YTHDF2 facilitates UBXN1 mRNA decay by recognizing METTL3-mediated m6A modification to activate NF-κB and promote the malignant progression of glioma

Abstract

Background: The prognosis for diffuse gliomas is very poor and the mechanism underlying their malignant progression remains unclear. Here, we aimed to elucidate the role and mechanism of the RNA N6,2'-O-dimethyladenosine (m⁶A) reader, YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), in regulating the malignant progression of gliomas.

Methods: YTHDF2 mRNA levels and functions were assessed using several independent datasets. Western blotting, quantitative polymerase chain reaction, and immunohistochemistry were used to evaluate the expression levels of YTHDF2 and other molecules in human and mouse tumor tissues and cells. Knockdown and overexpression were used to evaluate the effects of YTHDF2, methyltransferase-like 3 (METTL3), and UBX domain protein 1 (UBXN1) on glioma malignancy in cell and orthotopic xenograft models. RNA immunoprecipitation (RIP), methylated RIP, and RNA stability experiments were performed to study the mechanisms underlying the oncogenic role of YTHDF2.

Results: YTHDF2 expression was positively associated with a higher malignant grade and molecular subtype of glioma and poorer prognosis. YTHDF2 promoted the malignant progression of gliomas in both in vitro and in vivo models. Mechanistically, YTHDF2 accelerated UBXN1 mRNA degradation via METTL3-mediated m⁶A, which, in turn, promoted NF-κB activation. We further revealed that UBXN1 overexpression attenuated the oncogenic effect of YTHDF2 overexpression and was associated with better survival in patients with elevated YTHDF2 expression.

Conclusions: Our findings confirmed that YTHDF2 promotes the malignant progression of gliomas and revealed important insight into the upstream regulatory mechanism of NF-κB activation via UBXN1 with a primary focus on m⁶A modification.

Keywords: Glioblastoma; METTL3; N6,2′-O-Dimethyladenosine; NF-κB activation; YTHDF2.

Fig. 1

Expression and prognostic value of YTHDF2 in glioma. A–C The mRNA expression of YTHDF2 in primary gliomas with different WHO grade (A), IDH-mutant status (B), and classifications in CGGA microarray cohort. *P < 0.05; **P < 0.01; ****P < 0.0001. (D) The mRNA expression of YTHDF2 in primary and recurrent gliomas in CGGA microarray cohort. *P < 0.05. E–H The mRNA expression of YTHDF2 in gliomas stratified by different clinicopathological features in CGGA RNA-seq cohort (n = 693). **P < 0.01; ***P < 0.001; ****P < 0.0001. I Representative immunohistochemistry images of YTHDF2 protein in gliomas with different IDH status and histological grades. Bar = 50 µm. J The semi-quantitative for the immunohistochemistry results of YTHDF2 proteins. ***P < 0.001; ****P < 0.0001. K The protein expression of YTHDF2 in IDH-wildtype gliomas and in vitro cell models with different histological grades. L The protein expression of YTHDF2 in IDH-mutant gliomas with different histological grade. M–P Kaplan–Meier curves of gliomas and GBM from CGGA microarray cohort (M, N) and CGGA RNA-seq cohort (O, P) stratified by YTHDF2 expression. Q Venn diagram shows the overlap genes whose expression is positively correlated with YTHDF2 expression in primary gliomas form different datasets. R GO analysis terms of the 1493 genes whose expression are positively correlated with YTHDF2 expression in primary gliomas from four datasets

Fig. 2

YTHDF2 could facilitate the malignant phenotype of glioma cells. A YTHDF2 protein expression in glioma cells with or without YTHDF2 siRNA. All of raw images have been shown in Additional file 3. B, C Transwell analysis of cells with or without YTHDF2 siRNA. **P < 0.01 and ***P < 0.001. Bar = 50 μm. D CCK-8 assay (proliferation assay) of cells with or without YTHDF2 siRNA. YTHDF2 siRNA 1 versus scrambled siRNA. **P < 0.01; ***P < 0.001; ****P < 0.0001; YTHDF2 siRNA2 versus scrambled siRNA. ^#P < 0.05; ^####P < 0.0001. E Scratch assay of LN229 cells with or without YTHDF2 siRNA. Bar = 50 μm. F Gap closure rate of cells with or without YTHDF2 siRNA. G YTHDF2 protein expression in glioma cells with or without YTHDF2 overexpression. H, I Transwell analysis of cells with or without YTHDF2 overexpression. ***P < 0.001 and ****P < 0.0001. Bar = 50 μm. J, K CCK-8 assay (proliferation assay) of cells with or without YTHDF2 overexpression. *P < 0.05; **P < 0.01; and ****P < 0.0001. L Scratch assay of LN229 cells with or without YTHDF2 overexpression. Bar = 50 μm. M Gap closure rate of cells with or without YTHDF2 overexpression

