Erratum: Loss of lncRNA-SNHG7 Promotes the Suppression of Hepatic Stellate Cell Activation via miR-378a-3p and DVL2.

[This corrects the article DOI: 10.1016/j.omtn.2019.05.026.].

Expression of NOTCH receptors and ligands and prognosis of hepatocellular carcinoma.

Aim: To elucidate potential prognostic significance of NOTCH receptor and ligand expression in hepatocellular carcinoma. Materials & methods: NOTCH receptors and ligands were divided into increased and decreased expression groups by X-tile program. The association between NOTCH receptors/ligands and prognosis was analyzed by Kaplan-Meier method and log-rank test. Gene set enrichment analysis was performed to explore NOTCH receptors/ligands-related pathways via gsea-3.0. Results: DLL3 and DLL4 were independent prognostic factors for overall survival. Further studies showed that only DLL3 was significantly associated with tumor, node, metastasis stage. Gene set enrichment analysis analysis demonstrated that retinol metabolism, drug metabolism cytochrome P450 and tryptophan metabolism were significantly enriched in DLL3 expression phenotype. Conclusion: We demonstrate that DLL3 may be a prognostic biomarker in hepatocellular carcinoma.

Circular RNA cMTO1 Promotes PTEN Expression Through Sponging miR-181b-5p in Liver Fibrosis.

BACKGROUND: Circular RNAs (circRNAs) are considered as key regulators of cancer biology. Recently, cMTO1 (a circRNA derived from MTO1 gene, hsa_circ_0007874) has been demonstrated to act as a tumor suppressor in hepatocellular carcinoma (HCC). However, the roles of cMTO1 in liver fibrosis are largely unknown. METHODS: Expressions and roles of cMTO1 were examined in vivo and in vitro during liver fibrosis. The interaction between microRNA-181b-5p (miR-181b-5p) and cMTO1 was analyzed by luciferase activity assays and pull down assays. RESULTS: cMTO1 was shown to be reduced in the liver from patients with cirrhosis. In addition, cMTO1 was down-regulated in the mouse fibrotic livers as well as activated hepatic stellate cells (HSCs). Restoring of cMTO1 led to a reduction in HSC proliferation. Results of immunofluorescence analysis showed that cMTO1 suppressed the expressions of α-SMA and type I collagen. cMTO1 was found to be expressed in the cytoplasm of HSCs. Further studies confirmed that cMTO1 and miR-181b-5p were co-located in the cytoplasm. Interestingly, there was an interaction between cMTO1 and miR-181b-5p. Results of luciferase reporter assays and pull down assays confirmed that miR-181b-5p could bind to cMTO1. cMTO1-inhibited HSC activation was blocked down by miR-181b-5p or PTEN. Meanwhile, PTEN was a target of miR-181b-5p. CONCLUSION: cMTO1 inhibits HSC activation, at least in part, through miR-181b-5p-mediated PTEN expression. Our results also suggest that cMTO1 may be a novel therapeutic target in liver fibrosis.

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.

Liquiritigenin suppresses the activation of hepatic stellate cells via targeting miR-181b/PTEN axis.

BACKGROUND: Liquiritigenin (LQ), an aglycone of liquiritin in licorice, has demonstrated antioxidant, anti-inflammatory and anti-tumor activities. Previously, LQ was found to inhibit liver fibrosis progression. PURPOSE: Phosphatase and tensin homolog (PTEN) has been reported to act as a negative regulator of hepatic stellate cell (HSC) activation. However, the roles of PTEN in the effects of LQ on liver fibrosis have not been identified to date. METHODS: The effects of LQ on liver fibrosis in carbon tetrachloride (CCl4) mice as well as primary HSCs were examined. Moreover, the roles of PTEN and microRNA-181b (miR-181b) in the effects of LQ on liver fibrosis were examined. RESULTS: LQ markedly ameliorated CCl4-induced liver fibrosis, with a reduction in collagen deposition as well as α-SMA level. Moreover, LQ induced an increase in PTEN and effectively inhibited HSC activation including cell proliferation, α-SMA and collagen expression, which was similar with curcumin (a positive control). Notably, loss of PTEN blocked down the effects of LQ on HSC activation. PTEN was confirmed as a target of miR-181b and miR-181b-mediated PTEN was involved in the effects of LQ on liver fibrosis. LQ led to a significant reduction in miR-181b expression. LQ-inhibited HSC activation could be restored by over-expression of miR-181b. Further studies demonstrated that LQ down-regulated miR-181b level via Sp1. Collectively, we demonstrate that LQ inhibits liver fibrosis, at least in part, via regulation of miR-181b and PTEN. CONCLUSION: LQ down-regulates miR-181b level, leading to the restoration of PTEN expression, which contributes to the suppression of HSC activation. LQ may be a potential candidate drug against liver fibrosis.

