The significance of lipid metabolism reprogramming of tumor-associated macrophages in hepatocellular carcinoma.

In the intricate landscape of the tumor microenvironment, tumor-associated macrophages (TAMs) emerge as a ubiquitous cellular component that profoundly affects the oncogenic process. The microenvironment of hepatocellular carcinoma (HCC) is characterized by a pronounced infiltration of TAMs, underscoring their pivotal role in modulating the trajectory of the disease. Amidst the evolving therapeutic paradigms for HCC, the strategic reprogramming of metabolic pathways presents a promising avenue for intervention, garnering escalating interest within the scientific community. Previous investigations have predominantly focused on elucidating the mechanisms of metabolic reprogramming in cancer cells without paying sufficient attention to understanding how TAM metabolic reprogramming, particularly lipid metabolism, affects the progression of HCC. In this review article, we intend to elucidate how TAMs exert their regulatory effects via diverse pathways such as E2F1-E2F2-CPT2, LKB1-AMPK, and mTORC1-SREBP, and discuss correlations of TAMs with these processes and the characteristics of relevant pathways in HCC progression by consolidating various studies on TAM lipid uptake, storage, synthesis, and catabolism. It is our hope that our summary could delineate the impact of specific mechanisms underlying TAM lipid metabolic reprogramming on HCC progression and provide useful information for future research on HCC and the development of new treatment strategies.

Characterization of neutrophil extracellular traps related gene pair for predicting prognosis in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant disease with a high incidence rate, high mortality and poor prognosis. Neutrophil extracellular traps (NETs), as an extracellular reticular structure, promote the development and progression of cancer in the tumor microenvironment, and have a promising prospect as a prognostic indicator. In the present study, we elucidated the prognostic value of NET-related genes. METHODS: The NETs gene pair of The Cancer Genome Atlas cohort was constructed by least absolute shrinkage and selection operator analysis. Samples from the International Cancer Genome Consortium were performed to verify its feasibility. Kaplan-Meier analysis was used to compare the overall survival (OS) rate of the two subgroups. The independent predictors of OS were determined by univariate and multivariate Cox analysis. Furthermore, Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway were analyzed by gene set enrichment analysis. The single sample gene set enrichment analysis method was performed to deplore the relationship of risk score with tumor immune microenvironment. The GSE149614 dataset was applied as single cell RNA level validation. PCR was performed to the detect mRNA expression profiles of NETs-related genes. RESULTS: Our analysis of the NETs-related model provides a promising prospect as a prognostic indicator. The OS of high-risk group patients was significantly reduced. The risk score was an important independent predictor of HCC prognosis. The Nomogram model suggested a favorable classification performance. The drug resistance and sensitivity of tumor cells to chemotherapeutics was significantly correlated with the prognostic gene expression. The immune status of the two risk groups showed a marked difference. CONCLUSIONS: The novel prognostic gene pair and immune landscape could predict the prognosis of HCC patients and provide a new understanding of immunotherapy in HCC.

Association between triglyceride-glucose index and all-cause mortality in critically ill patients with ischemic stroke: analysis of the MIMIC-IV database.

BACKGROUND: The triglyceride-glucose (TyG) index was significantly associated with insulin resistance (IR). Several studies have validated the effect of TyG index on cerebrovascular disease. However, the value of TyG index in patients with severe stroke requiring ICU admission remains unclear. The aim of this study was to investigate the association between the TyG index and clinical prognosis of critically ill patients with ischemic stroke (IS). METHODS: This study identified patients with severe IS requiring ICU admission from the Medical Information Mart for Intensive Care (MIMIC-IV) database, and divided them into quartiles based on TyG index level. The outcomes included in-hospital mortality and ICU mortality. The association between the TyG index and clinical outcomes in critically ill patients with IS was elucidated using Cox proportional hazards regression analysis and restricted cubic splines. RESULTS: A total of 733 patients (55.8% male) were enrolled. The hospital mortality and intensive care unit (ICU) mortality reached 19.0% and 14.9%, respectively. Multivariate Cox proportional hazards analysis showed that the elevated TyG index was significantly related to all-cause death. After confounders adjusting, patients with an elevated TyG index had a significant association with hospital mortality (adjusted hazard ratio, 1.371; 95% confidence interval, 1.053-1.784; P = 0.013) and ICU mortality (adjusted hazard ratio, 1.653; 95% confidence interval, 1.244-2.197; P = 0.001). Restricted cubic splines revealed that a progressively increasing risk of all-cause mortality was related to an elevated TyG index. CONCLUSION: The TyG index has a significant association with hospital and ICU all-cause death in critically ill patients with IS. This finding demonstrates that the TyG index might be useful in identifying patients with IS at high risk of all-cause death.