Fig. 3

YTHDF2 may active NF-κB through suppressing its negative regulators. A KEGG pathways of the genes that positively correlated with YTHDF2 expression in gliomas from four datasets. The blue point shows the − log P value, and the red box shows the gene counts in each terms. B, C GSEA of hallmarks genes with increased expression in YTHDF2 high expression groups. D Immunostaining image showing NF-κB (p65) protein expression and localization in N33 cells with or without YTHDF2 siRNA. Bar = 10 μm. E Venn diagram shows the overlap genes whose expression is negatively correlated with YTHDF2 expression in primary gliomas form different datasets and negatively regulate NF-κB signaling. F Correlation of YTHDF2 expression and the 5 selected genes in gliomas from CGGA microarray and TCGA RNA-seq datasets

Fig. 4

YTHDF2 can activate NF-κB through suppressing UBXN1 expression. A UBXN1 mRNA expression in cells with or without YTHDF2 siRNA. *P < 0.05; **P < 0.01; ****P < 0.0001. B YTHDF2, p65, pp65, and UBXN1 protein expression in cells with or without YTHDF2 siRNA. C–E The quantified data of protein expression levels in cells with or without YTHDF2 siRNA. *P < 0.05; **P < 0.01; ***P < 0.001. F UBXN1 mRNA expression in cells with or without YTHDF2 overexpression (OE). **P < 0.01; ***P < 0.001. G YTHDF2, p65, pp65, and UBXN1 protein expression in cells with or without YTHDF2 OE

Fig. 5

YTHDF2 can facilitate UBXN1 mRNA decay through recognizing m⁶A modification on UBXN1 mRNA that mediated by METTL3. A MeRIP-sequencing data of UBXN1 in U87 cells with or without shRNA-mediated METTL3 knockdown (METTL3 KD). B MeRIP-PCR data shows the relative quantity of UBXN1 mRNA immunoprecipitated by the m⁶A antibody (m⁶A-IP) and IgG in cells with or without METTL3 KD. **P < 0.01; ***P < 0.001; **P < 0.0001. C, D RIP-PCR showing the content of UBXN1 mRNA immunoprecipitated by METTL3 and YTHDF2 antibodies. IgG antibodies were used as negative control. ****P < 0.0001. E RIP-qPCR showing the content of UBNX1 immunoprecipitated by YTHDF2 antibodies in U87 cells with or without METTL3 shRNA. F The stability of UBXN1 mRNA in U87 cells with or without YTHDF2 siRNA. G The stability of UBXN1 mRNA in U87 cells with or without YTHDF2 overexpression (OE). H The expression levels of UBXN1 mRNA in U87 cells with or without METTL3 shRNA. I The expression levels of UBXN1 mRNA in U87 cells with or without METTL3 OE. J The stability of UBXN1 mRNA in U87 cells with or without METTL3 shRNA. K The stability of UBXN1 mRNA in U87 cells with or without METTL3 OE. L METTL3, UBXN1, pp65, and p65 protein expression in U87 cells with or without YTHDF2 OE

Fig. 6

UBXN1 overexpression could attenuate malignant progression and NFKB activation that induced by YTHDF2 overexpression in glioma cells. A CCK-8 assay (proliferation assay) of U87 cells with or without YTHDF2 and UBXN1 OE ****P < 0.0001. B Transwell analysis of U87 cells with or without YTHDF2 and UBXN1 OE. ***P < 0.001. Bar = 50 μm. C YTHDF2, UBXN1, pp65, and p65 protein expression in U87 cells with or without YTHDF2 and UBXN1 OE. D–F Kaplan–Meier curves of gliomas with higher (larger than the median expression levels of respective cohorts) expression YTHDF2 stratified by UBXN1 expression in different datasets

Fig. 7

YTHDF2 and METTL3 promotes tumor growth and leads to a worse prognosis in an orthotopic xenograft model. A Representative tumor bioluminescence images of mice at 10, 20, and 30 days after tumor implantation in an orthotopic xenograft model generated by U87 cells transfected with an empty vector, YTHDF2 OE, and METTL3 OE vector. The representative magnetic resonance imaging of mice at 21 days after tumor implantation were also shown in the right panel. B The bioluminescence intensity of mice at 10, 20, and 30 days after tumor implantation. C Kaplan–Meier survival curve of nude mice with tumors. D Representative images of H&E and immunohistochemical staining for YTHDF2, UBXN1, and p65 in the tumors of nude mice. Bar = 3 mm in the left panel. Bar = 10 μm in the right panel. E The mechanistic scheme by which YTHDF2 facilitates UBXN1 mRNA decay via recognizing m⁶A modification that mediated by METTL3 to activate NF-κB and promote tumor malignancy