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.

Desmin Phosphorylation Triggers Preamyloid Oligomers Formation and Myocyte Dysfunction in Acquired Heart Failure.

RATIONALE: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. OBJECTIVE: We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF. METHODS AND RESULTS: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF. CONCLUSIONS: Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.

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.

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.

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.

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.

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.

Salvianolic acid B-induced microRNA-152 inhibits liver fibrosis by attenuating DNMT1-mediated Patched1 methylation.

Epithelial-mesenchymal transition (EMT) was reported to be involved in the activation of hepatic stellate cells (HSCs), contributing to the development of liver fibrosis. Epithelial-mesenchymal transition can be promoted by the Hedgehog (Hh) pathway. Patched1 (PTCH1), a negative regulatory factor of the Hh signalling pathway, was down-regulated during liver fibrosis and associated with its hypermethylation status. MicroRNAs (miRNAs) are reported to play a critical role in the control of various HSCs functions. However, miRNA-mediated epigenetic regulations in EMT during liver fibrosis are seldom studied. In this study, Salvianolic acid B (Sal B) suppressed the activation of HSCs in CCl4 -treated mice and mouse primary HSCs, leading to inhibition of cell proliferation, type I collagen and alpha-smooth muscle actin. We demonstrated that the antifibrotic effects caused by Sal B were, at least in part, via inhibition of EMT and the Hh pathway. In particular, up-regulation of PTCH1 was associated with decreased DNA methylation level after Sal B treatment. Accordingly, DNA methyltransferase 1 (DNMT1) was attenuated by Sal B in vivo and in vitro. The knockdown of DNMT1 in Sal B-treated HSCs enhanced PTCH1 expression and its demethylation level. Interestingly, increased miR-152 in Sal B-treated cells was responsible for the hypomethylation of PTCH1 by Sal B. As confirmed by the luciferase activity assay, DNMT1 was a direct target of miR-152. Further studies showed that the miR-152 inhibitor reversed Sal B-mediated PTCH1 up-regulation and DNMT1 down-regulation. Collectively, miR-152 induced by Sal B, contributed to DNMT1 down-regulation and epigenetically regulated PTCH1, resulting in the inhibition of EMT in liver fibrosis.

MicroRNA-17-5p activates hepatic stellate cells through targeting of Smad7.

A considerable amount of research has focused on the roles of microRNAs (miRNA) in the pathophysiology of liver fibrosis in view of their regulatory effects on hepatic stellate cell (HSC) functions, including proliferation, differentiation, and apoptosis. Recently, miR-17-5p was shown to promote cell proliferation and migration in liver. Transforming growth factor-ß1 (TGF-ß1) has been characterized as the master fibrogenic cytokine that stimulates HSC activation and promotes progression of liver fibrosis. The issue of whether miR-17-5p plays a role in TGF-ß1-induced hepatic fibrogenesis remains to be established. In this study, we demonstrated a dose-/time-dependent increase in miR-17-5p expression in TGF-ß1-treated HSCs. Enhanced miR-17-5p expression was additionally observed in CCl4-induced rat liver fibrosis. Inhibition of miR-17-5p led to suppression of HSC proliferation induced by TGF-ß1 without affecting cellular apoptosis. Notably, miR-17-5p was significantly associated with TGF-ß1-induced expression of type I collagen and α-SMA in HSC. Furthermore, Smad7, a negative regulator of the TGF-ß/Smad pathway, was confirmed as a direct target of miR-17-5p. Serum miR-17-5p levels were significantly higher in patients with cirrhosis, compared to healthy controls. Our results collectively indicate that miR-17-5p promotes HSC proliferation and activation, at least in part, via reduction of Smad7, supporting its potential utility as a novel therapeutic target for liver fibrosis.

Long non-coding RNA APTR promotes the activation of hepatic stellate cells and the progression of liver fibrosis.

In this study, we aimed at assessing a role of Alu-mediated p21 transcriptional regulator (APTR) in hepatofibrogenesis. APTR was upregulated in fibrotic liver samples and activated hepatic stellate cells (HSCs). Knockdown of APTR inhibited the activation of HSCs in vitro and mitigated the accumulation of collagen in vivo. Importantly, APTR silencing could abrogate TGF-ß1-induced upregulation of α-SMA in HSCs. In addition, inhibition of cell cycle and cell proliferation by APTR knockdown was attenuated by p21 siRNA1 in primary HSCs. Finally, serum APTR levels were increased in patients with liver cirrhosis, indicating a potential biomarker for liver cirrhosis. Collectively, evidence is proposed for a new biological role of APTR in hepatofibrogenesis.

