GPX8 deficiency–induced oxidative stress reprogrammed m6A epitranscriptome of oral cancer cells

Figures & data

Figure 1. The deletion of GPX8 induced oxidative stress in cells. (a) Editing of the GPX8 gene resulted in the insertion of a guanine nucleotide at codon 5 of the GPX8 coding sequence. (b) The edited GPX8 gene caused a frameshift mutation and premature termination of translation at codon 43. (c) GPX8 expression was not detected in GPX8-KO SCC-9 cells. (d, e) Compared with wild-type SCC-9 cells, GPX8-deficient SCC-9 cells increased ROS production. **P < 0.01.

Figure 2. Distribution of m⁶A peaks across the mRNA transcripts and the representative m⁶A motifs enriched. (a) The m⁶A peaks were highly enriched in 3`UTR and stop codon regions. (b) The m⁶A motifs with typical conserved sequence in SCC-9 cells. (c) The m⁶A motifs with typical conserved sequence in GPX8-KO SCC-9 cells. NC, SCC-9 cells; KO, GPX8-KO SCC-9 cells.

Figure 3. GO function and KEGG pathway enrichment of differentially methylated mRNA. (a) Top 20 significantly enriched GO terms. (b) Top 20 significantly enriched KEGG pathways.

Table 1. The 20 coding genes with the most significantly altered m⁶A peaks in GPX8-KO SCC-9 cells compared with SCC-9 cells.

Hyper-methylated		Hypo-methylated
Genes	Peak region	Genes	Peak region
TCAF1, TRPM8 channel associated factor 1	exonic	CDADC1, cytidine and dCMP deaminase domain containing 1	5’ UTR
EIF3C, eukaryotic translation initiation factor 3 subunit C	exonic	CXCL8, C-X-C motif chemokine ligand 8	exonic, 3’ UTR
RGPD6, RANBP2 like and GRIP domain containing 6	exonic, 3’ UTR	HSF2BP, heat shock transcription factor 2 binding protein	3’ UTR
SERF1B, small EDRK-rich factor 1B	3’ UTR	SP8, Sp8 transcription factor	5’ UTR
INHBE, inhibin subunit beta E	3’ UTR	SLC31A1, solute carrier family 31 member 1	5’ UTR
KLHL3, kelch like family member 3	3’ UTR	PBLD, phenazine biosynthesis like protein domain containing	3’ UTR
ROBO3, roundabout guidance receptor 3	5’ UTR	TANGO6, transport and golgi organization 6 homolog	intronic
CLEC18B, C-type lectin domain family 18 member B	3’ UTR	TCEANC2, transcription elongation factor A N-terminal and central domain containing 2	3’ UTR
RGPD2, RANBP2 like and GRIP domain containing 2	3’ UTR	MORF4L1, mortality factor 4 like 1	5’ UTR
CTAGE15, CTAGE family member 15	exonic	PKP4, plakophilin 4	5’ UTR

Table 2. Differential expression of oxidative stress-related genes in GPX8-KO SCC-9 cells.

Genes	log2FC	m^6A change
NCF2, neutrophil cytosolic factor 2	7.14	-
PTGS2, prostaglandin-endoperoxide synthase 2	4.70	-
STC2, stanniocalcin 2	3.84	-
ETV5, ETS variant transcription factor 5	2.80	3’ UTR, exonic/down
SLC7A11, solute carrier family 7 member 11	2.58	3’ UTR/down
NOX5, NADPH oxidase 5	2.16	-
PRNP, prion protein	1.88	3’ UTR/up
CYBB, cytochrome b-245 beta chain	1.83	-
RBPMS, RNA binding protein, mRNA processing factor	1.77	3’ UTR/up
SOD2, superoxide dismutase 2	1.76	3‘UTR, intronic/down; 3’ UTR/up
MCTP1, multiple C2 and transmembrane domain containing 1	1.76	-
ZC3H12A, zinc finger CCCH-type containing 12A	1.70	-
BCL2, BCL2 apoptosis regulator	1.65	-
DHRS2, dehydrogenase/reductase 2	1.33	-
NCOA7, nuclear receptor coactivator 7	1.30	3’ UTR, exonic/down
ATF4, activating transcription factor 4	1.26	5’ UTR/down
SLC1A1, solute carrier family 1 member 1	1.15	-
RBM11, RNA binding motif protein 11	1.10	-
SESN2, sestrin 2	1.10	-
CYBA, cytochrome b-245 alpha chain	−1.79	3‘UTR/up; 5’ UTR/down
PPARGC1A, PPARG coactivator 1 alpha	−1.73	3’ UTR/down
NQO1, NAD(P)H quinone dehydrogenase 1	−1.46	3‘UTR/up; 5’ UTR/down
PRKD1, protein kinase D1	−1.40	3’ UTR/up
GPX8, glutathione peroxidase 8	−1.40	5’ UTR/down
HSPA1A, heat shock protein family A (Hsp70) member 1A	−1.15	-
IDH1, isocitrate dehydrogenase (NADP(+)) 1	−1.10	3‘UTR/up; 5’ UTR/down
NR4A2, nuclear receptor subfamily 4 group A member 2	−1.10	3’ UTR/down
ENDOG, endonuclease G	−1.09	-

Figure 4. Transcriptional activity of the whole cell and differentially expressed genes. (a) GPX8-KO SCC-9 and SCC-9 cells have similar overall transcriptional activity. (b) Volcano plots of differentially expressed genes between GPX8-KO SCC-9 and SCC-9 cells. |log₂FC|≥1.0 and P < 0.05. KO, GPX8-KO SCC-9 cells; NC, SCC-9 cells.

