WTAP regulates Mitochondrial damage and Lipid oxidation in HCC by NOA1 mediated m6A modification

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. However, the molecular mechanism underlying the occurrence and development of HCC remains unclear. We are interested in the function of m6A methylation enzyme WTAP in the occurrence and development of HCC. Methods: Expression of the m6A methylation-associated enzymes in paired carcinoma and adjacent tissues (N=17) were detected by RT-PCR. Electron microscopy was adopted to observe the subcellular organelle. GPX4 levels in hepatoma cells were analyzed by Western blot and RT-PCR. The Fe²⁺ and GSH/GSSG levels were detected using the corresponding kits. Mass spectrometry (MS) was conducted to determine the altered protein types in hepatoma cells. Finally, methylated RNA immunoprecipitation (MeRIP) was used to analyze the m6A methylation of Nitric oxide-associated protein 1 (NOA1). Results: RT-PCR showed that there were no significant differences among tumor tissues and normal tissues in METTL3 (p=0.6485), FTO (p=0.1158), ALKBH (p=0.6148), YTH N6-Methyladenosine RNA binding protein F1 (YTHDF1) (p=0.3171), and YTH N6-Methyladenosine RNA binding protein F2 (YTHDF2) (p=0.1116). However, compared to normal tissue, WTAP (p=0.0011), METLL14 (p=0.0044) and YTH N6-Methyladenosine RNA binding protein F3 (YTHDF3) (p=0.0472) were obviously decreased in tumor tissues. The decrease of WTAP was most apparent. Conditional knockout of WTAP in Huh-7 and SNU-449 cells could induce mitochondria damage, which was manifested in smaller mitochondria and a compressed intermembrane space of mitochondria. The result was also confirmed by electron microscopy. Additionally, Huh-7 and SNU-449 cells with WTAP knockdown presented low mitochondrial membrane potential, while WTAP overexpression could reverse this effect. Interestingly, data from flow cytometry by Annexin V-FITC/PI and detection of pyroptosis-related marker Gasdermin D (GSDMD) by Western blot demonstrated that, overexpressing or knocking down WTAP will not affect cell apoptosis and pyroptosis in hepatoma cells. Furthermore, mRNA and protein levels of the key indicator GPX4 of ferroptosis in Huh-7 and SNU-449 cells with WTAP knockdown or overexpression were analyzed by RT-PCR and Western blot. It was shown that knockdown of WTAP promoted expressions of GPX4 in these cells (p<0.0001), but a distinct downregulation of GPX4 levels occurred in the WTAP overexpressing cells. Further study indicated that a significantly increase of GSH/GSSG levels and clearly decrease of Fe²⁺ concentrations appeared in Huh-7 and SNU-449 cells with WTAP knockdown (p<0.05). Opposite results were observed in the cells with WTAP overexpression (p<0.05). Moreover, we also clarified the effect of WTAP on modulating GSH synthesis might be independent of SLC7A11, not SLC3A2 or the Xc-system. Finally, mass spectrometry results showed that NOA1 might be related to WTAP. qPCR, WB and MeRIP-qPCR also confirmed WTAP regulated the m6A methylation of NOA1. It is supposed that NOA1 might be the molecule at the heart of the regulation mechanism by WTAP. Conclusion: WTAP may affect the m6A methylation of NOA1 to induce mitochondrial damage, meanwhile activate the GPX4-axis to inhibit the lipid oxidation, resulting in the development of HCC.

Keywords: HCC; Lipid oxidation; Mitochondrial damage; NOA1; WTAP.

Figure 1

mRNA expression of WTAP is decreased in tumors compared to normal tissues. 17 pairs of tissues (tumor and normal tissues) were collected. The total RNA was extracted by TRIzol. RT-PCR was used to detect the mRNA levels of genes related to the m6A methylation complex. Paired t-test was used to analyze the results. Compared with normal group.

