Yuhui Zang

MicroRNA‐101 suppresses liver fibrosis by targeting the TGFβ signalling pathway

Transforming growth factor‐β (TGFβ) is crucial for liver fibrogenesis and the blunting of TGFβ signalling in hepatic stellate cells (HSCs) or hepatocytes can effectively inhibit liver fibrosis. microRNAs (miRNAs) have emerged as key regulators in modulating TGFβ signalling and liver fibrogenesis. However, the regulation of TGFβ receptor I (TβRI) production by miRNA remains poorly understood. Here we demonstrate that the miR‐101 family members act as suppressors of TGFβ signalling by targeting TβRI and its transcriptional activator Kruppel‐like factor 6 (KLF6) during liver fibrogenesis. Using a mouse model of carbon tetrachloride (CCl4)‐induced liver fibrosis, we conducted a time‐course experiment and observed significant down‐regulation of miR‐101 in the fibrotic liver as well as in the activated HSCs and injured hepatocytes in the process of liver fibrosis. Meanwhile, up‐regulation of TβRI/KLF6 was observed in the fibrotic liver. Subsequent investigations validated that TβRI and KLF6 were direct targets of miR‐101. Lentivirus‐mediated ectopic expression of miR‐101 in liver greatly reduced CCl4‐induced liver fibrosis, whereas intravenous administration of antisense miR‐101 oligonucleotides aggravated hepatic fibrogenesis. Mechanistic studies revealed that miR‐101 inhibited profibrogenic TGFβ signalling by suppressing TβRI expression in both HSCs and hepatocytes. Additionally, miR‐101 promoted the reversal of activated HSCs to a quiescent state, as indicated by suppression of proliferation and migration, loss of activation markers and gain of quiescent HSC‐specific markers. In hepatocytes, miR‐101 attenuated profibrogenic TGFβ signalling and suppressed the consequent up‐regulation of profibrogenic cytokines, as well as TGFβ‐induced hepatocyte apoptosis and the inhibition of cell proliferation. The pleiotropic roles of miR‐101 in hepatic fibrogenesis suggest that it could be a potential therapeutic target for liver fibrosis. Copyright

The Autoregulatory Feedback Loop of MicroRNA-21/Programmed Cell Death Protein 4/Activation Protein-1 (MiR-21/PDCD4/AP-1) as a Driving Force for Hepatic Fibrosis Development

Background: MicroRNA-21 is important in hepatic fibrosis development, but the mechanism is unclear. Results: MicroRNA-21 is predominantly up-regulated in activated hepatic stellate cells and could form a double negative feedback loop that links fibrogenic machinery. Conclusion: The microRNA-21-mediated loop is a main driving force for hepatic fibrosis progression. Significance: It suggests a mechanism for how microRNA-21 contributes to hepatic fibrosis. Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.

MicroRNA-30 Protects Against Carbon Tetrachloride-induced Liver Fibrosis by Attenuating Transforming Growth Factor Beta Signaling in Hepatic Stellate Cells

Transforming growth factor beta (TGF-β) is crucial for transdifferentiation of hepatic stellate cells (HSCs) and the blunting of TGF-β signaling in HSCs can effectively prevent liver fibrosis. Krüppel-like factor 11 (KLF11) is an early response transcription factor that potentiates TGF-β/Smad signaling by suppressing the transcription of inhibitory Smad7. Using a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, we observed significant upregulation of KLF11 in the activated HSCs during liver fibrogenesis. Meanwhile, the downregulation of miR-30 was observed in the HSCs isolated from fibrotic liver. Adenovirus-mediated ectopic expression of miR-30 was under the control of smooth muscle α-actin promoter, showing that the increase in miR-30 in HSC greatly reduced CCl4-induced liver fibrosis. Subsequent investigations showed that miR-30 suppressed KLF11 expression in HSC and led to a significant upregulation of Smad7 in vivo. Mechanistic studies further confirmed that KLF11 was the direct target of miR-30, and revealed that miR-30 blunted the profibrogenic TGF-β signaling in HSC by suppressing KLF11 expression and thus enhanced the negative feedback loop of TGF-β signaling imposed by Smad7. Finally, we demonstrated that miR-30 facilitated the reversal of activated HSC to a quiescent state as indicated by the inhibition of proliferation and migration, the loss of activation markers, and the gain of quiescent HSC markers. In conclusion, our results define miR-30 as a crucial suppressor of TGF-β signaling in HSCs activation and provide useful insights into the mechanisms underlying liver fibrosis.

