[Knockdown of Cav1 inhibits mitochondrial function and mRNA m6A modification and expression of key genes in mouse hepatocytes].

OBJECTIVES: To investigate the role of Cav1 gene in regulating mitochondrial function and mRNA m6A modification and expressions of the key genes in mouse hepatocytes. METHODS: In mouse hepatocyte AML12 cells, the effects of lentivirus-mediated Cav1 knockdown and transfection with a negative control virus on mitochondrial membrane potential and mitochondrial respiratory function were analyzed using the TMRE fluorescent probe and the Seahorse extracellular flux analysis system. Methylated RNA immunoprecipitation (MeRIP) combined with m6A microarray was utilized to evaluate the changes in mRNA m6A modification and expression levels, followed by enrichment analysis to identify the functionally relevant genes. The m6A modification and expression levels of the mRNAs were validated by qPCR. RESULTS: Compared with the negative control group, the cells with Cav1 knockdown exhibited significantly reduced mitochondrial membrane potential and respiratory capacity. m6A microarray analysis revealed significant changes in m6A modification levels (fold change>1.5) of 7814 mRNAs, including 152 upregulated and 7662 downregulated mRNAs. Integrated expression analysis identified 2497 mRNAs showing coordinated changes in m6A modification and expression levels. These mRNAs were enriched in the oxidative phosphorylation pathway, with Usp15, Suclg2, and Ppa2 exhibiting the highest percent changes in m6A modification. Both microarray and qPCR results showed that the m6A modification and expression levels of Usp15, Suclg2 and Ppa2 mRNAs were significantly reduced in cells with Cav1 knockdown compared to the NC group. CONCLUSIONS: Cav1 knockdown induces significant alterations in mRNA m6A modification as well as their expression levels. The regulatory effects of Cav1 on mitochondrial function may be mediated by modulation of m6A modification of Usp15, Suclg2, and Ppa2 mRNAs.