tsRNA-08614 inhibits glycolysis and histone lactylation by ALDH1A3 to confer oxaliplatin sensitivity in colorectal cancer

Abstract

Background: Enhanced glycolysis contributes to the chemotherapy resistance of colorectal cancer (CRC). However, whether tRNA-derived small RNAs (tsRNAs) regulate CRC oxaliplatin sensitivity through glycolysis-mediated histone lactylation remains unclear.

Methods: By analyzing RNA-seq data from CRC samples in the TCGA database, we identified a glucose metabolism-related tsRNA. Overexpression of tsRNA-08614 was investigated to explore its impact on CRC sensitivity to oxaliplatin. The targeting gene of tsRNA-08614 was validated through a dual-luciferase reporter assay. The specific molecular mechanism of tsRNA-08614 regulating CRC oxaliplatin sensitivity was further revealed by ChIP-seq and RNA-seq.

Results: We found a down-regulated tsRNA-08614 targeted glycolysis-related gene, which was associated with chemotherapy resistance. Overexpression of tsRNA-08614 promoted oxaliplatin sensitivity of CRC cells. tsRNA-08614 inhibited the expression of the target gene ALDH1A3 and reduced glycolysis, whereas the glycolytic inducer reversed the enhanced sensitivity caused by tsRNA-08614. Interference of tsRNA-08614 increased H3K18la and pan-Kla levels, while the lactate inhibitor partially suppressed these effects. Furthermore, overexpression of tsRNA-08614 reprogrammed the transcription of genes mediated by histone lactylation modification, with EFHD2 showing the most significant differential expression. EFHD2 inhibited the sensitivity of CRC cells to oxaliplatin by upregulating CMPK2 and enhancing mitochondrial function. Finally, we demonstrated that tsRNA-08614 enhanced sensitivity to oxaliplatin in CRC by reducing histone lactylation levels in vivo.

Conclusion: tsRNA-08614 regulates ALDH1A3 to inhibit glycolysis and histone lactylation modification, thereby suppressing the transcriptional activity of EFHD2 and ultimately promoting the sensitivity of CRC to oxaliplatin. These findings suggest that tsRNA-08614 may represent a novel molecular target to combat oxaliplatin resistance in CRC chemotherapy.

Keywords: Colorectal cancer; Glycolysis; Histone lactylation; tsRNA.

Graphical abstract

Fig. 1

Glycolysis-associated tsRNA-08614 is related to oxaliplatin sensitivity in CRC. (A) The correlation heatmap displayed the significant correlations between gene expression and the IC50 values of three drugs. P-value < 0.05 & |r| > 0.3. (B) The heatmap depicted the nine genes related to glycolysis among the oxaliplatin sensitivity genes. P-value < 0.05 & |r| > 0.3. (C) The volcano plot displayed differentially expressed tsRNAs. P-value < 0.05, |log2FC| > 1. (D) The network diagram revealed the binding relationships between 56 tsRNAs and nine target genes. (E) The expression levels of tsRNA-08613 and tsRNA-08614 in HT-29 and SW480 cells treated with oxaliplatin were measured using RT-qPCR (N = 3). NC, PBS-treated group; oxaliplatin, oxaliplatin-treated group. ns, not significant. *P < 0.05; **P < 0.01.

Fig. 2

tsRNA-08614 promotes chemosensitivity in CRC. (A) The overexpression efficiency of tsRNA-08614 in HT-29 and SW480 cells was detected by RT-qPCR. (B) The effect of tsRNA-08614 overexpression on the apoptosis of HT-29 and SW480 cells was detected by flow cytometry. (C) The proliferation of HT-29 and SW480 cells with tsRNA-08614 overexpression was detected by colony formation assay. N = 3. NC, mimics NC group. **P < 0.01.

Fig. 3

tsRNA-08614 inhibits glycolysis by suppressing target genes. (A) The expression level of ALDH1A3 was measured by RT-qPCR in HT-29 cells. (B) WB analysis was performed to validate the effects of tsRNA-08614 overexpression on the expression of ALDH1A3. (C) The binding interaction between tsRNA-08614 and ALDH1A3 was validated by the luciferase reporter assay. (D) Lactate levels were measured in HT-29 and SW480 cells after overexpressing tsRNA-08614 and ALDH1A3. (E) ATP levels were measured in HT-29 and SW480 cells after overexpressing tsRNA-08614 and ALDH1A3. (F) Inducer of glycolysis partially inhibits tsRNA-08614-mediated CRC chemotherapy sensitivity. N = 3. *P < 0.05; **P < 0.01.