Salidroside ameliorates acetaminophen-induced acute liver injury through the inhibition of endoplasmic reticulum stress-mediated ferroptosis by activating the AMPK/SIRT1 pathway.

Acetaminophen (APAP) overdose has long been considered a major cause of drug-induced liver injury. Ferroptosis is a type of programmed cell death mediated by iron-dependent lipid peroxidation. Endoplasmic reticulum (ER) stress is a systemic response triggered by the accumulation of unfolded or misfolded proteins in the ER. Ferroptosis and ER stress have been proven to contribute to the progression of APAP-induced acute liver injury (ALI). It was reported that salidroside protects against APAP-induced ALI, but the potential mechanism remain unknown. In this study, male C57BL/6 J mice were intraperitoneally (i.p.) injected APAP (500 mg/kg) to induce an ALI model. Salidroside was i.p. injected at a dose of 100 mg/kg 2 h prior to APAP administration. Mice were sacrificed 12 h after APAP injection and the liver and serum of the mice were obtained for histological and biochemistry analysis. AML12 cells were used in in vitro assays. The results indicated that salidroside mitigated glutathione degradation via inhibiting cation transport regulator homolog 1 (CHAC1) to attenuate ferroptosis, and simultaneously suppressing PERK-eIF2α-ATF4 axis-mediated ER stress, thus alleviating APAP-induced ALI. However, PERK activator CCT020312 and overexpression of ATF4 inhibited the protective function of salidroside on CHAC1-mediated ferroptosis. Besides this, activation of the AMPK/SIRT1 signaling pathway by salidroside was demonstrated to have a protective effect against APAP-induced ALI. Interestingly, selective inhibition of SIRT1 ameliorated the protective effects of salidroside on ER stress and ferroptosis. Overall, salidroside plays a significant part in the mitigation of APAP-induced ALI by activating the AMPK/SIRT1 signaling to inhibit ER stress-mediated ferroptosis in the ATF4-CHAC1 axis.

Glycolysis-Related Gene Signature Can Predict Survival and Immune Status of Hepatocellular Carcinoma.

BACKGROUND: Concise and precise prognostic models are urgently needed due to the intricate genetic variations among hepatocellular carcinoma (HCC) cells. Disorder or change in glycolysis metabolism has been considered one of the "hallmarks" of cancer. However, the prognostic value of glycolysis-related genes in HCC remains elusive. METHODS: A multigene prognostic model was constructed by least absolute shrinkage and selection operator Cox regression analysis in the The Cancer Genome Atlas (TCGA) cohort with 365 HCC patients and validated in the International Cancer Genome Consortium (ICGC) cohort with 231 HCC patients. The Kaplan-Meier methodology and time-dependent receiver operating characteristic curve were employed to confirm its predictive capability. A predictive nomogram was established based on the stepwise multivariate regression model. The differential expression of prognostic genes between HCC tissues and normal tissues was verified by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry in an independent sample cohort with 30 HCC patients. RESULTS: The glycolysis-related gene signature and the nomogram model exhibited robust validity in predicting prognosis. The risk score was an independent predictor for overall survival (OS). Expression levels of immune checkpoint genes and cell cycle genes were significantly elevated in the high-risk group. The high-risk group presented high levels of immune exclusion. The risk score can distinguish the effect of immunotherapy in the IMvigor210 cohort. The prognostic gene expression showed a significant difference between HCC tissues and adjacent nontumorous tissues in the independent sample cohort. CONCLUSION: The currently established glycolysis-related gene signature can accurately predict prognosis and reflect immune status, which may be a therapeutic alternative.

Hypoxia induces the activation of hepatic stellate cells through the PVT1-miR-152-ATG14 signaling pathway.

