Dingkang Yao

The role of the miR-31/FIH1 pathway in TGF-β-induced liver fibrosis.

The miRNAs are small, non-coding RNAs that regulate various biological processes, including liver fibrosis. Hepatic stellate cells (HSCs) play a central role in the pathogenesis of liver fibrosis. By microarray profiling and real-time PCR, we noted that miR-31 expression in HSCs from rats, mice and humans was significantly increased during HSC activation in culture. Overall, miR-31 expression levels were unchanged in the whole-liver RNA extracts from fibrotic rat and human samples. Nevertheless, we found that miR-31 was particularly up-regulated in HSCs but not in hepatocytes during fibrogenesis. Thus, we hypothesized that miR-31 may mediate liver fibrosis. In the present study, we found that inhibition of miR-31 expression significantly inhibited HSC activation, whereas its over-expression obviously promoted HSC activation. Moreover, over-expression of miR-31 promoted HSC migration by enhancing matrix metalloproteinase (MMP)-2 expression whereas inhibition of miR-31 has an opposite effect. The biological function of miR-31 during HSC activation might be through targeting FIH1, a suppressor of hypoxia-inducible factor (HIF-1), because a knockdown of FIH1 by shRNA could mimic the effects of miR-31. In addition, primary rat HSCs were isolated and treated with different cytokines, such as transforming growth factor β (TGF-β), vascular endothelial growth factor and platelet-derived growth factor-BB, to evaluate upstream regulators of miR-31. We found that only TGF-β, a pivotal regulator in liver fibrosis, remarkably increased miR-31 expression in HSCs. And the effects of TGF-β on HSCs can be partially counteracted by inhibition of miR-31. In addition, chromatin immunoprecipitation experiments and the luciferase reporter assay demonstrated that Smad3, a major TGF-β-downstream transcription factor, stimulated the transcription activity of miR-31 by binding directly to miR-31s promoter. In conclusion, the miR-31/FIH1 pathway associates with liver fibrosis, perhaps by participation in the TGF-β/Smad3 signalling of HSCs.

Overexpression of miR-126 Inhibits the Activation and Migration of HSCs through Targeting CRK

Background & Aims: MicroRNAs (miRNAs) have been shown to play essential roles in HSCs activation which contributes to hepatic fibrosis. Our previous miRNA microarray results suggested that miR-126 might be decreased during HSCs activation as other studies. The aim of this study is to investigate the role of miR-126 during HSCs activation. Methods: In this study, the expression of miR-126 during HSCs activation was measured and confirmed by qRT-PCR. Then, miR-126 expression was restored by transfection of lentivirus vector encoding miR-126. Futhermore, cell proliferation was assayed by the cell counting kit-8 (CCK-8), cell migration was assayed by transwell assay, and the markers of activation of HSCs, α-SMA and collagen type I, were assayed by qRT-PCR, Western Blotting, Immunostaining and ELISA. Luciferase reporter assay was used to find the target of miR-126, and Western Blotting and Immunostaining was used to validate the target of miR-126. Then, the expression and the role of the target of miR-126 during HSCs activation was further assessed. Results: The expression of miR-126 was confirmed to be significantly decreased during HSCs activation. Overexpression of miR-126 significantly inhibited HSCs migration but did not affect HSCs proliferation. The expression of α-SMA and collagen type I were both obviously decreased by miR-126 restoration. CRK was found to be the target of miR-126 and overexpression of miR-126 significantly inhibited CRK expression. And it was found that overexpression of CRK also significantly decreased miR-126 expression and promoted HSCs activation. Conclusions: Our study showed that overexpression of miR-126 significantly inhibited the activation and migration of HSCs through targeting CRK which can also decrease miR-126 expression and promote HSCs activation.

MiR-9a-5p regulates proliferation and migration of hepatic stellate cells under pressure through inhibition of Sirt1.

AIM To reveal the functions of microRNAs (miRNAs) with respect to hepatic stellate cells (HSCs) in response to portal hypertension. METHODS Primary rat HSCs were exposed to static water pressure (10 mmHg, 1 h) and the pressure-induced miRNA expression profile was detected by next-generation sequencing. Quantitative real-time polymerase chain reaction was used to verify the expression of miRNAs. A potential target of MiR-9a-5p was measured by a luciferase reporter assay and Western blot. CCK-8 assay and Transwell assay were used to detect the proliferation and migration of HSCs under pressure. RESULTS According to the profile, the expression of miR-9a-5p was further confirmed to be significantly increased after pressure overload in HSCs (3.70 ± 0.61 vs 0.97 ± 0.15, P = 0.0226), which resulted in the proliferation, migration and activation of HSCs. In vivo, the up-regulation of miR-9a-5p (2.09 ± 0.91 vs 4.27 ± 1.74, P = 0.0025) and the down-regulation of Sirt1 (2.41 ± 0.51 vs 1.13 ± 0.11, P = 0.0006) were observed in rat fibrotic liver with portal hypertension. Sirt1 was a potential target gene of miR-9a-5p. Through restoring the expression of Sirt1 in miR-9a-5p transfected HSCs on pressure overload, we found that overexpression of Sirt1 could partially abrogate the miR-9a-5p mediated suppression of the proliferation, migration and activation of HSCs. CONCLUSION Our results suggest that during liver fibrosis, portal hypertension may induce the proliferation, migration and activation of HSCs through the up-regulation of miR-9a-5p, which targets Sirt1.

