| Objective: Glioma is a type of primary brain tumor that starts in the glial cells.According to the WHO classification system,gliomas could be basically divided into low-grade gliomas(LGGs)[WHO grade â… -â…¡],indicating a better prognosis and high-grade gliomas(HGGs)[WHO grades â…¢-â…£],indicating a worse prognosis.Glioblastomas(GBMs)are the most common and aggressive malignant gliomas of WHO grade IV,the 5-year survival rate was reported to be less than 7%.Nowadays,besides histology,molecular framework has also been introduced into the diagnoses of central nervous system tumors,and many potential diagnostic,prognostic,and predictive biomarkers have been explored,including isocitrate dehydrogenase(IDH)mutation and 1p/19 q co-deletion,the methylation status of O6-methylguanine-DNA methyl-transferase(MGMT),mutations in telomerase reverse transcriptase(TERT)and the α thalassemia/mental retardation syndrome X-linked(ATRX)gene et al.Nevertheless,it remains a challenge to improve the prognosis due to the gene heterogeneity of gliomas,which calls for some more specific and more effective biomarkers.Heterogeneous nuclear ribonucleoprotein F(hnRNP F),as a critical member of heterogeneous nuclear ribonucleoprotein family,plays important roles in regulating alternative splicing,pre-mRNA processing,mRNA transcription and translation regulation as well as maintaining telomere stability.It was widely reported to be involved in the tumorigenesis and cancer progression.But its roles in gliomas remain undiscovered to this day.In the present study,we detected the expression of hnRNP F in human glioma tissues and cell lines.Its effects on the biological behavior of glioma cells and the intrinsic mechanisms were also explored and discussed.Methods: 1.To explore the differential expression of HNRNPF gene in gliomas of different grades and different histology types,bioinformatics analysis was carried out by the high-throughput sequencing data of GBM and LGG obtained from TCGA and CGGA databases.The correlation between the HNRNPF expression level and the overall survival time was also estimated.2.The expression level of hnRNP F protein was examined by western blot using clinical samples and glioma cell lines.3.Hn RNP F overexpression and knockdown lentivirus as well as the control lentiviruses were constructed and produced by Genechem(Shanghai,China).Stable transfected cell lines were screened using puromycin(3 μg/μL;Sigma)for at least 10 days after infection.4.The effects on proliferation,migration and invasion were investigated by Cell Counting Kit 8(CCK8)assay,5-ethynyl-2-deoxyuridine(Ed U)staining,colony formation assay,matrigel invasion assay and wound healing assay respectively.5.VEGF concentration in cell culture supernatants was detected with Human VEGF ELISA Kit(Proteintech,Wuhan,China).The proangiogenesis ability of glioma cells after transfection was evaluated by tube formation assay.6.The protein levels of epithelial-mesenchymal transition(EMT)related proteins were detected by western blot.7.Transcriptome sequencing and RNA immunoprecipitation and high-throughput sequencing(RIP-seq)were conducted with hnRNP F-knockdown U251 cells.As a result,the mRNAs that were binded and regulated by hnRNP F were screened out.The related pathways were also explored.8.We underwent western blot to evaluate the protein levels of PI3K-Akt pathway.Besides,the specific inhibitor LY294002 and THBS2 overexpression plasmid were used for further validation.9.Subcutaneous tumor xenografts in the nude mice were established to detect the tumorigenic ability in vivo.With the excised tumor tissues from the mice,immunohistochemical analyses were conducted to detect the expression of hnRNP F,EMT-related proteins and Ki-67.10.All the experiments were repeated more than 3 times independently.Graphpad prism 8.0 was used for plotting and statistical analyses(Graph Pad Software,San Diego,CA,USA).T-test was used for comparison,and P < 0.05 was considered to be statistically different.Results: 1.Bioinformatics analysis of gliomas sequencing data obtained from TCGA and CGGA databases showed that the expression of HNRNPF gene was lower in grade I and II gliomas and higher in grade III and IV gliomas.Classified by the histopathologic subtypes and mutant status,we found that in oligoastrocytoma the HNRNPF expression level was the lowest,while in GBM it was the highest.Besides,the expression in IDH mutant and 1p19 q co-deletion group was lower,while the expression in IDH wild type and1p19 q non-deletion group was higher.2.Immunohistochemical study on paraffin sections of clinical samples showed that the hnRNP F expression was overexpressed in GBM.Similarly,in glioma cell lines,we found that the hnRNP F expression was the higher in high-grade gliomas.3.CCK-8 assay,Ed U staining,colony formation assay,matrigel invasion assay and wound healing assay were underwent,confirming that the hnRNP F expression level correlated with the proliferation,migration and invasion capabilities of glioma cells.4.It was revealed that the concentration of VEGF decreased in the medium of hnRNP F-knockdown U251 cells and increased in the medium of hnRNP Foverexpressed U87 cells.Similarly,tube formation assays showed that the proangiogenesis ability of hnRNP F-knockdown U251 cells was decreased,while it was increased in hnRNP F-overexpressed U87 cells.5.Western blot showed that there were significant changes in expression levels of EMT-related proteins like vimentin,MMP-2,N-cadherin and E-cadherin,indicating that hnRNP F was involved in the regulation of EMT process in gliomas.6.The transcriptome sequencing of hnRNP F-knockdown U251 cells was underwent,showing that 216 genes were up-regulated and 52 genes were downregulated.Most of the down-regulated genes were enriched in PI3K-Akt signal pathway.Through RIP-seq assay,10193 genes binding with hnRNP F protein were obtained,only30 genes of which decreased simultaneously at the transcriptome level.THBS2,as one of the 30 genes,encodes THBS2 protein,which is an upstream regulator of the PI3K-Akt pathway.Therefore,it was reasonable to assume that hnRNP F could bind the THBS2 mRNA and regulate its expression,then regulate the biological behaviors of glioma cells via the PI3K-Akt pathway.7.Western blot confirmed that the expression of PI3 K and phosphorylated AKT decreased in hnRNP F-knockdown U251 cells and increased in hnRNP F-expressed U87 cells.LY294002 treatment can suppress the EMT process,cell proliferation,cell invasion and migration caused by hnRNP F-overexpression.q PCR confirmed that the mRNA transcription level of THBS2 changed along with the hnRNP F expression.Meanwhile,transfection of THBS2 plasmid could rescue the inhibitory effects on PI3K-Akt pathway in hnRNP F-knockdown U251 cells,which confirmed its modulatory effects.8.Subcutaneous tumor xenografts in the nude mice showed that silencing hnRNP F reduced the tumorigenicity and overexpression of hnRNP F enhanced the tumorigenicity.Immunohistochemical assays showed the expression of E-cadherin decreased,the expression of N-cadherin increased,which further confirmed the modulatory function of hnRNP F in vivo.Conclusion: 1.hnRNP F was highly expressed in human gliomas and positively correlated with the degree of malignancy.Besides,its expression is associated with the overall survival of patients.2.hnRNP F can promote the proliferation,migration,invasion,EMT process and angiogenesis of human glioma cells.3.hnRNP F could enhance the proliferation,migration and EMT process of human glioma cells via activating PI3K-Akt signaling pathway.4.hnRNP F could regulate the PI3K-Akt pathway activity through modulating the THBS2 expression. |
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