Increasing studies have indicated that hypoxia serves as a pivotal microenvironmental factor that facilitates activation of hepatic stellate cells (HSCs). However, the mechanism by which hypoxia activates HSCs is not clear. Here, we demonstrated that plasmacytoma variant translocation 1 (PVT1) and autophagy were overexpressed in liver fibrotic specimens. In primary mouse HSCs, both PVT1 and autophagy were induced by hypoxia. Further study showed that hypoxia-induced autophagy depended on expression of PVT1 and miR-152 in HSCs. Luciferase reporter assay indicated that autophagy-related gene 14 (ATG14) was a direct target of miR-152. In addition, inhibition of autophagy by 3-methyladenine and Beclin-1 siRNA impeded activation of HSCs cultured in 1% O2. Taken together, autophagy induction via the PVT1-miR-152-ATG14 signaling pathway contributes to activation of HSCs under hypoxia condition.

The value of the atherogenic index of plasma in non-obese people with non-alcoholic fatty liver disease: a secondary analysis based on a cross-sectional study.

BACKGROUND AND OBJECTIVES: The atherogenic index of plasma (AIP) is elevated in fatty liver disease, but its value in non-obese people with non-alcoholic fatty liver disease (NAFLD) is unclear. This study aimed to investigate the relationship between AIP and NAFLD as well as to determine whether AIP might be used as an indicator of NAFLD in non-obese individuals. METHODS: The present study involved non-obese Chinese and Japanese participants. Risk factors are evaluated using univariate and multivariate analysis. The performance of risk factors was compared according to the area under the receiver operating characteristic curve. RESULTS: In the unadjusted model, the odds ratio (OR) for every 1 standard deviation (SD) increase in AIP was 52.30. In adjusted models I and II, the OR for every 1 SD increase in AIP was 36.57 and 50.84, respectively. The area under the receiver operating characteristic curve for AIP was 0.803 and 0.802 in the development and validation groups, respectively. The best cut-off value of AIP for discrimination between NAFLD and non-NAFLD was 0.005 in the Chinese group and - 0.220 in the Japanese group. CONCLUSIONS: AIP and NAFLD are positively correlated in Chinese and Japanese populations. Therefore, AIP can be used as a new screening indicator for non-obese people with NAFLD in different nations.

Long noncoding RNA HOTTIP mediates SRF expression through sponging miR-150 in hepatic stellate cells.

HOXA transcript at the distal tip (HOTTIP) has been shown to be up-regulated in a variety of cancers and is identified as an oncogenic long noncoding RNA. However, the biological role of HOTTIP in liver fibrosis is unclear. Here, we reported that HOTTIP was specifically overexpressed in activated hepatic stellate cells (HSCs). HOTTIP knockdown suppressed the activation and proliferation of HSCs. Luciferase reporter assay showed that HOTTIP and serum response factor (SRF) were targets of miR-150. RNA binding protein immunoprecipitation assay indicated the interaction between miR-150 and HOTTIP. Further study revealed that HOTTIP increased SRF expression as a competing endogenous RNA for miR-150, thus prompting HSC activation. Taken together, we provide a novel HOTTIP-miR-150-SRF signalling cascade in liver fibrosis.

CircMTO1 inhibits liver fibrosis via regulation of miR-17-5p and Smad7.

Circular RNAs (circRNAs), often dysregulated in a variety of human diseases, participate in the initiation and development of cancers. Recently, circMTO1 (a circRNA derived from MTO1 gene), identified as a tumor suppressor, has been shown to contribute to the suppression of hepatocellular carcinoma. The present study aimed to explore the clinical significance and roles of circMTO1 in liver fibrosis. Here, we found that serum circMTO1 was significantly down-regulated in chronic hepatitis B (CHB) patients. Interestingly, serum circMTO1 was negatively correlated with fibrosis stages as well as HAI scores. Receiver operating characteristic curve analysis revealed that serum circMTO1 may serve as a diagnostic biomarker for liver fibrosis in CHB patients. Notably, overexpression of circMTO1 led to the suppression of transforming growth factor-ß1-induced hepatic stellate cells (HSCs) activation. Bioinformatic analysis and luciferase activity assays indicated that circMTO1 was a target of mircoRNA-17-5p (miR-17-5p). Data from RNA pull-down assay further confirmed that circMTO1 interacted with miR-17-5p. The inhibitory effects of circMTO1 on HSC activation were suppressed by miR-17-5p mimics. Further studies showed that Smad7 was a target of miR-17-5p. Moreover, circMTO1-inhibited HSC activation was also blocked down by loss of Smad7. Taken together, we demonstrate that circMTO1 inhibits liver fibrosis via regulation of miR-17-5p and Smad7, and serum circMTO1 may be a novel promising biomarker of liver fibrosis.

