High immunocompetence in chronic hepatitis patients with normal alanine transaminase levels and and negative hepatitis B e-antigen for the progression of liver fibrosis.

INTRODUCTION: This study aimed to investigate the role of immunocompetence in chronic hepatitis B (CHB) patients with normal alanine transaminase (ALT) levels and negative hepatitis B e antigen (HBeAg) in the risk assessments of the progression of liver fibrosis. METHODS: We collected the clinical data of 57 patients with CHB, with normal ALT levels and negative HBeAg from December 2020 to December 2022. With hepatitis B virus (HBV) DNA > 20 IU/mL and ALT ≤ 40 U/L, these patients had never undergone antiviral therapy. The levels of CD4+ , CD4+ CD25+ , CD8+ , and CD4+ CD25+ CD127LOW regulatory T cells (Tregs) in the patients were detected using flow cytometry; the liver stiffness measurement (LSM) values of the patients were detected using Fibroscan. RESULTS: There was a statistically significant difference between the levels of fibrosis-4 (FIB-4) and hepatitis B surface antigen (HBsAg) when the cutoff point was HBsAg ≥ 1500 (p < .001). FIB-4 was negatively correlated with HBsAg (R = -0.291, p = .028) and positively correlated with age (R = 0.787, p < .001). LSM was negatively correlated with Treg but this correlation was not statistically significant (p > .05). Findings based on the analysis using logistic regression were as follows: (i) age was the independent risk factor when FIB-4 was used as the indicator for assessing liver fibrosis; (ii) Treg was the independent risk factor when LSM was used as the indicator for assessing liver fibrosis. When Treg was used to predict liver fibrosis, the cutoff value, diagnostic efficacy, area under the receiver operating characteristic (ROC) curve, and p value of the ROC curve were 6.875, 0.641, 0.84, and .027, respectively. CONCLUSION: Age and Treg are independent risk factors for progressive liver fibrosis. The cutoff value of Treg > 6.81 indicates the need for timely antiviral treatment and can serve as an indicator for evaluating liver fibrosis.

The upregulation of CLGN in hepatocellular carcinoma is potentially regulated by hsa-miR-194-3p and associated with patient progression.

Background: Patients with hepatocellular carcinoma (HCC) have poor prognosis, especially in advanced stages. Targeted therapy is the main treatment for advanced HCC patients, but the optimal targets for HCC remain poorly understood. The main purpose of this study was to identify potential novel prognostic markers and therapeutic targets. Methods: Firstly, differentially expressed genes (DEGs) in HCC were identified from the Gene Expression Omnibus (GEO) database. The expression, significance in prognosis, and potential mechanisms of DEGs were analyzed using GEPIA, TIMER, HPA, Kaplan Meier Plotter, CBioPortal, miRWalk, TargetScan, and ENCORI databases. Immunohistochemical staining was used to determine the protein expression levels of potential candidate genes. Results: The mRNA levels of MND1, STXBP6, and CLGN were significantly increased in HCC (p< 0.01). HCC patients with elevated CLGN mRNA levels had poorer overall survival (OS), disease-free survival (DFS), progression-free survival (PFS), and disease-specific survival (DSS) (p < 0.05). Higher MND1 mRNA levels significantly correlated with poorer DFS in HCC patients (p< 0.05). However, there was no significant correlation between STXBP6 expression and prognosis of HCC (p> 0.05). Further analysis revealed that patients with elevated CLGN mRNA expression in advanced pathology stages had poorer prognosis (p< 0.01). In addition, CLGN protein levels were elevated in HCC compared to their levels in normal tissues. The mRNA levels of CLGN had no significant correlation with the abundance of six common tumor infiltrating lymphocytes in HCC (COR < 0.5). Moreover, the mutation rate of CLGN was less than 1% in HCC patients (10/1089). Finally, the expression level of hsa-miR-194-3p in HCC was significantly lower than that in normal tissues (p < 0.05), and prognosis of HCC with low expression of hsa-miR-194 was poor (p < 0.05). Conclusion: The upregulation of CLGN in HCC is significantly associated with poor patient prognosis, especially in the advanced stages, and may be regulated by hsa-miR-194-3p. These findings suggest that CLGN may be closely related to the progression of HCC, and is a potential therapeutic target and prognostic indicator for patients with advanced HCC.

Activation of human hepatic stellate cells enhances the metastatic ability of hepatocellular carcinoma cells via up-regulation of interleukin-1ß.

