increased expression level of FGFR1-s can promote the secretion of chemokines such as NDGR1(N-myc Downstream Regulated Gene 1)by activating the PI3K-AKT signaling pathway,and then promote the polarization and recruitment of M2 tumor-associated macrophages in the tumor microenvironment.In this study,we demonstrated the molecular mechanism of CELF2 affecting tumor malignant progression through alternative splicing,that is,regulating the expression level of M2 macrophage chemokines by mediating FGFR1 to produce different splicing subtypes,further remodeling the tumor microenvironment and affecting the PMT process.Targeting FGFR1-s and its downstream chemokines may reverse the polarization and recruitment process of M2 TAMs in the immune microenvironment,providing a new strategy for improving the success rate of GBM precision therapy and improving the prognosis of patients.