MicroRNA-30a Suppresses the Activation of Hepatic Stellate Cells by Inhibiting Epithelial-to-Mesenchymal Transition.

BACKGROUND/AIMS: The activation of hepatic stellate cells (HSCs) is considered as a pivotal event in liver fibrosis and epithelial-mesenchymal transition (EMT) process has been reported to be involved in HSC activation. It is known that microRNAs (miRNAs) play a pro-fibrotic or anti-fibrotic role in HSC activation. Recently, emerging studies show that miR-30a is down-regulated in human cancers and over-expression of miR-30a inhibits tumor growth and invasion via suppressing EMT process. However, whether miR-30a could regulate EMT process in HSC activation is still unclear. METHODS: miR-30a expression was quantified using real-time PCR in carbon tetrachloride (CCl4)-induced rat liver fibrosis, activated HSCs and patients with cirrhosis. Roles of miR-30a in liver fibrosis in vivo and in vitro were also analyzed. Luciferase activity assays were performed to examine the binding of miR-30a to the 3'-untranslated region of snail family transcriptional repressor 1 (Snai1). RESULTS: miR-30a was down-regulated in human cirrhotic tissues. In CCl4 rats, reduced miR-30a was found in fibrotic liver tissues as well as isolated HSCs. There was a significant reduction in miR-30a in primary HSCs during culture days. miR-30a over-expression resulted in the suppression of CCl4-induced liver fibrosis. Restoration of miR-30a led to the inhibition of HSC activation including cell proliferation, α-SMA and collagen expression. Notably, miR-30a inhibited EMT process, with a reduction in TGF-ß1 and Vimentin as well as an increase in GFAP and E-cadherin. miR-30a induced a significant reduction in Snai1 protein expression when compared with the control. Interestingly, Snail protein expression was increased during liver fibrosis, indicating that there may be a negative correlation between miR-30a level and Snai1 protein expression. Further studies demonstrated that Snai1 was a target of miR-30a. CONCLUSION: Our results suggest that miR-30a inhibits EMT process, at least in part, via reduction of Snai1, leading to the suppression of HSC activation in liver fibrosis.

HOTAIR Epigenetically Modulates PTEN Expression via MicroRNA-29b: A Novel Mechanism in Regulation of Liver Fibrosis.

Homeobox transcript antisense RNA (HOTAIR), as a long intergenic non-coding RNA (lincRNA), is upregulated in various cancers and involved in diverse cellular functions. However, its role in liver fibrosis is unclear. In this study, HOTAIR expression was upregulated in hepatic stellate cells (HSCs) in vivo and in vitro during liver fibrosis. HOTAIR knockdown suppressed HSC activation including α-smooth muscle actin (α-SMA) and typeIcollagen in vitro and in vivo. Both HSC proliferation and cell cycle were inhibited by HOTAIR knockdown. Notably, inhibition of HOTAIR led to an increase in PTEN, associated with the loss of DNA methylation. miR-29b-mediated control of PTEN methylation was involved in the effects of HOTAIR knockdown. HOTAIR was confirmed a target of miR-29b and lack of the miR-29b binding site in HOTAIR prevented the suppression of miR-29b, suggesting HOTAIR contributes to PTEN expression downregulation via sponging miR-29b. Interestingly, increased HOTAIR was also observed in hepatocytes during liver fibrosis. Loss of HOTAIR additionally led to the increase in PTEN and the reduction in typeIcollagen in hepatocytes. Collectively, we demonstrate that HOTAIR downregulates miR-29b expression and attenuates its control on epigenetic regulation, leading to enhanced PTEN methylation, which contributes to the progression of liver fibrosis.

Epigenetically-Regulated MicroRNA-9-5p Suppresses the Activation of Hepatic Stellate Cells via TGFBR1 and TGFBR2.

