Jie Zhu

miR-30 inhibits TGF-β1-induced epithelial-to-mesenchymal transition in hepatocyte by targeting Snail1

Epithelial-to-mesenchymal transition (EMT) has been implicated in embryonic development and various pathological events. Snail1 is a well-known E-cadherin-transcriptional repressor that is significantly upregulated during the TGF-β1-induced EMT in hepatocyte. However, the functional involvement of microRNA during the EMT process in hepatocyte remains to be determined. Here, we revealed that while the expression of Snail1 increased during the TGF-β1-induced EMT in AML12 murine hepatocytes, the expression of miR-30 family members exhibited significant downregulation. Computational microRNA target predictions detected a conserved sequence matching to the seed region of miR-30 in the 3UTR of Snail1 mRNA. Our results demonstrated that miR-30 could negatively regulate the expression of Snail1 by direct targeting the predicted binding site. More importantly, transfection of miR-30b mimics significantly inhibited the TGF-β1-induced EMT in AML12 cells as assessed through cell morphology changes and the expression profiles of Snail1, E-cadherin and other fibroblast markers. Finally, we demonstrated that TGF-β1-induced hepatocyte migration was greatly suppressed in cells transfected with miR-30b mimics. Our results provide a new insight into the role of miR-30 in regulating EMT, which could be of importance in understanding the related physiologic and pathologic processes.

MicroRNA-31 activates the RAS pathway and functions as an oncogenic MicroRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1).

Background: MicroRNAs are important for colorectal cancer signal transduction. Results: miR-31 stimulates colorectal cancer cell proliferation and tumorigenesis by directly targeting RASA1. Conclusion: miR-31 activates the RAS pathway and functions as an oncogenic microRNA in human colorectal cancer. Significance: Learning how miRNAs participate in tumor signaling is crucial for understanding tumor signal transduction and cancer therapy. MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3′-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.

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

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.

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.

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.

High-level expression of recombinant human paraoxonase 1 Q in silkworm larvae (Bombyx mori)

Human serum paraoxonase 1 (hPON1) belongs to a family of enzymes that catalyze the hydrolysis of a broad range of esters and lactones. Although the very first identification of hPON1 might have been as a calcium-dependent paraoxonase/arylesterase, PON1 is in fact a lactonase associated with high-density lipoprotein and strongly stimulated by apoA-I. PON1 hydrolyzes various organophosphates, including insecticides and nerve gases. PON1 also plays a key role in prevention of atherosclerosis. Mediation of cholesterol efflux from macrophage is a key in vivo function of PON1. In present study, the hPON1 Q gene was cloned into baculovirus transfer vector pVL1392 and expressed in silkworm expression system. The rhPON1 Q presented two bands with every near molecular weight of about 40 and 43xa0kDa according to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis. The expression level was up to 1,256xa0mg/L in haemolymph, about 50 times as high as that from BmN cells (24.8xa0mg/L). After purified by two chromatography steps (DEAE-Sepharose and HiTrap Chelating HP), the purity of rhPON1 Q was up to 90%, and the enzymatic properties are similar to serum hPON1.

Knockdown of N-Acetylglucosaminyl Transferase V Ameliorates Hepatotoxin-Induced Liver Fibrosis in Mice

Aberrant N-glycosylation caused by altered N-acetyl glucosaminyltransferase V (GnT-V) expression is known to regulate tumor invasion and metastasis by modulating multiple cytokine signaling pathways. However, the exact role of GnT-V in the development of liver fibrosis has not been clearly defined. Here, we induced mouse liver fibrosis by ip injections of carbon tetrachloride (CCl4) or thioacetamide (TAA) and observed significant increase of hepatic GnT-V during the processes of liver fibrogenesis. Meanwhile, upregulations of GnT-V were detected in the activated hepatic stellate cells (HSCs) and injured hepatocytes. To knock down hepatic GnT-V expression, adenovirus that expressed the GnT-V siRNA was injected via the tail vein. Adenovirus-mediated delivery of GnT-V siRNA dramatically reduced the GnT-V expression in fibrotic liver and activated HSC in vivo and consequently alleviated CCl4- or TAA-induced liver fibrosis as assessed through collagen deposition and profiles of profibrogenic markers. Furthermore, knockdown of GnT-V in HSCs reduced transforming growth factor beta (TGF-β)/Smad signaling and blunted the activated HSC phenotype. The suppression of TGF-β/Smad signaling in HSCs correlated with the decrease of GnT-V-modified β1,6-branched N-glycan on TGF-β receptors. Knockdown of GnT-V also suppressed platelet-derived growth factor (PDGF)-induced HSC proliferation and migration through inhibiting PDGF/Erk signaling. Finally, we demonstrated that knockdown of GnT-V profoundly suppressed TGF-β1-induced epithelial-mesenchymal transition (EMT) in hepatocytes by morphological assessment and reversal of EMT markers. In conclusion, this study demonstrates that GnT-V is implicated in hepatotoxin-induced liver fibrosis, and targeting GnT-V may be a feasible and promising approach for treating liver fibrosis.

Expression of a novel recombinant dual human stem cell factor in insect cells

Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. A dual human stem cell factor (dhSCF) cDNA was constructed, which consisted of a full-length human stem cell factor cDNA plus a truncated hSCF cDNA (1-145aa), linked by a peptide (GGGGSGGGGSGG) coding region. The dhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the control of polyhedrin promoter. The Sf9 cells infected with the recombinant virus expressed rdhSCF up to 6000 U/10(6) cell in flask and 8300 U/10(6) cell in spinner flask. The rdhSCF was purified by two-step chromatography. The molecular mass of rdhSCF was examined by western blotting and HPLC analysis. The specific activity of rdhSCF was up to 3.1x10(6) U/mg, about 8.7 times as high as that of monomer rhSCF from Escherichia coli.