Hepatic stellate cell is activated by microRNA-181b via PTEN/Akt pathway.

Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. MicroRNAs have been shown to play a pivotal role in regulating HSC functions such as cell proliferation, differentiation, and apoptosis. Recently, miR-181b has been reported to promote HSCs proliferation by targeting p27. But whether alpha-smooth muscle actin (α-SMA) or collagens could be promoted by miR-181b in activated HSCs is still not clear. Therefore, the understanding of the role of miR-181b in liver fibrosis remains limited. Our results showed that miR-181b expression was increased much higher than miR-181a expression in vitro in transforming growth factor-ß1-induced HSC activation as well as in vivo in carbon tetrachloride-induced rat liver fibrosis. Of note, overexpression of miR-181b significantly increased the expressions level of α-SMA and type I collagen, and further promoted HSCs proliferation. Furthermore, phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of PI3K/Akt pathway, were confirmed as a direct target of miR-181b. We demonstrated that miR-181b could suppress PTEN expression and increase Akt phosphorylation in HSCs. Interestingly, the effects of miR-181b on the activation of HSCs were blocked down by Akt inhibitor LY294002. Our results revealed a profibrotic role of miR-181b in HSC activation and demonstrated that miR-181b could activate HSCs, at least in part, via PTEN/Akt pathway.

Serum miR-181b Is Correlated with Hepatitis B Virus Replication and Disease Progression in Chronic Hepatitis B Patients.

BACKGROUND: Previously, we reported that microRNA-181b (miR-181b) activates hepatic stellate cells partly through the phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/Akt pathway. AIMS: The main objective of this study was to ascertain whether serum miR-181b expression is correlated with that of liver hepatitis B virus (HBV) DNA and disease progression in chronic hepatitis B (CHB) patients. METHODS: Serum miR-181b and liver HBV DNA levels were quantified in 64 CHB patients with real-time PCR. Liver fibrosis and necroinflammation were graded according to the Ishak scoring system. RESULTS: Serum miR-181b levels were evaluated in the CHB group, compared with healthy controls. Expression in patients with HBsAg (+) was higher than that in patients with HBsAg (-). Notably, serum miR-181b and liver HBV DNA levels were significantly correlated (P < 0.05). Serum miR-181 levels were higher in patients with serum HBV DNA > 10(3) IU/ml (P = 0.017), histologic activity index (HAI) >8 (P = 0.001) and fibrosis score >4 (P < 0.0001). Liver HBV DNA levels were higher in patients with abnormal alanine aminotransferase (ALT) values (P = 0.004), serum HBV DNA levels > 10(3) IU/ml (P = 0.034) and fibrosis score >4 (P = 0.006). Using multivariate logistic regression analysis, serum miR-181b was identified as an independent predictor of disease progression (OR 4.172, 95 % CI 1.838-9.473, P = 0.009 for HAI >8; OR 5.387, 95 % CI 2.067-14.036, P = 0.001 for fibrosis score >4). CONCLUSIONS: Serum miR-181b is correlated with liver and serum HBV DNA levels as well as disease progression in CHB.

High expression of serum miR-17-5p associated with poor prognosis in patients with hepatocellular carcinoma.

BACKGROUND: MicroRNAs (miRNAs) have been shown to play pivotal roles in diverse biological processes. Altered expressions of miR-17-5p in several tumor types have been reported. However, the expression and clinical significance of serum miR-17-5p in patients with hepatocellular carcinoma (HCC) are unclear. METHODOLOGY: The expression of miR-17-5p was measured in the serum of paired pre-operative and post-operative groups (n=96) as well as non-relapsed and relapsed groups (n=40) by qRT-PCR. Further study was performed to analyze the correlation of miR-17-5p expression with clinicopathologic variables and the relationship between miR-17-5p expression and the prognosis of HCC patients. RESULTS: The expression of serum miR-17-5p was significantly down-regulated in post-operative group and upregulated in relapsed group. Moreover, the expression of serum miR-17-5p was remarkably associated with the metastasis status and TNM stages (P<0.0001). Importantly, Kaplan-Meier curve analysis revealed that HCC patients with high expression of serum miR-17-5p had a significantly shortened overall survival (P=0.003). Univariate and Multivariable Cox regression analysis indicated that the expression of serum miR-17-5p was an independent risk factor for overall survival (P=0.005 and P=0.043, respectively). CONCLUSIONS: The level of serum miR-17-5p is associated with development of HCC and can serve as a non-invasive biomarker for the prognostic prediction of HCC patients.