BACKGROUND/AIMS: Recently, microRNAs (miRNAs) have been demonstrated to act as regulators of activation of hepatic stellate cells (HSCs). It is well known that the main profibrogenic inducer transforming growth factor-ß1 (TGF-ß1) contributes to HSC activation, which is a key event in liver fibrosis. Increasing studies show that miR-9-5p is down-regulated in liver fibrosis and restoration of miR-9-5p limits HSC activation. However, the role of miR-9-5p in TGF-ß1-induced HSC activation is still not clear. METHODS: miR-9-5p expression was quantified using real-time PCR in chronic hepatitis B (CHB) patients and TGF-ß1-treated LX-2 cells. In CHB patients, histological activity index (HAI) and fibrosis stages were assessed using the Ishak scoring system. Effects of miR-9-5p on liver fibrosis in vivo and in vitro were analyzed. Luciferase activity assays were performed to examine the binding of miR-9-5p to the 3'-untranslated region of type I TGF-ß receptor (TGFBR1) as well as TGFBR2. RESULTS: Compared with healthy controls, miR-9-5p was reduced in CHB patients. There was a lower miR-9-5p expression in CHB patients with higher fibrosis scores or HAI scores. miR-9-5p was down-regulated by TGF-ß1 in a dose-dependent manner. TGF-ß1-induced HSC activation including cell proliferation, α-SMA and collagen expression was blocked down by miR-9-5p. Notably, miR-9-5p ameliorates carbon tetrachloride-induced liver fibrosis. As determined by luciferase activity assays, TGFBR1 and TGFBR2 were targets of miR-9-5p. Further studies demonstrated that miR-9-5p inhibited TGF-ß1/Smads pathway via TGFBR1 and TGFBR2. Interestingly, promoter methylation was responsible for miR-9-5p down-regulation in liver fibrosis. The relationship between miR-9-5p expression and methylation was confirmed in CHB patients and TGF-ß1-treated cells. CONCLUSION: Our results demonstrate that miR-9-5p could inhibit TGF-ß1-induced HSC activation through TGFBR1 and TGFBR2. Loss of miR-9-5p is associated with its methylation status in liver fibrosis.

LincRNA-p21 Inhibits the Wnt/ß-Catenin Pathway in Activated Hepatic Stellate Cells via Sponging MicroRNA-17-5p.

BACKGROUND/AIMS: It is known that the activation of hepatic stellate cells (HSCs) is a pivotal step in the initiation and progression of liver fibrosis. Aberrant activated Wnt/ß-catenin pathway is known to accelerate the development of liver fibrosis. microRNAs (miRNAs)-mediated Wnt/ß-catenin pathway has been reported to be involved in HSC activation during liver fibrosis. However, whether long noncoding RNAs (lncRNAs) regulate Wnt/ß-catenin pathway during HSC activation still remains unclear. METHODS: Long intergenic noncoding RNA-p21 (lincRNA-p21) expression was detected in Salvianolic acid B (Sal B)-treated cells. Effects of lincRNA-p21 knockdown on HSC activation and Wnt/ß-catenin pathway activity were analyzed in Sal B-treated cells. In lincRNA-p21-overexpressing cells, effects of miR-17-5p on HSC activation and Wnt/ß-catenin pathway activity were examined. RESULTS: LincRNA-p21 expression was up-regulated in HSCs after Sal B treatment. In primary HSCs, lincRNA-p21 expression was down-regulated at Day 5 relative to Day 2. Sal B-inhibited HSC activation including the reduction of cell proliferation, α-smooth muscle actin (α-SMA) and type I collagen was inhibited by lincRNA-p21 knockdown. Sal B-induced Wnt/ß-catenin pathway inactivation was blocked down by loss of lincRNA-p21. Notably, lincRNA-p21, confirmed as a target of miR-17-5p, suppresses miR-17-5p level. Lack of the miR-17-5p binding site in lincRNA-p21 prevents the suppression of miR-17-5p expression. In addition, the suppression of HSC activation and Wnt/ß-catenin pathway induced by lincRNA-p21 overexpression was almost inhibited by miR-17-5p. CONCLUSION: We demonstrate that lincRNA-p21-inhibited Wnt/ß-catenin pathway is involved in the effects of Sal B on HSC activation and lincRNA-p21 suppresses HSC activation, at least in part, via miR-17-5p-mediated-Wnt/ß-catenin pathway.

Serum lincRNA-p21 as a potential biomarker of liver fibrosis in chronic hepatitis B patients.