Fig. 4

tsRNA-08614 inhibits histone lactylation modification in CRC cells. tsRNA-08614 was knocked down in HT-29 and SW480 cells, and they were incubated with 10 mM oxamate (a lactate inhibitor) for 4 h. Immunofluorescence was performed to detect the level of H3K18la in HT-29 (A) and SW480 cells (B). WB was conducted to detect H3K18la and pan-Kla in HT-29 (C) and SW480 cells (D). N = 3. NC, inhibitor NC group. *P < 0.05; **P < 0.01.

Fig. 5

tsRNA-08614 reprograms the transcription of genes associated with histone lactylation modification. (A) tsRNA-08614 was overexpressed in HT-29 cells and ChIP-seq analysis was performed with H3K18la antibody. The circos plot illustrated the distribution of histone lactylation modification peaks between the tsRNA-08614 overexpression group and the control group. (B) The Venn diagram showed the number of lactylation modification peaks (left) and genes (right). (C) MA plot displayed the changes in differential peaks of lactylation modification in the tsRNA-08614 group and the control group (|M.value| > 1, P < 0.05). (D) A density distribution analysis was conducted to assess the lactylation modification levels between the tsRNA-08614 group and the control group. (E) KEGG analysis was performed on the differentially lactylated genes. NC, mimics NC group. mimics, tsRNA-08614 mimics group.

Fig. 6

tsRNA-08614 inhibits EFHD2 transcription through lactylation modification, thereby promoting CRC sensitivity to oxaliplatin. (A) The visualization of lactylation modification peaks on the seven selected genes. (B) ChIP-PCR was conducted to validate the H3K18la levels of seven candidate target genes (N = 3). (C) The overexpression efficiency of EFHD2 in HT-29 cells was verified by WB (N = 3). NC, vector group; EFHD2-OE, EFHD2 overexpression group. (D) The effects of tsRNA-08614 and EFHD2 on CRC cell proliferation were assessed through clone formation assay (N = 3). *P < 0.05; **P < 0.01.

Fig. 7

RNA-seq revealed that the transcriptome landscape of HT-29 cells changes after EFHD2 overexpression. (A) EFHD2 was overexpressed in HT-29 cells and then transcriptome sequencing was performed. The heatmap displayed gene expression levels in the EFHD2-OE group and control group. (B) The volcano plot showed DEGs between the EFHD2-OE group and the control group (log2FC > 1 or < -1, FDR < 0.05). (C) GO functional enrichment analysis was conducted on DEGs following EFHD2 overexpression. (D) KEGG analysis revealed the signaling pathways enriched with DEGs.

Fig. 8

EFHD2 enhances mitochondrial function and promotes CRC oxaliplatin resistance by upregulating CMPK2. (A) RT-qPCR was used to detect the expression level of CMPK2 after EFHD2 overexpression in HT-29 cells. (B) The effect of EFHD2 and CMPK2 on HT-29 cell proliferation was verified through colony formation assays. (C) The effects of EFHD2 overexpression and CMPK2 knockdown on DNA damage marker γ-H2AX were detected by WB. (D) JC-1 staining was used to detect changes in mitochondrial membrane potential in HT-29 cells. The green fluorescence represents the JC-1 monomer and the red fluorescence represents the JC-1 aggregate. *P < 0.05; **P < 0.01.

Fig. 9

The promotion of CRC sensitivity to oxaliplatin by tsRNA-08614 was validated in vivo. (A) The subcutaneous xenograft tumor model was established, with tsRNA-08614 antagomir injected intratumorally, followed by the intraperitoneal administration of oxaliplatin. The changes in tumor volume during tumor formation were detected (N = 5). (B) The effect of tsRNA-08614 on the weight of isolated tumors (N = 5). (C) Immunofluorescence analysis was performed to assess the effect of tsRNA-08614 on H3K18la levels in the tumor (N = 3). tsRNA-08614 antagomir, tsRNA-08614 knockdown group. Oxamate, lactate inhibitor. *P < 0.05; **P < 0.01.