Loss of lncRNA-SNHG7 Promotes the Suppression of Hepatic Stellate Cell Activation via miR-378a-3p and DVL2.

Small nuclear RNA host gene 7 (SNHG7), a novel long non-coding RNA (lncRNA), acts as an oncogene in cancers. However, whether SNHG7 is involved in hepatic stellate cell (HSC) activation during liver fibrosis is still unclear. In this study, upregulation of SNHG7 was found in vivo and in vitro during liver fibrosis. Silencing of SNHG7 led to the suppression of HSC activation, with a reduction in cell proliferation and collagen expression. SNHG7 knockdown also resulted in the suppression of liver fibrosis in vivo. Interestingly, miR-378a-3p was a target of SNHG7. SNHG7 and miR-378a-3p were co-located in the cytoplasm. Downregulation of miR-378a-3p blocked down the effects of loss of SNHG7 on HSC activation. Notably, SNHG7 could enhance Wnt/ß-catenin pathway activation to contribute to liver fibrosis, with an increase in T cell factor (TCF) activity and a reduction in P-ß-catenin level. It was found that miR-378a-mediated dishevelled segment polarity protein 2 (DVL2) was responsible for SNHG7-activated Wnt/ß-catenin pathway. DVL2 was confirmed as a target of miR-378a-3p. SNHG7-induced HSC activation was almost blocked down by DVL2 knockdown. Accordingly, enhanced Wnt/ß-catenin by SNHG7 was suppressed by loss of DVL2. Collectively, we demonstrate that SNHG7 reduces miR-378a-3p and attenuates its control on DVL2, leading to aberrant Wnt/ß-catenin activity, which contributes to liver fibrosis progression.

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.

LncRNA-MEG3 inhibits activation of hepatic stellate cells through SMO protein and miR-212.

Activation of hepatic stellate cells (HSCs), a pivotal event in liver fibrosis, is considered as an epithelial-mesenchymal transition (EMT) process. Deregulation of long noncoding RNAs (lncRNAs) has been reported to be involved in a series of human diseases. LncRNA-maternally expressed gene 3 (MEG3) functions as a tumor suppressor in cancers and has been shown to play a vital role in EMT process. However, the biological role of MEG3 in liver fibrosis is largely unknown. In this study, MEG3 was reduced in vivo and in vitro during liver fibrosis. Restoring of MEG3 expression led to the suppression of liver fibrosis, with a reduction in α-SMA and type I collagen. Notably, MEG3 overexpression inhibited HSC activation through EMT, associated with an increase in epithelial markers and a reduction in mesenchymal markers. Further studies showed that Hedgehog (Hh) pathway-mediated EMT process was involved in the effects of MEG3 on HSC activation. Smoothened (SMO) is a member of Hh pathway. Using bioinformatic analysis, an interaction between MEG3 and SMO protein was predicted. This interaction was confirmed by the results of RNA immunoprecipitation and deletion-mapping analysis. Furthermore, MEG3 was confirmed as a target of microRNA-212 (miR-212). miR-212 was partly responsible for the effects of MEG3 on EMT process. Interestingly, MEG3 was also reduced in chronic hepatitis B (CHB) patients with liver fibrosis when compared with healthy controls. MEG3 negatively correlated with fibrosis stage in CHB patients. In conclusion, we demonstrate that MEG3 inhibits Hh-mediated EMT process in liver fibrosis via SMO protein and miR-212.

