Nlrp6 overexpression inhibits lipid synthesis to suppress proliferation of hepatocellular carcinoma cells by regulating the AMPK-Srebp1c axis

doi:10.12122/j.issn.1673-4254.2024.10.09

Abstract

Abstract:

Objective To investigate the mechanism of Nlrp6 for regulating hepatocellular carcinoma (HCC) progression in light of lipid synthesis regulation. Methods Nlrp6 expression level in HCC tissues of different pathological grades was investigated using RNA-seq data from The Cancer Genome Atlas (TCGA) database, and its correlation with the patients' survival was analyzed with Kaplan-Meier survival analysis. HepG2 cells with adenovirus-mediated Nlrp6 overexpression or knockdown were treated with palmitic acid (PA), and the changes in lipid deposition and cell proliferation were evaluated using Oil Red O staining, CCK-8 assay, EdU staining, and colony formation assay. RT-qPCR and Western blotting were used to detect the changes in expression of lipid synthesis-related genes and the proteins in the AMPK-Srebp1c axis. In a mouse model of hepatic steatosis established in liver-specific Nlrp6 knockout mice by high-fat diet feeding for 24 weeks, liver fibrosis was examined with histological staining, and the changes in expressions of HCC markers and the AMPK-Srebp1c signaling pathway were detected. Results Nlrp6 expression was significantly reduced in HCC tissues with negative correlations with the pathological grades and the patients' survival (P<0.0001). In HepG2 cells, Nlrp6 overexpression significantly inhibited lipid deposition and cell proliferation, whereas Nlrp6 knockdown produced the opposite effects. Nlrp6 overexpression strongly suppressed the expression of lipid synthesis-related genes, promoted AMPK phosphorylation, and inhibited Srebp1c expression. The mice with liver-specific Nlrp6 knockout and high-fat feeding showed increased hepatic steatosis, collagen deposition, and AFP expression with reduced AMPK phosphorylation and increased Srebp1c expression. Conclusion Nlrp6 overexpression inhibits lipid synthesis in HCC cells by regulating the AMPK-Srebp1c axis, which might be a key pathway for suppressing HCC cell proliferation.

Key words: Nlrp6, hepatocellular carcinoma cells, proliferation, lipid synthesis, hepatic steatosis

Cuiyuan HUANG, Yunping SUN, Wenqiang LI, Li LIU, Wei WANG, Jing ZHANG. Nlrp6 overexpression inhibits lipid synthesis to suppress proliferation of hepatocellular carcinoma cells by regulating the AMPK-Srebp1c axis[J]. Journal of Southern Medical University, 2024, 44(10): 1910-1917.

Figures/Tables 6

Fig.1 Reduced Nlrp6 expression in liver tissues is associated with poor survival outcomes of hepatocellular carcinoma (HCC) patients. A: Nlrp6 expression levels in HCC patients with varying pathological grades based on RNA-seq and clinical data of patients from The Cancer Genome Atlas (TCGA) dataset. B: Kaplan-Meier survival curve analysis based on TCGA dataset (n=371). C: Spearman correlation analysis between Nlrp6 expression and tumor proliferation markers using TCGA dataset (n=371). D: Comparative analysis of Nlrp6, lipid metabolism, and proliferation-related gene expressions in HCC tissues (n=371) versus adjacent tissues (n=50) using TCGA dataset. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.2 Nlrp6 overexpression inhibits lipid accumulation in HepG2 cells. A: Validation of Nlrp6 overexpression and knockdown (n=3). B: Oil Red O staining after stimulation with 0.5 μmol/L palmitic acid (PA) for 24 h. *P<0.05 vs Ad-NC; #P<0.05 vs Ad-shNC.

Fig.3 Nlrp6 overexpression inhibits proliferation of HepG2 cells. A: CCK-8 assay for detecting cell proliferation (n=5). B: Colony formation assay. C: EdU staining. **P<0.01 vs Ad-shNC; #P<0.05 vs Ad-NC.

Fig.4 Nlrp6 regulates HepG2 cell proliferation by modulating lipid metabolism. A: Q-PCR assay for assessing the effect of Nlrp6 down-regulation on expressions of genes related to lipid synthesis in HepG2 cells. B: Effect of Nlrp6 overexpression on expressions of genes related to lipid synthesis in HepG2 cells (n=3). *P<0.05 vs Ad-shNC; ##P<0.01 vs Ad-NC.

Fig. 5 The AMPK-Srebp1c axis mediates Nlrp6 overexpression-induced inhibition of HepG2 cell proliferation. A: Western blotting for assessing the effect of Nlrp6 downregulation on the AMPK-Srebp1c signaling pathway in HepG2 cells. B: Western blotting for assessing the effect of Nlrp6 upregulation on the AMPK-Srebp1c signaling pathway in HepG2 cells. *P<0.05 vs Ad-shNC; #P<0.05 vs Ad-NC.

Fig.6 Liver-specific Nlrp6 knockout promotes lipid metabolism disorder-induced injury via the AMPK-Srebp1c axis. A: HE staining, Masson staining and Sirius scarlet staining of mouse livers after modeling with high-fat diet (HFD) in wild-type mice (Loxp) and Nlrp6 liver-specific knockout mice (LKO). B: RT-qPCR for detecting expressions of the relevant genes in the liver tissues. C: Western blotting for assessing the effect of liver-specific NLRP6 knockout on the AMPK-Srebp1c signaling pathway. *P<0.05, **P<0.01 vs Loxp-HFD.

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