Figure 2

Overexpressed plasmid and siRNA were used to regulate WTAP expression. Two siRNAs of WTAP were designed and synthesized. The overexpressed plasmid pcDNA3.1-WTAP was constructed. Huh-7 and SNU-449 cells were transfected with the siRNAs or plasmid by Lipofectamine™ 3000. 48h later, the total RNA or cell lysates were collected. Western blot (A, D) and RT-PCR (B-C, E-F) were used to detect the mRNA and protein levels of WTAP. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.001 compared with control groups.

Figure 3

Mitochondrial damage occurred in SNU-449 treated with siRNA of WTAP. SNU-449 cells were transfected with the siRNAs or plasmid with Lipofectamine™ 3000. (A) 48h later cells were collected and fixed, then electron microscopy was used to view the mitochondrial morphology, scale bar=5μm (left), scale bare=1μm (right). (B) JC-1 was used to detect the mitochondrial membrane potential, scale bar=100μm, CCCP (10 mM) is added to the cell culture medium in the ratio of 1:1000 as positive control. CCCP, Carbonyl cyanide 3-chlorophenylhydrazone, a proton carrier (H+ ionophore), is a potent uncoupler of mitochondrial oxidative phosphorylation that induces permeability of the inner mitochondrial membrane to H+, leading to a loss of membrane potential on both sides of the inner mitochondrial membrane.

Figure 4

WTAP is independent of the apoptosis of SNU-449 cells. The siRNAs and plasmid were co-transfected into SNU-449 cells by lipo3000. 48h later, cells were digested with trypsin and stained with Annexin V-FITC and PI using the apoptosis detection kit, then cells were analyzed by flow cytometry. (A) Representative flow cytometry images. (B) The apoptosis level of SNU-449 cells transfected with siRNA. (C) The apoptosis level of SNU-449 cells transfected with pcDNA3.1 and pcDNA3.1-WTAP. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.001 compared with control groups; ns, no significance.

Figure 5

WTAP is independent of the pyroptosis of Huh-7 and SNU-449 cells. The siRNAs and plasmid were co-transfected to Huh-7 (A) and SNU-449 (B) cells by Lipofectamine™ 3000. 48h later, cell lysates were collected and WB was used to detect the expression of GSDMD, which was activated in pyroptosis.

Figure 6

WTAP is related to GPX4 expression to regulate the lipid oxidation in hepatoma cells. Huh-7 and SNU-449 cells were transfected with the siRNAs or plasmid by Lipofectamine™ 3000. 48h later, total RNA or cell lysates were collected, RT-PCR and Western blot were used to detect the mRNA and protein levels of GPX4 (A-F). Fe2+ (G-J), LPO (K-N), GSH (O-P), and GSSG (Q-R) levels were detected. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.001 compared with control groups.

Figure 7

WTAP is independent of SLC7A11, not SLC3A2 or Xc^- system in Huh-7 and SNU-449 cells. The siRNAs or plasmid were co-transfected to Huh-7 and SNU-449 cells by Lipofectamine™ 3000. 48h later, the total RNA or cell lysates were collected. RT-PCR and Western blot were used to detect the mRNA and protein levels of SLC7A11 (A-F) and SLC3A2 (G-L). *p < 0.05 compared with control groups.

Figure 8

WTAP affects the m6A methylation of NOA1. (A) SNU-449 cells were transfected with siRNAs or plasmid using Lipofectamine™ 3000. 48h later, LC/MS was used to analyze the protein expression of NOA1. (B-G) Huh-7 and SNU-449 cells were transfected with the siRNAs or plasmid by Lipofectamine™ 3000. 48h later, total RNA or cell lysates were collected. RT-PCR and Western blot were used to detect the mRNA and protein levels of NOA1. (H-I) SNU-449 cells were transfected with siRNAs or plasmid using Lipofectamine™ 3000. 48h later, MeRIP-qPCR was used to detect the m6A mRNA methylation of NOA1. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.001 compared with control groups.

Figure 9

The putative mechanism of WTAP in this study. WTAP affects mRNA m6A methylation of NOA1. Lower WTAP finally induces mitochondrial damage, affects GPX4 expression, Fe²⁺ and GSH/GSSH levels to inhibit lipid oxidation, resulting in modulating the pathogenesis and development of HCC.