Kupffer cells (KCs) play a crucial role in the pathogenesis of acute-on-chronic liver failure (ACLF) which is characterized by acute and severe disease in patients with preexisting liver disease and shows high mortality. Long noncoding RNAs (lncRNAs) are recently found to be involved in gene regulation. However, the mechanisms of how KCs are regulated by inflammatory factors, tumor necrosis factor-α (TNF-α), and whether lncRNAs are involved in the process remain largely unknown. Hence, we investigated the role of lncRNAs in the cytotoxicity of TNF-α on KCs.lncRNA array (The lncRNAs in the array are apoptosis-related lncRNAs reported in some research papers.) was used to identify lncRNAs related with liver fibrosis. Annexin V/protease inhibitor (PI) staining was used for detection of cell apoptosis. Real time-polymerase chain reaction was utilized for analysis of mRNA levels of lncRNA hypoxia-inducible factor 1 alpha-antisense RNA 1 (HIF1A-AS1) and apoptosis-related genes. Western blot was implied to the determination of lymphoid enhancer factor-1 (LEF-1).In this study, we found that HIF1A-AS1 could be upregulated by TNF-α by lncRNA array analysis and knockdown of HIF1A-AS1 significantly rescued cell apoptosis induced by TNF-α. Moreover, inhibition of HIF1A-AS1 markedly reduced mRNA level of caspase 3 which can be significantly enhanced by TNF-α. Furthermore, HIF1A-AS1 showed binding sites for LEF-1 and siRNA-mediated downregulation of LEF-1 decreased HIF1A-AS1 level in KCs treated with TNF-α.This study elucidates a new role of HIF1A-AS1 in TNF-α-induced cell apoptosis and provides potential therapeutic targets for ACLF.
Hypoxia serves a critical role in the pathogenesis of liver fibrosis. Hypoxia‑inducible factor 1α (HIF1‑α) is induced when cells are exposed to low O2 concentrations. Recently, it has been suggested that Rho‑associated coiled‑coil‑forming kinase 1 (ROCK1) may be an important HIF1‑α regulator. In the present study, it was analyzed whether crosstalk between HIF1‑α and ROCK1 regulates cell proliferation and collagen synthesis in hepatic stellate cells (HSCs) under hypoxic conditions. For this purpose, a rat hepatic HSC line (HSC‑T6) was cultured under hypoxic or normoxic conditions, and HIF1‑α and ROCK1 expression was measured at different time points. Additionally, HSC‑T6 cells were transfected with HIF1‑α small interfering RNA (siHIF1‑α), and measured protein expression and mRNA transcript levels of α‑smooth muscle actin, collagen 1A1 and ROCK1. Collagen 3A1 secretion was also measured by ELISA. Cell proliferation was assessed by the MTT assay under these hypoxic conditions. The results indicated that a specific ROCK inhibitor, Y‑27632, increased HIF1‑α and ROCK1 expression over time in HSC‑T6 cells in response to hypoxia. In addition, knockdown of HIF1‑α inhibited HSC‑T6 proliferation, suppressed collagen 1A1 expression, decreased collagen 3A1 secretion and attenuated ROCK1 expression. Notably, ROCK1 inhibition caused HSC‑T6 quiescence, suppressed collagen secretion and downregulated HIF1‑α expression. Collectively, these findings indicated that the interplay between HIF1‑α and ROCK1 may be a critical factor that regulates cell proliferation and collagen synthesis in rat HSCs under hypoxia.
Abstract. Activation of hepatic stellate cells (HSCs) is a pivotal event during hepatic fibrogenesis. Activated HSCs are the main source of collagen and other extracellular matrix (ECM) components, and emerging antifibrotic therapies are aimed at preventing ECM synthesis and deposition. MicroRNAs (miRNAs) have been demonstrated to exert regulatory effects on HSC activation and ECM synthesis. In the present study, the HSC-T6 rat hepatic stellate cell line was transiently transfected with a miRNA (miR)-203 mimic, which is an artificial miRNA that enhances the function of miR-203, with a miR-203 inhibitor or with a scramble miRNA negative control. mRNA and protein expression levels of collagen (COL) 1A1, COL3A1, α-smooth muscle actin (α-SMA) and mothers against decapentaplegic homolog 3 (SMAD3) were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The interaction between miR-203 and the 3'-untranslated region (UTR) of SMAD3 mRNA was examined using a dual-luciferase reporter assay. The proliferative capabilities of activated HSCs were measured using an MTT assay. The present results demonstrated that the mRNA and protein expression levels of COL1A1, COL3A1, α-SMA and SMAD3 were significantly upregulated following transfection of HSC-T6 cells with the miR-203 inhibitor. Conversely, COL1A1, COL3A1, α-SMA, and SMAD3 mRNA and protein expression appeared to be downregulated in rat HSCs transfected with miR-203 mimics. Notably, the inhibition of miR-203 expression was revealed to promote HSC proliferation, whereas increased miR-203 expression suppressed the proliferative capabilities of HSC-T6 cells. Furthermore, SMAD3 was revealed to be a direct target of miR-203. The present study suggested that miR-203 may function to prevent the synthesis and deposition of ECM components, including COL1A1, COL3A1 and α-SMA, and to inhibit the proliferation of HSCs through a SMAD3-dependent mechanism. Therefore, it may be hypothesized that miR-203 has potential as a novel target for the development of alternative therapeutic strategies for the treatment of patients with hepatic fibrosis in clinical practice.
There is currently a lack of reliable, reproducible, and easily applied methods for assessing changes in liver histology in patients in the gray zone phase of chronic hepatitis B (CHB). Therefore, we aimed to develop a novel predictive scoring system to detect significant liver histological changes in these patients.A total of 388 patients in the gray zone phase of CHB who underwent liver biopsy were divided into a training group and a validation group, and their clinical and routinely available laboratory parameters were analyzed using univariate analysis, Spearman correlation analysis, and logistic modeling. A novel scoring system, termed the Significant Histological Model (SHM), was constructed using logistic modeling. The diagnostic accuracy of our novel scoring system was evaluated by the receiving operating characteristic (ROC) method, sensitivity, specificity, and positive and negative predictive values (NPVs).We established the novel SHM scoring system using serum aspartate transaminase (AST), platelet counts (PLTs), albumin (ALB), and hepatitis B virus (HBV) DNA (log10 IU/mL) levels. The area under the ROC curve of the SHM scoring system was 0.763 in the training group and 0.791 in the validation group. For patients with a score of −1.0 or less and no significant histological changes, the sensitivity was 78.9%, specificity was 51.5%, positive predictive value (PPV) was 46.4%, and NPV was 82.0%. In the validation set, the sensitivity, specificity, PPV, and NPV were 80.0%, 66.6%, 56.3%, and 86.2%, respectively.This novel scoring system using AST, PLT, ALB, and HBV DNA (log10 IU/mL) levels identifies patients in the gray zone phase of CHB with and without histological changes with a high degree of accuracy. Here, we provide the experimental basis for the initiation of clinical antiviral treatment without the need for liver biopsy.
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