An orally administrated nucleotide-delivery vehicle targeting colonic macrophages for the treatment of inflammatory bowel disease.

Tumor necrosis factor-alpha (TNF-α) plays a central role in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF-α therapies have shown protective effects against colitis, but an efficient tool for target suppression of its secretion - ideally via oral administration - remains in urgent demand. In the colon tissue, TNF-α is mainly secreted by the colonic macrophages. Here, we report an orally-administrated microspheric vehicle that can target the colonic macrophages and suppress the local expression of TNF-α for IBD treatment. This vehicle is formed by cationic konjac glucomannan (cKGM), phytagel and an antisense oligonucleotide against TNF-α. It was given to dextran sodium sulfate (DSS) colitic mice via gastric perfusion. The unique swelling properties of cKGM enabled the spontaneous release of cKGM& antisense nucleotide (ASO) nano-complex from the phytagel scaffold into the colon lumen, where the ASO was transferred into colonic macrophages via receptor-mediated phagocytosis. The treatment significantly decreased the local level of TNF-α and alleviated the symptoms of colitis in the mice. In summary, our study demonstrates a convenient, orally-administrated drug delivery system that effectively targets colonic macrophages for suppression of TNF-α expression. It may represent a promising therapeutic approach in the treatment of IBD.

Transgene expression of human PON1 Q in mice protected the liver against CCl4-induced injury.

Oxidative stress, often in association with decreased antioxidant defenses, plays a pathogenetic role in both initiation and progression of liver injuries, leading to almost all clinical and experimental conditions of chronic liver diseases. Human paraoxonase 1 (hPON1) is a liver‐synthesized enzyme possessing antioxidant properties. Here, we investigate the effects of transgene‐expressed hPON1 Q on alleviating lipid peroxidation and preventing liver injury in a mouse model.

Loss of MicroRNA-101 Promotes Epithelial to Mesenchymal Transition in Hepatocytes

Epithelial‐to‐mesenchymal transition (EMT) has been implicated in embryonic development and various pathological events. However, the involvement of microRNA in the process of EMT remains to be fully defined in hepatocyte. ZEB1 is a well‐known transcriptional repressor of E‐cadherin and plays a major role in triggering EMT during organ fibrosis and cancer cell metastasis. Computational microRNA target predictions detect a conserved sequence matching to miR‐101 in the 3′UTR of ZEB1 mRNA. Our results confirm that miR‐101 suppresses ZEB1 expression by targeting the predicted site of ZEB1 3′UTR. Subsequent investigations show that miR‐101 is significantly downregulated in the cultured hepatocytes undergoing EMT and in the hepatocytes isolated from fibrotic liver. Along with the loss of miR‐101, the ZEB1 expression increases simultaneously in hepatocytes. In addition, miR‐101 levels in HCC cell lines are negatively associated with the ZEB1 productions and the metastatic potentials of tumor cells. Mechanistically, we demonstrate that miR‐101 significantly inhibits the TGF‐β1‐induced EMT in hepatocytes, whereas inhibition of miR‐101 promotes the EMT process as indicated by the changes of morphology, cell migration, and the expression profiles of EMT markers. In the fibrotic liver, ectopic expression of miR‐101 can significantly downregulate ZEB1 in the hepatocyte and thereby reduces the mesenchymal marker expression. Moreover, miR‐101 significantly inhibits the proliferation and migration of HCC cell. Our results demonstrate that miR‐101 regulates HCC cell phenotype by upregulating the epithelial marker genes and suppressing the mesenchymal ones. J. Cell. Physiol. 9999: 2706–2717, 2015.

3,3'-Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting miR-21 expression.

Hepatic fibrosis is a type of liver disease characterized by excessive collagen deposition produced by activated hepatic stellate cells (HSCs), and no appropriate drug treatment is available clinically. The microRNA, miR‐21 exhibits an important role in the pathogenesis and progression of hepatic fibrosis. 3,3′‐Diindolylmethane (DIM) is a natural autolytic product in plants and can down‐regulate miR‐21 expression. Here we have assessed the therapeutic effects of DIM against hepatic fibrosis and investigated the underlying mechanisms.

