Carnitine palmitoyltransferase-II inactivity promotes malignant progression of metabolic dysfunction-associated fatty liver disease via liver cancer stem cell activation

Figure 1 Carnitine palmitoyl transferase-II is downregulated in human hepatocellular carcinoma tissues. A: The carnitine palmitoyl transferase-II gene (CPT2) is located on the inner mitochondrial membrane; B: CPT2 in normal liver (n = 50) or LIHC (n = 269) tissues from TCGA database; C: CPT2 in LIHC (n = 50) and self-control normal liver (n = 50) tissues from TCGA database; D: Carnitine palmitoyl transferase-II (CPT-II) was evaluated via immunohistochemistry (IHC), and the relative ratio of metabolic dysfunction-associated fatty liver disease (MAFLD)-related hepatocellular carcinoma (HCC) (n = 12) to self-control non-HCC tissues (n = 12) was determined; E: CPT-II in livers: IHC (up) & specific concentration (ng/mg protein, down) between MAFLD-related HCC (n = 12) and their self-controlled non-HCC tissues (n = 12). ^bP < 0.01, compared with non-HCC tissues. HCC: Hepatocellular carcinoma; IHC: Immunohistochemistry; CoA: Coenzyme A; IMM: Inner mitochondrial membrane; OMM: Outer mitochondrial membrane; CPT2: The carnitine palmitoyl transferase-II gene; CPT1: The carnitine palmitoyl transferase-I gene.

Figure 2 Metabolic dysfunction-associated fatty liver disease models, pathological alterations, and specific concentrations. A: Gross specimen of a liver; B: Liver Hematoxylin & eosin staining (100 ×); C: Oil red O staining (100 ×); D: Liver carnitine palmitoyl transferase-II specific concentration (ng/mg protein); E: Liver CD44 levels; F: Liver CD24 levels; G: Liver alpha-fetoprotein levels. ^aP < 0.05, ^bP < 0.01, compared with the normal control group; NC: Normal control; HCC: Hepatocellular carcinoma; MAFL: Metabolic dysfunction-associated fatty liver; MASH: Metabolic dysfunction-associated steato-hepatitis; LC: Liver cirrhosis; CPT-II: Carnitine palmitoyl transferase-II; AFP: Alpha-fetoprotein; H&E: Hematoxylin & eosin.

Figure 3 carnitine palmitoyl transferase-II in T cells or the mitochondrial membrane potential in metabolic dysfunction-associated fatty liver disease hepatocarcinogenesis. A: Ratios of blood CD3+ T cells to peripheral blood mononuclear cells; B: Blood CD3+ T cell percentages; C: Blood CD4+ T cell percentages; D: Blood CD8+ T cells; E: Carnitine palmitoyl transferase-II (CPT-II) was evaluated by Western blot analysis at specific concentrations; F: CPT-II in CD4+ T cells; G: CPT-II in CD8+ T cells; H: Mitochondrial membrane potential median fluorescence intensity (MFI) in blood CD3+ T cells; I: MFI in spleen CD4+ T cells; J: MFI in spleen CD8+ T cells. ^aP < 0.05, compared with the normal control group; NC: Normal control; HCC: Hepatocellular carcinoma; MAFL: Metabolic dysfunction-associated fatty liver; MASH: Metabolic dysfunction-associated steato-hepatitis; LC: Liver cirrhosis; CPT-II: Carnitine palmitoyl transferase-II.

Figure 4 Enrichment analysis of T cell differentially expressed genes in metabolic dysfunction-associated fatty liver disease hepatocarcinogenesis. A: Differentially expressed genes (DEGs) in IL-CD4+ T cells. Left, metabolic dysfunction-associated fatty liver (MAFL) vs normal control (NC); Right, hepatocellular carcinoma (HCC) vs metabolic dysfunction-associated steatohepatitis (MASH); B: DEGs in IL-CD8+ T cells. Left, MAFL vs NC; Right, HCC vs MASH; C: Top 10 DEGs in IL-CD4+ T cells. Left, MAFL vs NC; Right, HCC vs MASHc; D: Top 10 DEGs in IL-CD8+ T cells. Left, MAFL vs NC; Right, HCC vs MASH; E: Gene Ontology (GO) analysis of DEGs; F: KEGG pathway with distribution of the corresponding GO terms of the top 332 features in hepatocarcinogenesis. ^aP < 0.05, compared with the NC group; NC: Normal control.

Figure 5 Validation of liver cancer stem cell activation in human metabolic dysfunction-associated fatty liver disease. A: Blood monocytes and CD44+ cells in healthy individuals; B: Blood monocytes and CD44+ cells from hepatocellular carcinoma (HCC) patients; C: Blood monocytes and CD24+ cells in healthy individuals; D: Blood monocytes and CD24+ cells in HCC patients; E: Average number of CD44+ cells in the blood of metabolic dysfunction-associated fatty liver disease (MAFLD) or HCC patients; F: Average number of CD24+ blood cells in MAFLD and HCC patients; G: A possible mechanism of hepatic carnitine palmitoyl transferase-II inactivity in MAFLD-related hepatocarcinogenesis involves the activation of LCSCs. ^bP < 0.01, compared with the normal control; LCSC: Liver cancer stem cell; MAFLD: Metabolic dysfunction-associated fatty liver disease; HFD: High-fat diet; NC: Normal control.