Serum long non-coding RNAs (lncRNAs) are emerging as promising biomarkers for various human diseases. The aim of this study was to investigate the feasibility of using serum long intergenic non-coding RNA-p21 (lincRNA-p21) as a biomarker for chronic hepatitis B patients. Serum lincRNA-p21 levels were quantified using real-time PCR in 417 CHB patients and 363 healthy controls. The promoter methylation level of lincRNA-p21 was detected using bisulphite-sequencing analysis in primary hepatic stellate cells (HSCs). Sera from hepatitis B-infected patients contained lower levels of lincRNA-p21 than sera from healthy controls. Serum lincRNA-p21 levels negatively correlated with stages of liver fibrosis in infected patients. Receiver operating characteristic (ROC) curve analyses suggested that serum lincRNA-p21 had a significant diagnostic value for liver fibrosis in these patients. It yielded an area under the curve of ROC of 0.854 with 100% sensitivity and 70% specificity in discriminating liver fibrosis from healthy controls. There was additionally a negative correlation between serum lincRNA-p21 level and the markers of liver fibrosis including α-SMA and Col1A1. However, there was no correlation of serum lincRNA-p21 level with the markers of viral replication, liver inflammatory activity, and liver function. Notably, during primary HSCs culture, loss of lincRNA-p21 expression was associated with promoter methylation. Serum lincRNA-p21 could serve as a potential biomarker of liver fibrosis in CHB patients. Down-regulation of lincRNA-p21 in liver fibrosis may be associated with promoter methylation.

Long Non-coding RNA Growth Arrest-specific Transcript 5 (GAS5) Inhibits Liver Fibrogenesis through a Mechanism of Competing Endogenous RNA.

Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat, and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. A pulldown assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs, GAS5 had no effect on primary miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. Quantitative RT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a competing endogenous RNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs cross-talk by competing for shared microRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis.

Activation of Hepatic Stellate Cells is Inhibited by microRNA-378a-3p via Wnt10a.

BACKGROUND/AIMS: Wnt/ß-catenin pathway is involved in liver fibrosis and microRNAs (miRNAs) are considered as key regulators of the activation of hepatic stellate cells (HSCs). A recent study showed the protective role of miR-378a-3p against cardiac fibrosis. However, whether miR-378a-3p suppresses Wnt/ß-catenin pathway in liver fibrosis is largely unknown. METHODS: miR-378a-3p expression was detected in carbon tetrachloride-induced liver fibrosis and activated HSCs. Effects of miR-378a-3p overexpression on HSC activation and Wnt/ß-catenin pathway were analyzed. Bioinformatic analysis was employed to identify the potential targets of miR-378a-3p. Serum miR-378a-3p expression was analyzed in patients with cirrhosis. RESULTS: Reduced miR-378a-3p expression was observed in the fibrotic liver tissues and activated HSCs. Up-regulation of miR-378a-3p inhibited HSC activation including cell proliferation, α-smooth muscle actin (α-SMA) and collagen expression. Moreover, miR-378a-3p overexpression resulted in Wnt/ß-catenin pathway inactivation. Luciferase reporter assays demonstrated that Wnt10a, a member of Wnt/ß-catenin pathway, was confirmed to be a target of miR-378a-3p. By contrast, miR-378a-3p inhibitor contributed to HSC activation, with an increase in cell proliferation, α-SMA and collagen expression. But all these effects were blocked down by silencing of Wnt10a. Notably, sera from patients with cirrhosis contained lower levels of miR-378a-3p than sera from healthy controls. Receiver operating characteristic curve analysis suggested that serum miR-378a-3p differentiated liver cirrhosis patients from healthy controls, with an area under the curve of ROC curve of 0.916. CONCLUSION: miR-378a-3p suppresses HSC activation, at least in part, via targeting of Wnt10a, supporting its potential utility as a novel therapeutic target for liver fibrosis.

The Epigenetically-Regulated microRNA-378a Targets TGF-ß2 in TGF-ß1-Treated Hepatic Stellate Cells.

