AP001885.4 promotes the proliferation of esophageal squamous cell carcinoma cells by histone lactylation- and NF-κB (p65)-dependent transcription activation and METTL3-mediated mRNA stability of c-myc

Abstract

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignant neoplasm, and up to now, the role of long non-coding RNA (lncRNA) AP001885.4 in cancer, including ESCC, is absolutely unclear. The GEPIA database was applied to identify differentially expressed and prognosis-associated genes in esophageal cancer (ESCA). CCK-8, colony formation, Western blot, and qRT-PCR methods were harnessed to investigate the role and mechanism of AP001885.4 in esophageal carcinogenesis. By analyzing TCGA data in the GEPIA database, two lncRNAs were selected. AP001885.4 was overexpressed and positively associated with the unfavorable outcome of ESCC patients, and LINC001786 was under-expressed and negatively linked with the poor prognosis. Knockdown of AP001885.4 suppressed the proliferation and colony formation of ESCC cells. Importantly, the silence of AP001885.4 downregulated c-myc. Mechanically, the knockdown of AP001885.4 reduced METTL3 expression and m6A modification in c-myc mRNA, and METTL3 positively regulated c-myc. Furthermore, the knockdown of AP001885.4 diminished histone lactylation and NF-κB (p65) expression, and the protein lactylation inhibitors (2-DG, 2-deoxy-D-glucose and oxamate) and the NF-κB inhibitor (JSH-23) also lessened c-myc expression. Consequently, our findings suggested that AP001885.4 promoted the proliferation of esophageal squamous cell carcinoma cells by histone lactylation- and NF-κB (p65)-dependent transcription activation and METTL3-mediated mRNA stability of c-myc.

Keywords: AP001885.4; C-myc; ESCC; NF-κB; lactylation.

Figure 1.

AP001885.4 is elevated and positively correlated with poor prognosis in esophageal cancer. (A) Method of identifying differentially-expressed and prognosis-associated lncRNAs in ESCA. (B-C) The expression level and prognostic value of AP001885.4 (also known as RP11-157K17.5 and ENSG00000179038.8) in ESCA. (D) Pan-cancer expression of AP001885.4. (E) Amplification of the KDM2A gene in cancers was analyzed using the TCGA Copy Number portal database. (F) The expression level of AP001885.4 in ESCC tissues (T) and adjacent normal tissues (N). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 2.

Knockdown of AP001885.4 suppresses the proliferation and colony formation of esophageal squamous cell carcinoma cells. (A) Genomic location of AP001885.4. (B) Sub-cellular location prediction of AP001885.4 using the lncLocator database. (C) Coding potential prediction of AP001885.4 using the CPC2.0 (Coding Potential Calculator 2) database. (D) Expression levels of AP001885.4 two transcripts in KYSE150, KYSE450, and EC109 cells. (E) Expression level of AP001885.4 in 8 ESCC cell lines. (F) Confirmation of knockdown efficiency using qRT-PCR assay. The proliferation of KYSE150 (G) and KYSE450 (H) was detected using CCK-8 assay. (I) Detection of colony formation ability after AP001885.4 knockdown. mRNA levels of CCND1 (J) and CCNE1 (K) were determined by the qRT-PCR method. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3.

Knockdown of AP001885.4 downregulates c-myc in a METTL3-dependent way. After transfection of AP001885.4 siRNAs, the RNA level of AP001885.4 (A) and protein as well as mRNA levels of c-myc (B and C) were detected using qRT-PCR and Western blot assays. After AP001885.4 knockdown, the protein expression of METTL3, ALKBH5 and ELAVL1 (D) and the mRNA level of METTL3 (E) were measured. The m6A modification of c-myc mRNA was evaluated using RIP assay (F and G). GSE53622 and GSE53624 datasets and TCGA data (in ENCORI and GEPIA databases) were harnessed to analyze METTL3 expression level and correlation between METTL3 and c-myc (H-L). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 4.

Knockdown of AP001885.4 downregulates c-myc via restraining histone lactylation. After AP001885.4 knockdown, ATP level (A), lactate level (B), H3K18 lactylation level (C), and protein pan-lactylation level (D) were detected using cellular ATP, lactate, and Western blot assays. (E-G) 2-DG and oxamate treatment inhibited H3K18 lactylation and protein pan-lactylation. (H-K) Protein and mRNA levels of c-myc in 2-DG- and oxamate-treated cells were measured using Western blot and qRT-PCR assays. (L-N) mRNA and protein levels of c-myc were detected in LDHA-silenced cells. (O) The histone lactylation in the promoter and coding sequence of MYC was detected using ChIP-PCR. (P-R) Correlation analysis was performed using TCGA data in the GEPIA database. (S and T) The RNA level of AP001885.4 after 2-DG and oxamate treatment was detected by qRT-PCR technology. (U and V) The H3K18la level in the promoter of AP001885.4 was analyzed using the ChIP-PCR method. ns, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 5.

AP001885.4 interacted with LDHA and LDHB in ESCC cells. (A) AP001885.4 interacted proteins including LDHA and LDHB were predicted using the catRAPID software. (B) The complex of AP001885.4 and LDHA/LDHB was analyzed using the AlphaFold 3 software. (C) The interaction of AP001885.4 and LDHA/LDHB was detected using the RIP method. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6.

Knockdown of AP001885.4 downregulates c-myc via inactivating NF-κB signaling pathway. (A) Protein and mRNA levels of NF-κB (p65; gene name: RELA) after AP001885.4 knockdown were detected using Western blot and qRT-PCR methods. (C and D) Protein and mRNA levels of c-myc were measured by Western blot and qRT-PCR assays. (E-G) The binding ability between NF-κB (p65) and the promoter of MYC was detected using the ChIP-PCR assay. (H and I) The m6A level of NF-κB (p65) was detected using the RIP-PCR method. (J) Correlation between NF-κB (p65) and c-myc was analyzed using the GEPIA database. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 7.

The main findings of this article.