HFD-induced TRAF6 upregulation promotes liver cholesterol accumulation and fatty liver development via EZH2-mediated miR-429/PPARα axis.

Despite the increasing prevalence of fatty liver diseases worldwide, the molecular mechanism underlying their pathogenesis remains poorly defined. This study examines the expression and significance of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in the high-fat diet (HFD)-induced mouse obesity model and the oleic acid/palmitic acid (OA/PA)-induced cell model. After developing these models, we measured the expressions of TRAF6, enhancer of the zeste homolog 2 (EZH2), and peroxisome proliferator activated receptor alpha (PPARα). The expression of TRAF6, EZH2, and PPARα was manipulated to investigate their roles in cholesterol accumulation through evaluating the plasma levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Co-immunoprecipitation (coIP) assay was used to determine the interaction between TRAF6 and EZH2 and chromatin immunoprecipitation (ChIP) assay to detect the enrichment of EZH2 and H3K27me3 in microRNA-429 (miR-429) promoter. We found that HFD resulted in elevated TRAF6 and miR-429 in fatty liver and reduced EZH2 and PPARα. TRAF6 mediated the ubiquitination of EZH2 and increased miR-429 expression, and miR-429 targeted PPARα. TRAF6 increased cholesterol accumulation in liver cells in vitro via the EZH2/miR-429/PPARα axis. Collectively, HFD upregulates TRAF6 and ubiquitinates EZH2 to promote the miR-429-dependent inhibition of PPARα, leading to cholesterol accumulation in liver and the occurrence of fatty liver.

CDKN2A deregulation in fatty liver disease and its accelerative role in the process of lipogenesis.

Previous literature has indicated that cyclin-dependent kinase inhibitor 2 A (CDKN2A) is upregulated, while the Protein Inhibitor of Activated STAT1 (PIAS1) is downregulated in the liver tissues of obese mice. The current study aimed to investigate the relationship between CDKN2A and PIAS1 in the lipogenesis of fatty liver disease. In the C57BL/6J db/db mouse model and hepatocyte model of fatty liver, the expression pattern of CDKN2A, PIAS1, Protein arginine methyltransferase 1 (PRMT1) and CASP8 and FADD-like apoptosis regulator (CFLAR) was characterized by RNA quantitative and Western blot analysis. The lipogenesis-related genes (Srebp1c and Fas) in the liver tissues and cells were employed in the assessment of lipogenesis in response to gain- or loss-of-function of CDKN2A, PIAS1, PRMT1, and CFLAR, while triglyceride and fat content were evaluated in relation to fat accumulation. Western blot analysis was conducted to determine c-Jun amino-terminal kinase (JNK) phosphorylation, while the ubiquitination of CFLAR and SUMOylation of PIAS1 was examined by immunoprecipitation. PIAS1 and CFLAR were downregulated, while CDKN2A, PRMT1, and phosphorylation of JNK was elevated in the tissues and cells of the fatty liver models. Our results suggested that CDKN2A enhanced the SUMOylation of PIAS1 to reduce the expression of PIAS1. PRMT1 downregulated CFLAR by triggering its ubiquitination, while CFLAR repressed phosphorylation of JNK. The in vitro and in vivo results indicated that CDKN2A silencing prevented lipogenesis and fat accumulation by impairing the PRMT1-dependent ubiquitination of CFLAR and blocking the phosphorylation of JNK. Taken together, the central observations of our study demonstrate that targeting CDKN2A contributes to the suppression of lipogenesis and fat accumulation in fatty liver disease. The findings of our study highlight the potential of CDKN2A as a promising target against fatty liver.

Circulating Tumor-Cell-Associated White Blood Cell Clusters in Peripheral Blood Indicate Poor Prognosis in Patients With Hepatocellular Carcinoma.

