| BackgroundsChanges in liver metabolism pathways play an important role in the occurrence and development of liver cancer and other liver diseases.Exploring the regulatory mechanisms of liver disease metabolism pathways is of great significance for the prevention,diagnosis,and treatment of liver diseases.m6 A RNA methylation is a widely present RNA modification in various organisms.METTL14,as one of the m6 A RNA methyltransferases,is involved in the development of various liver diseases,including liver cancer.Low expression of METTL14 in liver cancer patients often indicates poor prognosis.Additionally,downregulation of METTL14 is also associated with the proliferation,invasion,and metastasis of liver cancer cells.Recent studies have shown that m6 A modification plays an important role in regulating liver metabolism.Specifically,m6 A family molecules can change the m6 A modification levels of key metabolic enzymes in liver cells,thereby affecting the expression of these genes to regulate liver metabolism.Therefore,the purpose of this study is to explore the regulatory role of METTL14 in the glucose metabolism pathway of liver cancer and its downstream regulatory factors.The aim is to gain a deeper understanding of the relationship between m6 A modification and liver cancer,and to evaluate the potential value of METTL14 in the diagnosis and treatment of liver cancer by combining clinical data and pathological tissue samples,providing a new theoretical basis for developing relevant treatment strategies.MethodsOur team used CRISPR technology to construct a Mettl14 liver-specific knockout mouse in the early stage of our study.We first evaluated the liver function of the knockout mice and confirmed the histopathological changes in liver tissue through HE staining.We then assessed the degree of liver fibrosis in the Mettl14 knockdown mice using staining methods such as Sirius Red,while also examining liver cell apoptosis and mitochondrial damage.Through comprehensive analysis of RNA methylation sequencing(Me RIP-seq)and proteomic multi-omics analysis of the Mettl14 knockout mouse liver tissue,we revealed metabolic changes and identified the related target gene glucose-6-phosphatase(G6pc).We further clarified the role of G6 pc in the process of Mettl14 regulating liver glucose metabolism through immunoblotting,immunohistochemistry,fluorescence quantification experiments,and slow virus infection experiments.Additionally,we used m6A-RIP experiments to determine the specific regulatory mechanism of Mettl14 on G6 pc.We also observed changes in the proliferative capacity of liver cells in the knockout mouse through Ki67 staining.Based on the single-cell sequencing results of liver cancer tissue in the public database,we systematically analyzed the expression correlation of METTL14 and G6 PC in liver cancer and their metabolic effects.We then confirmed the expression correlation between METTL14 and G6 PC in clinical primary liver cancer samples through immunoblotting,immunohistochemistry,and slow virus infection experiments,as well as the role of G6 PC in METTL14-regulated substance energy metabolism in liver cancer.Furthermore,we analyzed the relationship between the expression of METTL14 and G6 PC and the prognosis of liver cancer survival.ResultsWe found that liver-specific knockout of Mettl14 in mice led to significant liver dysfunction.HE staining showed the presence of inflammatory cell infiltration in the liver of the knockout mice.Sirius Red staining and α-SMA staining experiments showed that Mettl14 deficiency promoted liver fibrosis formation,as well as liver cell apoptosis and mitochondrial damage.Based on the protein mass spectrometry results of Mettl14 liverspecific knockout mice,we discovered that the loss of Mettl14 function altered liver substance metabolism,manifested as significantly suppressed glucose metabolism levels and enhanced fatty acid β-oxidation in c KO mice.Meanwhile,c KO mice exhibited decreased peripheral blood glucose levels and increased glycogen synthesis in liver cells.Through comprehensive analysis of m6A-RNAseq sequencing results and protein mass spectrometry results of knockout mouse liver tissue,we screened downstream target gene G6 pc,which is related to glucose metabolism.In vivo and in vitro experiments revealed that Mettl14 may be involved in the regulation of glucose metabolism in liver cells by regulating the expression of G6 pc,and m6A-RIP experiments further confirmed that Mettl14 participated in the post-transcriptional regulation of G6 pc.Single-cell sequencing data of liver cancer tissue and clinical liver cancer tissue showed a positive correlation between METTL14 and G6 PC expression.Additionally,low expression of both METTL14 and G6 PC was closely related to poor prognosis of liver cancer.At the cellular level,we further confirmed that METTL14 plays an important role in regulating glucose metabolism in liver cancer cells by regulating the expression of G6 PC.ConclusionWe found for the first time that METTL14 can regulate the expression of glucose metabolism-related gene G6 PC,which may be closely related to the occurrence and development of liver diseases and participate in the malignant progression of hepatocellular carcinoma.The expression levels of METTL14 and G6 PC are positively correlated in hepatocellular carcinoma,and their decreased expression levels indicate poor prognosis for patients.Furthermore,we found that the loss of Mettl14 function downregulates the m6 A methylation level of the 1201 st purine in G6pc’s transcript,leading to a decrease in its expression level and further affecting the glucose metabolism process of liver cells and liver cancer cells.These results provide new insights into the molecular mechanisms underlying the development of hepatocellular carcinoma and the development of related therapeutic targets. |