PURPOSE: The purpose was to investigate the effect of activated human hepatic stellate cell (HSC) microenvironment on the metastatic capacity of hepatocellular carcinoma (HCC) cells and its underlying mechanism. METHODS: LX-2 HSCs were stimulated with Human Transforming Growth Factor-Beta 1(TGF-ß1), and protein expression of α-smooth muscle actin (α-SMA) and filamentous actin (F-actin) were determined to verify the activation of LX-2 cells. Next, SMMC7721 HCC cells were cultured in the conditioned medium originating from activated LX-2 cells. Wound healing and Transwell assays were performed to examine cell migration and invasion. The expression of metastasis-related genes Matrix Metalloproteinase9 (MMP9), N-cadherin, and Vascular endothelial growth factor (VEGF) was detected. ELISA was carried out to determine the interleukin (IL) -1ß level. Finally the inhibitors of TGF-ß1 and IL-1ß were employed to investigate the roles of LX-2 activation and IL-1ß in the metastasis-related gene alterations. RESULTS: TGF-ß1 activated LX-2 cells, as evidenced by up-regulated α-SMA and F-actin expression. Compared with the control medium, the conditioned medium derived from LX-2 cells significantly promoted the migration and invasion of SMMC7721 cells. And it also up-regulated mRNA and protein expression of the metastasis-related genes in SMMC7721 cells. Furthermore, it resulted in a significant increase in the IL-1ß level in SMMC7721 cells. Importantly, TGF-ß1 inhibitor and IL-1ß inhibitor either individually or synergistically abolished the up-regulated expression of conditioned medium-induced metastasis-related gene in SMMC7721 cells. CONCLUSIONS: The conditioned medium generating from TGF-ß1-activated LX2 cells can enhance the metastatic ability of SMMC7721 cells through up-regulating IL-1 expression.

MicroRNA-122 inhibits epithelial-mesenchymal transition of hepatic stellate cells induced by the TGF-ß1/Smad signaling pathway.

Transforming growth factor (TGF)-ß1 may stimulate the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. As micro RNA (miRNA)-122 is known to be associated with liver inflammation, its effects on the epithelial-mesenchymal transition (EMT) of HSCs through the inhibition of the TGF-ß1/drosophila mothers against decapentaplegic protein 4 (Smad4) signaling pathway were investigated. The MTT assay was performed to explore the optimum TGF-ß1 concentration suitable for HSC stimulation. Fluorescence microscopy was used to observe the transfection efficiency and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to observe gene and protein expression levels of α-smooth muscle actin (α-SMA), E-cadherin, N-cadherin and Smad4, respectively, in HSCs treated with TGF-ß1 or TGF-ß1 and miRNA-122. MTT assay results indicated that the concentration of 10 µg/l TGF-ß1 was suitable for maximum growth and survival of HSCs. Notably, the mRNA expression levels of N-cadherin and α-SMA were significantly increased (each, P<0.05), but the expression levels of E-cadherin were decreased following 10 µg/l TGF-ß1 treatment. Similar results were observed regarding the protein expression levels of N-cadherin, α-SMA and E-cadherin. Furthermore, the expression of F-actin was increased in the 10 µg/l TGF-ß1 treated group compared with the 0 µg/l TGF-ß1 treaded group and stretching of the muscle fiber filament was observed. miRNA-122 lentiviral vector transfection significantly decreased the mRNA expression of N-cadherin and increased the mRNA expression of E-cadherin in HSCs stimulated with TGF-ß1, as evident from RT-qPCR results. Similar results were also observed regarding the protein expression levels of N-cadherin and E-cadherin. The expression levels of Smad4, the primary component of the TGF-ß1 signaling pathway, were significantly lower in cells treated with TGF-ß1 and miRNA-122 (P<0.01) compared those treated with TGF-ß1. Thus, miRNA-122 may inhibit the activation and EMT of HSCs stimulated by TGF-ß1.

Up-regulation of thy-1 promotes invasion and metastasis of hepatocarcinomas.

Increasing evidence has revealed that thy-1 was a potential stem cell marker of liver cancer, but no data have been shown on how thy-1 regulates the pathophysiology of liver cancer, such as proliferation, apoptosis, invasion and migration. We previously demonstrated that thy-1 was expressed in about 1% of hepg2 cells, thy-1+ hepg2 cells, but not thy-1-, demonstrating high tumorigenesis on inoculation 0.5x105 cells per BACA/LA mouse after 2 months. In the present study, our results showed that higher expression of thy-1 occurs in 72% (36/50 cases) of neoplastic hepatic tissues as compared to 40% (20/50 cases) of control tissues, and the expression of thy-1 is higher in poorly differentiated liver tumors than in the well-differentiated ones. In addition, thy-1 expression was detected in 85% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis or hepatitis. There was a significant negative correlation between thy-1 expression and E-cadherin expression (a marker of invasion and migraton), but not between thy-1 expression and AFP expression in all the liver cancer and blood samples. We further investigated the relationship between thy-1 and E- cadherin in liver cancer hepg2 cell line which was transfected with pReceiver-M29/thy-1 eukaryotic expression vector followed by aspirin treatment. Lower expression of E- cadherin but higher expressions of thy-1 were detected in hepg2 cells transfected with pReceiver-M29/thy-1. Taken together, our study suggested that thy-1 probably regulates liver cancer invasion and migration.