BACKGROUND/AIMS: In liver fibrosis, the activation of hepatic stellate cells (HSCs) is considered as a pivotal event. It is well known that transforming growth factor-ß1 (TGF-ß1) is the main stimuli factor responsible for HSC activation. microRNAs (miRNAs), regulating various biological processes, have recently been shown to be involved in HSC activation. A recent study reported that deficiency of miR-378a contributes to cardiac fibrosis via TGF-ß1-dependent paracrine mechanism. However, the involvement of miR-378a and its roles in TGF-ß1-induced HSC activation remains largely unknown. METHODS: miR-378a expression was detected in TGF-ß1-treated cells and patients with cirrhosis. Then, effects of miR-378a overexpression on cell proliferation and HSC activation were analyzed. We also analyzed the binding of miR-378a to the 3'-untranslated region of TGF-ß2. RESULTS: In response to TGF-ß1, miR-378a expression was down-regulated in a dose-dependent manner. miR-378a overexpression suppressed both cell proliferation and cell cycle in TGF-ß1-treated LX-2 cells. Moreover, miR-378a overexpression inhibited TGF-ß1-induced HSC activation including the reduction of α-smooth muscle actin (α-SMA) and type I collagen. Similarly, miR-378a resulted in a reduction in cell proliferation, and the expressions of α-SMA and Col1A1 in TGF-ß1-treated primary HSCs. Notably, TGF-ß2 was confirmed as a target of miR-378a by luciferase reporter assays. Interestingly, miR-378a promoter methylation may be responsible for miR-378a down-regulation in TGF-ß1-treated LX-2 cells and TGF-ß1-treated primary HSCs. Further studies confirmed that reduced miR-378a was associated with promoter methylation in patients with cirrhosis compared with healthy controls. CONCLUSION: Our results demonstrate that miR-378a expression is associated with its methylation status in TGF-ß1-treated cells, and epigenetically-regulated miR-378a inhibits TGF-ß1-induced HSC activation, at least in part, via TGF-ß2.

MicroRNA-125a-5p Contributes to Hepatic Stellate Cell Activation through Targeting FIH1.

BACKGROUND/AIMS: Emerging evidence shows that microRNAs (miRNAs) play a crucial role in the regulation of activation, proliferation and apoptosis of hepatic stellate cells (HSCs). Previous studies have indicated that miR-125a-5p is correlated with hepatitis B virus replication and disease progression. However, little is known about the biological role and underlying mechanism of miR-125a-5p in liver fibrosis. METHODS: We analyzed the level of miR-125a-5p in carbon tetrachloride-induced liver fibrosis and activated HSCs. We analyzed the effects of miR-125a-5p down-regulation on HSC activation and proliferation. We also analyzed the binding of miR-125a-5p to the 3'-untranslated region of factor inhibiting hypoxia-inducible factor 1 (FIH1) mRNA. RESULTS: Up-regulation of miR-125a-5p was observed in the liver tissues of fibrotic mice and activated HSCs. Down-regulation of miR-125a-5p prevented the activation and proliferation of HSCs. FIH1, a negative modulator of hypoxia inducible factor 1, was confirmed to be a target of miR-125a-5p using the luciferase reporter assay. Further studies demonstrated that miR-125a-5p prompted the activation and proliferation of HSCs, at least in part, by down-regulating FIH1. CONCLUSION: Our findings shed new light on miRNAs as a promising therapeutic target in liver fibrosis.

Identification of a Novel lincRNA-p21-miR-181b-PTEN Signaling Cascade in Liver Fibrosis.

Previously, we found that long intergenic noncoding RNA-p21 (lincRNA-p21) inhibits hepatic stellate cell (HSC) activation and liver fibrosis via p21. However, the underlying mechanism of the antifibrotic role of lincRNA-p21 in liver fibrosis remains largely unknown. Here, we found that lincRNA-p21 expression was significantly downregulated during liver fibrosis. In LX-2 cells, the reduction of lincRNA-p21 induced by TGF-ß1 was in a dose- and time-dependent manner. lincRNA-p21 expression was reduced in liver tissues from patients with liver cirrhosis when compared with that of healthy controls. Notably, lincRNA-p21 overexpression contributed to the suppression of HSC activation. lincRNA-p21 suppressed HSC proliferation and induced a significant reduction in α-SMA and type I collagen. All these effects induced by lincRNA-p21 were blocked down by the loss of PTEN, suggesting that lincRNA-p21 suppressed HSC activation via PTEN. Further study demonstrated that microRNA-181b (miR-181b) was involved in the effects of lincRNA-p21 on HSC activation. The effects of lincRNA-p21 on PTEN expression and HSC activation were inhibited by miR-181b mimics. We demonstrated that lincRNA-p21 enhanced PTEN expression by competitively binding miR-181b. In conclusion, our results disclose a novel lincRNA-p21-miR-181b-PTEN signaling cascade in liver fibrosis and suggest lincRNA-p21 as a promising molecular target for antifibrosis therapy.