Comparative evaluation of the protective potentials of human paraoxonase 1 and 3 against CCl4-induced liver injury

We previously reported that electroporation mediated hPON1 or hPON3 gene delivery could protect against CCl(4)-induced liver injury. However, substantial evidence supported that the in vivo physiological functions of hPON1 and hPON3 were distinct. To compare the protective efficacies of hPON1 and hPON3 against liver injury, recombinant adenovirus AdPON1 and AdPON3, which were capable of expressing hPON1 and hPON3 respectively, were intravenously injected into mice before they were given CCl(4). Adenovirus mediated expression of hPON1 and hPON3 were demonstrated by elevated serum esterase activity, hepatic lactonase activity, and hPON1/hPON3 mRNA expression in liver. Serum transaminase assay, histological observation and TUNEL analysis revealed that the extent of liver injury and hepatocyte apoptosis in AdPON1 or AdPON3 treated mice was significantly ameliorated in comparison with control. Meanwhile, overexpression of hPON1 and hPON3 reduced the hepatic oxidative stress and strengthen the total antioxidant capabilities in liver through affecting the hepatic malondialdehyde (MDA), glutathione (GSH) and total antioxidant capability (T-AOC) levels, regardless of the exposure to CCl(4) or corn oil. Administration of AdPON1 or AdPON3 also suppressed inflammatory response by decreasing TNF-alpha and IL-1beta levels in CCl(4) mice. In this study, hPON1 exhibited a slightly higher efficacy than hPON3 in alleviating liver injury, but the difference between them were not significant.

Protective effects of transgene expressed human PON3 against CCl4-induced subacute liver injury in mice

Oxidative stress plays a crucial role in both initiation and progression of liver injury in almost all experimental and clinical liver diseases. Antioxidative therapy is therefore an effective means of preventing and attenuating oxidative stress related liver diseases. Human paraoxonase 3 (hPON3) is a lipid-associated enzyme with antioxidant activity. In the present study, hPON3 cDNA gene was cloned into pcDNA3.1 plasmid and electro-transferred into mouse skeletal muscle to maintain a higher serum PON3 activity. After gene delivery, serum PON3 activity was about 1.4 times higher than those of control and PON3 mRNA expression was also detected in mouse skeletal muscle. To investigate the role of hPON3 in protecting mice against liver injury, subacute liver injury model was induced by repeated CCl(4) administration and hPON3 gene was delivered into mouse skeletal muscle before progression or recovery phase, respectively, of liver injury. Afterwards, the mice were euthanized to evaluate liver marker enzymes, degrees of oxidative stress and liver histological architecture in order to reveal the effects of PON3 on subacute liver injury. In both damage phases, delivery of hPON3 gene significantly reduced serum aminotransferase level and improved liver histological architecture. Moreover, transgene expression of hPON3 attenuated oxidative stress by increasing hepatic glutathione content, superoxide dismutase (SOD) activity, total antioxidant capability (T-AOC), and reducing malondialdehyde (MDA) level.

Targeted delivery of let-7b to reprogramme tumor-associated macrophages and tumor infiltrating dendritic cells for tumor rejection.

Both tumor associated macrophages (TAMs) and tumor infiltrating dendritic cells (TIDCs) are important components in the tumor microenvironment that mediate tumor immunosuppression and promote cancer progression. Targeting these cells and altering their phenotypes may become a new strategy to recover their anti-tumor activities and thereby restore the local immune surveillance against tumor. In this study, we constructed a nucleic acid delivery system for the delivery of let-7b, a synthetic microRNA mimic. Our carrier has an affinity for the mannose receptors on TAMs/TIDCs and is responsive to the low-pH tumor microenvironment. The delivery of let-7b could reactivate TAMs/TIDCs by acting as a TLR-7 agonist and suppressing IL-10 production in vitro. In a breast cancer mouse model, let-7b delivered by this system efficiently reprogrammed the functions of TAMs/TIDCs, reversed the suppressive tumor microenvironment, and inhibited tumor growth. Taken together, this strategy, designed based upon TAMs/TIDCs-targeting delivery and the dual biological functions of let-7b (TLR-7 ligand and IL-10 inhibitor), may provide a new approach for cancer immunotherapy.