Jianjian Zheng

Curcumin up‐regulates phosphatase and tensin homologue deleted on chromosome 10 through microRNA‐mediated control of DNA methylation – a novel mechanism suppressing liver fibrosis

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has been reported to play a role in the suppression of activated hepatic stellate cells (HSCs). Moreover, it has been demonstrated that hypermethylation of the PTEN promoter is responsible for the loss of PTEN expression during HSC activation. Methylation is now established as a fundamental regulator of gene transcription. MicroRNAs (miRNAs), which can control gene expression by binding to their target genes for degradation and/or translational repression, were found to be involved in liver fibrosis. However, the mechanism responsible for miRNA‐mediated epigenetic regulation in liver fibrosis still remained unclear. In the present study, curcumin treatment significantly resulted in the inhibition of cell proliferation and an increase in the apoptosis rate through the up‐regulation of PTEN associated with a decreased DNA methylation level. Only DNA methyltransferase 3b (DNMT3b) was reduced in vivo and in vitro after curcumin treatment. Further studies were performed aiming to confirm that the knockdown of DNMT3b enhanced the loss of PTEN methylation by curcumin. In addition, miR‐29b was involved in the hypomethylation of PTEN by curcumin. MiR‐29b not only was increased by curcumin in activated HSCs, but also was confirmed to target DNMT3b by luciferase activity assays. Curcumin‐mediated PTEN up‐regulation, DNMT3b down‐regulation and PTEN hypomethylation were all attenuated by miR‐29b inhibitor. Collectively, it is demonstrated that curcumin can up‐regulate miR‐29b expression, resulting in DNMT3b down‐regulation in HSCs and epigenetically‐regulated PTEN involved in the suppression of activated HSCs. These results indicate that miRNA‐mediated epigenetic regulation may be a novel mechanism suppressing liver fibrosis.

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

Background: Long non-coding RNAs function as competing endogenous RNAs (ceRNAs). Whether growth arrest-specific transcript 5 (GAS5) acts as a ceRNA for microRNA-222 in liver fibrosis remains undefined. Results: GAS5 increases p27 expression as a ceRNA for microRNA-222, thereby inhibiting liver fibrosis progression. Conclusion: The GAS5/microRNA-222/p27 axis underlies the pathogenesis of liver fibrosis. Significance: The ceRNA network helps to understand liver fibrogenesis. 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.

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-β1 (TGF-β1) 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-β1-induced hepatic fibrogenesis remains to be established. In this study, we demonstrated a dose-/time-dependent increase in miR-17-5p expression in TGF-β1-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-β1 without affecting cellular apoptosis. Notably, miR-17-5p was significantly associated with TGF-β1-induced expression of type I collagen and α-SMA in HSC. Furthermore, Smad7, a negative regulator of the TGF-β/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.

MALAT1 functions as a competing endogenous RNA to mediate Rac1 expression by sequestering miR-101b in liver fibrosis

Emerging evidence shows that Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a pivotal role in cell proliferation, migration, and invasion in tumors. However, the biological role and underlying mechanism of MALAT1 in liver fibrosis remains undefined. In this study, up-regulation of MALAT1 was observed in fibrotic liver tissues and in activated hepatic stellate cells (HSCs). In addition, depletion of MALAT1 inhibited the activation of HSCs in vitro and attenuated collagen deposits in vivo. Our results demonstrated that MALAT1 expression is negatively correlated with microRNA-101b (miR-101b) expression. Furthermore, there was a negative feedback loop between the levels of MALAT1 and miR-101b. Luciferase reporter assay indicated that MALAT1 and RAS-related C3 botulinum substrate 1 (Rac1) are targets of miR-101b. We uncovered that MALAT1 regulates Rac1 expression through miR-101b as a competing endogenous RNA (ceRNA), thereby influencing the proliferation, cell cycle and activation of primary HSCs. Collectively, The ceRNA regulatory network may prompt a better understanding of liver fibrogenesis and contribute to a novel therapeutic strategy for liver fibrosis.

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.

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-β1-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.

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.

lincRNA-p21 inhibits hepatic stellate cell activation and liver fibrogenesis via p21

Long non‐coding RNAs are involved in various biological processes and diseases. The biological role of long intergenic non‐coding RNA‐p21 (lincRNA‐p21) in liver fibrosis remains unknown before this study. In this study, we observed marked reduction of lincRNA‐p21 expression in mice liver fibrosis models and human cirrhotic liver. Over‐expression of lincRNA‐p21 suppressed activation of hepatic stellate cells (HSCs) in vitro. Lentivirus‐mediated lincRNA‐p21 transfer into mice decreased the severity of liver fibrosis in vivo. Additionally, lincRNA‐p21 reversed the activation of HSCs to their quiescent phenotype. The mRNA levels of lincRNA‐p21 and p21 were positively correlated. Our results show that over‐expression of lincRNA‐p21 promotes up‐regulation of p21 at both the mRNA and protein levels. Furthermore, lincRNA‐p21 inhibited cell‐cycle progression and proliferation of primary HSCs through enhancement of p21 expression. Compared with healthy subjects, serum lincRNA‐p21 levels were significantly lower in patients with liver cirrhosis, especially those with decompensation. These findings collectively indicate that lincRNA‐p21 is a mediator of HSC activation, supporting its utility as a novel therapeutic target for 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-β1 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.

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

Background/Aims: Wnt/β-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/β-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/β-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/β-catenin pathway inactivation. Luciferase reporter assays demonstrated that Wnt10a, a member of Wnt/β-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.