Aim: Circulating tumor cells (CTC) are a precursor to metastasis in several types of cancer and are occasionally found in the bloodstream in association with immune cells, such as white blood cells (WBCs). CTC-associated WBC (CTC-WBC) clusters can promote CTC appreciation and metastasis, suggesting that patients with CTC-WBC clusters found in the peripheral blood may have a worse prognosis. However, it is unclear whether CTC-WBC clusters are present in the peripheral blood of patients with hepatocellular carcinoma (HCC) and suggest a poor prognosis for HCC. Methods: We collected peripheral blood from 214 patients with HCC from January 2014 to December 2016. CanPatrol™ CTC analysis technology was used to isolate and count CTCs and CTC-WBC clusters in the patients' peripheral blood. Chi-squared analysis was used to calculate the correlation between the CTC-WBC clusters and clinicopathological characteristics. Kaplan-Meier survival analysis and Cox regression analysis were used to assess patient prognosis. Results: We used CanPatrol™ CTC analysis technology to count different types of CTCs and CTC-WBC clusters. The results showed that CTC-WBC clusters and tumor size (P = 0.001), tumor number (P = 0.005), portal vein tumor thrombus (P = 0.026), BCLC stage (P < 0.001), AFP level (P = 0.002), and total number of CTCs (P < 0.001) were statistically related. Cox regression analysis revealed that CTC-WBC clusters are an independent prognostic indicator of DFS (HR = 1.951, 95%CI:1.348-2.824, P < 0.001) and OS (HR = 3.026, 95%CI:1.906-4.802, P < 0.001) in HCC patients. Using Kaplan-Meier analysis, we found that positive CTC-WBC cluster patients had significantly shorter DFS and OS than patients with negative CTC-WBC (P < 0.001 and P < 0.001, respectively). Conclusions: CTC-WBC clusters in the peripheral blood are an independent predictor of DFS and OS, and their presence indicates poor prognosis in patients with HCC.

Correction to: Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1.

An amendment to this paper has been published and can be accessed via the original article.

Downregulated microRNA-129-5p by Long Non-coding RNA NEAT1 Upregulates PEG3 Expression to Aggravate Non-alcoholic Steatohepatitis.

Long non-coding RNAs (lncRNAs) have recently emerged as inflammation-associated biological molecules with a specific role in the progression of liver fibrosis conditions including non-alcoholic steatohepatitis (NASH). The aim of this study was to elucidate the effects of lncRNA nuclear enriched abundant transcript 1 (NEAT1), microRNA-129-5p (miR-129-5p), and paternally expressed gene 3 (PEG3) on the biological activities of hepatic stellate cells (HSCs) subjected to NASH. First, microarray-based analysis revealed upregulated PEG3 in NASH. Liver tissues from mice fed a methionine-choline-deficient (MCD) diet exhibited increased expression of NEAT1 and PEG3 along with lower miR-129-5p expression. A series of in vitro and in vivo assays were then performed on HSCs after transfection with shPEG3, miR-129-5p mimic, or treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of the nuclear factor-kappa B (NF-κB) signaling pathway. Results confirmed the alleviated fibrosis by restoring miR-129-5p, while depleting PEG3 or NEAT1, as evidenced by the inactivation of HSCs. To sum up, NEAT1 can bind specifically to miR-129-5p and consequently regulate miR-129-5p and PEG3 expression in relation to the HSC activation occurring in NASH. Thus, NEAT1-targeted inhibition against miR-129-5p presents a promising therapeutic strategy for the treatment of NASH.

Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1.

BACKGROUND: Circulating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches. METHODS: Detection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188-9, and specific blockade with CXCR4 antibody were explored. RESULTS: The number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo, mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci. CONCLUSIONS: These findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.

Correlation Between Postoperative Early Recurrence of Hepatocellular Carcinoma and Mesenchymal Circulating Tumor Cells in Peripheral Blood.

BACKGROUND: Circulating tumor cells (CTCs) have been actively studied for their functions in hepatocellular carcinoma (HCC) recurrence. However, the relationship between circulating tumor cells subtypes and hepatocellular carcinoma recurrence is still unclear. METHODS: CTCs were collected from the peripheral blood of 62 postoperative HCC patients. The CTCs were isolated with a filtration-based method. Multiplex fluorescence in situ hybridization was used to characterize the CTCs based on mRNA expression levels of epithelial and mesenchymal markers. RESULTS: Of the 62 HCC patients, 26 were diagnosed with early recurrence (ER) and 36 did not experience recurrence. Comparison between the recurrence group and the non-recurrence group showed the total number of CTCs, mesenchymal CTCs, and mixed CTCs in the recurrence group was significantly higher than in the non-recurrence group. Receiver operator characteristic (ROC) curve analysis was performed to define the positive cutoff values as follows: total number of CTCs ≥ 4, mesenchymal CTCs ≥ 1, and mixed CTCs ≥ 3. Analysis showed that portal vein tumor thrombus (hazard ratio [HR] = 2.905, P = 0.023) and mesenchymal CTC positivity (HR = 3.453, P = 0.007) were independent risk factors for ER. The correlation between the presence of mesenchymal CTCs and time to recurrence was further examined, and the results showed significantly shortened postoperative disease-free survival in patients positive for mesenchymal CTCs (P < 0.001). CONCLUSIONS: HCC patients with positive peripheral mesenchymal CTCs have a more serious risk of ER, which could be a potential biomarker in HCC prognosis monitoring.