Increased Siglec-1 Expression in Monocytes of Patients with Primary Biliary Cirrhosis

Objectives: To evaluate Siglec-1 protein (CD169) and mRNA levels in peripheral blood monocytes of patients with primary biliary cirrhosis (PBC) and investigate its role in PBC pathogenesis by looking for correlations between Siglec-1 expression and key PBC associated biochemical indices. Methods: FACS analysis was used to identify the percentage of peripheral blood monocytes positive for both CD14 and Siglec-1 in (a) 45 PBC patients, (b) 40 patients with liver cirrhosis after hepatitis B infection and (c) 36 healthy controls. Siglec-1 mRNA was measured by real-time RT-PCR and serum biomarkers by routine biochemistry. Results: The percentage of CD14-Siglec-1 double positive cells was significantly higher (p< 0.01) in PBC patients than in healthy controls or cirrhosis post-hepatitis patients (13.68 ± 2.44%, 1.0 ± 0.2 %, and 4.1 ± 0.5 %, respectively). Siglec-1 mRNA expression in the PBC group was 3.42 times higher than in healthy controls (p < 0.01). Conclusion: We investigated the role of Siglec-1 in PBC by assessing its expression in mononuclear cells of PBC patients and levels of secreted cytokines in cell supernatants after Siglec-1 RNA interference. It is possible that elevated Siglec-1 expression in peripheral blood monocytes of PBC patients is correlated with monocyte-mediated inflammatory responses during the development of PBC.

Resistance to activation-induced cell death and elevated FLIPL expression of CD4+ T cells in a polyI:C-induced primary biliary cirrhosis mouse model

Primary biliary cirrhosis (PBC) is a type of organ-specific autoimmune disease in which immune tolerance is impaired by an unknown mechanism. We established a PBC animal model by injecting C57BL/6 mice with polyI:C to study activation-induced cell death (AICD) in CD4+ T lymphocytes and changes of apoptosis-associated molecules as a first step to understand the immune tolerance of PBC mice. Obvious inflammatory cell infiltration was observed in the portal area of the liver tissues in model mice and antimitochondrial antibodies (AMA) positive rate was 80%. The AICD level in both splenic and hepatic CD4+ T cells in the model group were all lower than those in controls, and in the model group the level for hepatic CD4+ T cells were significantly lower than that for splenic CD4+ T cells. Quantitative PCR revealed that FasL mRNA and TRAIL expression in CD4+ T cells in the model group decreased significantly compared with that in the control group. Western blots revealed that the expression of the anti-apoptotic protein FLIPL in the model group increased significantly with the FLIPL expression in hepatic CD4+ T cells significantly higher than that in splenic CD4+ T cells. There was a positive linear correlation between the number of infiltrated portal areas and relative expression of FLIPL in splenic CD4+ T cells in model group. There were no obvious changes for caspase-8 in either group. These results show that the anti-apoptotic ability of CD4+ T lymphocytes play an important role in immune tolerance in the PBC mouse model, and elevated FLIPL expression may enhance this ability. The inhibition of FasL and TRAIL expression may also help enhance this anti-apoptotic ability in CD4+ T lymphocytes and contribute to the aggravation of portal area inflammation.

TNFSF9 expression in primary biliary cirrhosis and its clinical significance

Primary biliary cirrhosis (PBC) is a TH1/Th17 biased autoimmune disease of the medium and small bile ducts. The role of the costimulatory TNFSF9 (4-1BBL) in PBC progress was investigated by comparing its cell surface expression in peripheral blood mononuclear cells (PBMC) by flow cytometry, its mRNA expression in PBMCs by QRT-PCR and its serum concentrations in PBC patients vs. healthy controls. The TNFSF9 expression levels were compared with Mayo risk scores, PBC stages, IL-18 serum levels, total bilirubin (TBIL), and gamma glutamyltransferase (gamma-GT). The PBC patients expressed significantly greater levels of membrane bound TNFSF9, mRNA on peripheral blood mononuclear cells (PBMC), and soluble TNFSF9 (P<0.05) than healthy controls. Stage III and IV PBC subjects showed significantly reduced TNFSF9 mRNA than stage I and II. The TBIL, gamma-GT, and IL-18 were greatly increased in PBC patients compared with healthy controls. Stage II, III, and IV patients exhibited significantly higher IL-18 levels than stage I subjects. TNFSF9 mRNA significantly correlated with serum TBIL, gamma-GT, and IL-18 (P<0.05, P<0.01, P<0.01). Thus, TNFSF9 mRNA levels in PBMC may be associated with PBC progression, provide new clues for monitoring its condition and pathogenesis.