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.

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.

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.

NEAT1 accelerates the progression of liver fibrosis via regulation of microRNA-122 and Kruppel-like factor 6.

Long non-coding RNAs (lncRNAs) have been reported to be involved in many important biological processes including proliferation, apoptosis, differentiation, and survival. Recently, nuclear paraspeckle assembly transcript 1 (NEAT1), a novel lncRNA, serves as a crucial regulator in tumors. However, the biological role of NEAT1 in liver fibrosis is largely unknown. In this study, the role of NEAT1 was explored in primary mouse hepatic stellate cells (HSCs) and carbon tetrachloride (CCl4)-induced mouse liver fibrosis models. We found that NEAT1 expression was significantly increased in CCl4-induced mice and activated HSCs. Loss of NEAT1 suppressed liver fibrosis in vivo and in vitro. Conversely, NEAT1 overexpression accelerated HSC activation, including increased cell proliferation and collagen expression. Further studies indicated that the microRNA-122 (miR-122)-Kruppel-like factor 6 (KLF6) axis was involved in the effects of NEAT1 on HSC activation. The effects of NEAT1 on HSC activation were almost blocked down by miR-122 mimics or KLF6 knockdown. Interestingly, both NEAT1 and KLF6 are targets of miR-122. In addition, miR-122 led to a significant reduction in NEAT1 level while NEAT1 overexpression resulted in the suppression of miR-122 expression. Pull-down assay confirmed a direct interaction between miR-122 and NEAT1. NEAT1 contributes to HSC activation via the miR-122-KLF6 axis. In human fibrotic liver samples, increased NEAT1 levels positively correlated with liver fibrosis markers. In conclusion, we disclose a novel NEAT1-miR-122-KLF6 signaling cascade and its implication in liver fibrosis. KEY MESSAGES: NEAT1 was significantly increased in CCl4-induced mice and activated HSCs. Loss of NEAT1 suppressed liver fibrosis in vivo and in vitro. KLF6 and miR-122 were required for the effects of NEAT1 on HSC activation. NEAT1 contributes to HSC activation via competitively binding miR-122. We disclose a novel NEAT1-miR-122-KLF6 signaling cascade.

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.

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.

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.

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.

Long non-coding RNA PVT1 activates hepatic stellate cells through competitively binding microRNA-152.

Epithelial-mesenchymal transition (EMT) process is considered as a key event in the activation of hepatic stellate cells (HSCs). Hedgehog (Hh) pathway is known to be required for EMT process. Long non-coding RNAs (lncRNAs) have been reported to be involved in a wide range of biological processes. Plasmacytoma variant translocation 1 (PVT1), a novel lncRNA, is often up-regulated in various human cancers. However, the role of PVT1 in liver fibrosis remains undefined. In this study, PVT1 was increased in fibrotic liver tissues and activated HSCs. Depletion of PVT1 attenuated collagen deposits in vivo. In vitro, PVT1 down-regulation inhibited HSC activation including the reduction of HSC proliferation, α-SMA and type I collagen. Further studies showed that PVT1 knockdown suppressed HSC activation was through inhibiting EMT process and Hh pathway. Patched1 (PTCH1), a negative regulator factor of Hh pathway, was enhanced by PVT1 knockdown. PTCH1 demethylation caused by miR-152 was responsible for the effects of PVT1 knockdown on PTCH1 expression. Notably, miR-152 inhibitor reversed the effects of PVT1 knockdown on HSC activation. Luciferase reporter assays and pull-down assays showed a direct interaction between miR-152 and PVT1. Collectively, we demonstrate that PVT1 epigenetically down-regulates PTCH1 expression via competitively binding miR-152, contributing to EMT process in liver fibrosis.