Table 3. The top 20 differentially expressed coding genes in GPX8-KO SCC-9 cells compared with SCC-9 cells.

Upregulated	log2FC	Downregulated	log2FC
CXCL10, C-X-C motif chemokine ligand 10	∞	RANBP3L, RAN binding protein 3 like	−7.49
CCL20, C-C motif chemokine ligand 20	∞	C2CD4A, C2 calcium dependent domain containing 4A	−5.10
IFNL1, interferon lambda 1	∞	VAV3, vav guanine nucleotide exchange factor 3	−5.02
CXCL1, C-X-C motif chemokine ligand 1	9.62	LGSN, lengsin, lens protein with glutamine synthetase domain	−4.48
IL13RA2, interleukin 13 receptor subunit alpha 2	8.28	NOG, noggin	−4.40
CCL5, C-C motif chemokine ligand 5	8.17	FRMD3, FERM domain containing 3	−4.00
OASL, 2’−5’-oligoadenylate synthetase like	8.14	PRG4, proteoglycan 4	−3.90
CXCL11, C-X-C motif chemokine ligand 11	7.51	SLC12A3, solute carrier family 12 member 3	−3.80
CCND1, cyclin D1	7.46	PLK2, polo like kinase 2	−3.70
IL33, interleukin 33	7.40	IGFBP5, insulin like growth factor binding protein 5	−3.63

Note: FPKM>2 in up-regulated genes.

Figure 5. Distribution of differentially expressed genes with differential m⁶A peaks. Hyper-up, m⁶A peak upregulated and mRNA expression upregulated; Hyper-down, m⁶A peak upregulated and mRNA expression downregulated; Hypo-up, m⁶A peak downregulated and mRNA expression upregulated; Hypo-down, m⁶A peak downregulated and mRNA expression downregulated.

Figure 6. GO function and KEGG pathway enrichment of differentially expressed genes with differential m⁶A peaks. (A) Top 20 significantly enriched GO terms. (B) Top 20 significantly enriched KEGG pathways.

Table 4. The first 10 differentially expressed genes with differential m⁶A peaks in GPX8-KO SCC-9 cells compared with SCC-9 cells.

Hypo-methylated genes	log2FC	Hyper-methylated genes	log2FC
Upregulation of expression
CXCL1, C-X-C motif chemokine ligand 1	9.62	CCL5, C-C motif chemokine ligand 5	8.17
DNER, delta/notch like EGF repeat containing	7.05	SLC6A15, solute carrier family 6 member 15	5.92
IFIT2, interferon induced protein with tetratricopeptide repeats 2	6.79	ALCAM, activated leukocyte cell adhesion molecule	5.23
FN1, fibronectin 1	6.56	RAET1L, retinoic acid early transcript 1 L	5.03
RSAD2, radical S-adenosyl methionine domain containing 2	6.30	INHBA, inhibin subunit beta A	4.55
IL6, interleukin 6	5.93	ANTXR2, ANTXR cell adhesion molecule 2	4.54
IFIT1, interferon induced protein with tetratricopeptide repeats 1	5.92	NRP1, neuropilin 1	4.37
SP8, Sp8 transcription factor	5.64	SERPINE1, serpin family E member 1	4.29
CXCL8, C-X-C motif chemokine ligand 8	5.45	DUSP6, dual specificity phosphatase 6	3.83
ADM2, adrenomedullin 2	4.77	CFB, complement factor B	3.74
Downregulation of expression
LGSN, lengsin, lens protein with glutamine synthetase domain	−4.48	FRMD3, FERM domain containing 3	−4.00
PLK2, polo like kinase 2	−3.70	PRG4, proteoglycan 4	−3.90
IGFBP5, insulin like growth factor binding protein 5	−3.63	GLP2R, glucagon like peptide 2 receptor	−3.55
DAB2, DAB adaptor protein 2	−2.71	SFRP1, secreted frizzled related protein 1	−3.47
TSHZ2, teashirt zinc finger homeobox 2	−2.53	KCNH5, potassium voltage-gated channel subfamily H member 5	−3.14
MTARC1, mitochondrial amidoxime reducing component 1	−2.50	MVD, mevalonate diphosphate decarboxylase	−2.97
STEAP4, STEAP4 metalloreductase	−2.33	GATD3B, glutamine amidotransferase like class 1 domain containing 3B	−2.80
ATP1B1, ATPase Na+/K+ transporting subunit beta 1	−2.29	DAB2, DAB adaptor protein 2	−2.71
CDK10, cyclin dependent kinase 10	−2.19	H3C10, H3 clustered histone 10	−2.49
SPIRE2, spire type actin nucleation factor 2	−2.18	H3C8, H3 clustered histone 8	−2.47

Note: FPKM>2 in up-regulated genes.

Figure 7. Expression of m⁶A regulatory genes detected by real-time RT-PCR. (A) Expression of m⁶A regulators in GPX8-KO SCC-9 and SCC-9 cells. (B) Expression of m⁶A regulators after hydrogen peroxide treatment within the indicated time. All bars indicate the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Data availability statement

All data generated or analysed during this study are available from the corresponding author upon reasonable request (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?) with GEO accession number GSE224718.