Increased Granulysin Expression in Peripheral Blood Cells of Patients with Primary Biliary Cirrhosis and Its Clinical Implications

IntroductionGranulysin is a cytotoxic molecule involved in cellular immune reactions.Materials and methodsThe levels of granulysin mRNA in the peripheral blood and serum granulysin of patients with primary biliary cirrhosis (PBC) were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of granulysin mRNA and serum protein in PBC was increased compared to the controls.ResultsThe expression of granulysin mRNA or serum protein showed close associations, respectively, with natural killer cell population in PBC patients. Serum granulysin was down-regulated by steroid and ursodeoxycholic therapy for PBC according to the improvement of severity of PBC. In addition, the expression of serum granulysin were related to serum total bilirubin and Mayo Clinic risk score. The serum granulysin reflected the cellular immune status of patients with PBC, and the expression correlated with the severity of PBC.ConclusionTherefore, there is a clinical benefit to monitoring granulysin as a biomarker of the prognosis of patients with PBC.

Potentials of the elevated circulating miR-185 level as a biomarker for early diagnosis of HBV-related liver fibrosis

Early diagnosis of liver fibrosis is critical for early intervention and prognosis of various chronic liver diseases. Conventional repeated histological assessment is impractical due to the associated invasiveness. In the current study, we evaluated circulating miR-185 as a potential biomarker to predict initiation and progression of liver fibrosis. We found that miR-185 was significantly up-regulated in blood specimens from patients with HBV-liver fibrosis and rats with liver fibrosis, the miR-185 levels were correlated with liver fibrosis progression, but not with the different viral loads in HBV-infected patients. miR-185 was observed in collagen deposition regions during advanced liver fibrosis. We found that differences in miR-185 levels facilitated the discrimination between early-staged or advanced-staged liver fibrosis and the healthy controls with high specificity, sensitivity, and likelihood ratio using receiver-operator characteristic analysis. miR-185 targeted SREBF1, and increased expression of COL1A1 and a-SMA genes that are hallmarks of liver fibrosis. Our data supported that circulating miR-185 levels could be used as potential biomarkers for the early diagnosis of liver fibrosis.

Value of baseline platelet count for prediction of complications in primary biliary cirrhosis patients treated with ursodeoxycholic acid

Abstract Background. Decreased platelet count has been observed in various liver diseases, but its significance in primary biliary cirrhosis (PBC) remains unknown. The present study aimed to evaluate the predictive value of the platelet count at diagnosis for PBC-related complications in patients newly diagnosed with PBC and treated with ursodeoxycholic acid (UDCA). Methods. Ninety-six PBC patients without complications treated with UDCA immediately after diagnosis were retrospectively reviewed. All hematologic and chemical parameters, Mayo risk score and PBC-related complications including upper gastrointestinal hemorrhage, presence of ascites, serum bilirubin concentration > 102.6 μmol/L and onset of hepatic encephalopathy were extracted. The associations between these parameters at diagnosis and complications were determined and the prognostic value of the platelet count was evaluated by receiver operating characteristics (ROC) analysis, Kaplan-Meier method and Cox proportional hazard model with the hazard ratio (HR) and 95% confidence interval (CI) calculated. Results. Patients with PBC-related complications had significantly decreased platelet count and serum bilirubin concentration, prolonged prothrombin time, and increased Mayo risk score compared to those without complications. A platelet count of ≤ 132.5 × 109/L was associated with the occurrence of complications, with an area under the ROC curve of 0.74 (95% CI: 0.64–0.85). The association remained even after adjustment for Mayo risk score (HR: 2.85; 95% CI: 1.46–5.54; p < 0.01), as shown in the Cox proportional hazard model. Conclusions. Decreased platelet count is a predictive factor for PBC-related complications. A cut-off value of ≤ 132.5 × 109/L is recommended for the baseline platelet count to predict complications in patients newly diagnosed with PBC and treated with UDCA.

Expression of granulysin in patients with primary biliary cirrhosis and its clinical significance

AIM: To explore the serum expression of granulysin mRNA and protein in patients with primary biliary cirrhosis (PBC). METHODS: Patients with PBC (n = 60), hepatitis B related cirrhosis (n = 60), and healthy controls (n = 100) were included in this study. Based on TaqMan fluorescent probe technique, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of granulysin mRNA, using 18S rRNA as the internal control. The level of granulysin protein was measured by immunohistochemistry. RESULTS: The mean copy number of granulysin mRNA was significantly higher in PBC patients than that in healthy controls [(2.7 ± 2.5) × 10 vs (3.0 ± 1.9) × 10, P < 0.01) or patients with hepatitis B related cirrhosis [(2.7 ± 2.5) × 10 vs (4.7 ± 3.6) × 10, P < 0.001). Meanwhile, the serum level of granulysin protein was also markedly higher in PBC patients than that in healthy controls (15.48 ± 3.24 μg/L vs 4.76 ± 2.32 μg/L) or patients with hepatitis B related cirrhosis (15.48 ± 3.24 μg/L vs 2.57 ± 1.84 μg/L, P < 0.01). CONCLUSION: Granulysin expression is associated with the occurrence and progression of PBC, which can help to make diagnosis for PBC.