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

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.