Tumor suppressor function of ezrin-radixin-moesin-binding phosphoprotein-50 through β-catenin/E-cadher in pathway in human hepatocellular cancer

AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used.We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50.Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells.In vitro cell proliferation was assessed with a Cell Counting Kit-8(CCK-8) assay.Cell cycle distribution was assessed with flow cytometry.Invasion and migration ability of before and after the transfection were determined with a transwell assay.Cell apoptosis was demonstrated with Annexin V-FITC.The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.RESULTS:The transfection efficiency was confirmed with Western blotting(1.36 ± 0.07 vs 0.81 ± 0.09,P < 0.01).The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells(P < 0.01).Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50(61.3% ± 3.1% vs 54.0% ± 2.4%,P < 0.05).The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells(5.8 ± 0.8 vs 21.6 ± 1.3,P < 0.01).Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells(14.8% ± 2.7% vs 3.4% ± 1.3%,P < 0.05).The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells(0.28 ± 0.07 vs 0.56 ± 0.12,P < 0.05;0.55 ± 0.08 vs 0.39 ± 0.07,P < 0.05).In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells(     

Generation of functional hepatocyte-like cells from human bone marrow mesenchymal stem cells by overexpression of transcription factor HNF4α and FOXA2

Background: Our previous study showed that overexpression of hepatocyte nuclear factor 4α(HNF4α) could directly promote mesenchymal stem cells(MSCs) to differentiate into hepatocyte-like cells. However, the efficiency of hepatic differentiation remains low. The purpose of our study was to establish an MSC cell line that overexpressed HNF4α and FOXA2 genes to obtain an increased hepatic differentiation efficiency and hepatocyte-like cells with more mature hepatocyte functions. Methods: Successful establishment of high-level HNF4α and FOXA2 co-overexpression in human induced hepatocyte-like cells(hi Hep cells) was verified by flow cytometry, immunofluorescence and RT-PCR. Measurements of albumin(ALB), urea, glucose, indocyanine green(ICG) uptake and release, cytochrome P450(CYP) activity and gene expression were used to analyze mature hepatic functions of hi Hep cells. Results: hi Hep cells efficiently express HNF4α and FOXA2 genes and proteins, exhibit typical epithelial morphology and acquire mature hepatocyte-like cell functions, including ALB secretion, urea production, ICG uptake and release, and glycogen storage. hi Hep cells can be activated by CYP inducers. The percentage of both ALB and α-1-antitrypsin(AAT)-positive cells was approximately 72.6%. The expression levels of hepatocyte-specific genes( ALB, AAT, and CYP1A1) and liver drug transport-related genes( ABCB1, ABCG2, and SLC22A18) in hi Hep cells were significantly higher than those in MSCs-Vector cells. The hi Hep cells did not form tumors after subcutaneous xenograft in BALB/c nude mice after 2 months. Conclusion: This study provides an accessible, feasible and efficient strategy to generate hi Hep cells from MSCs.     

Involvement of 90-kuD ribosomal S6 kinase in collagen type Ⅰ expression in rat hepatic fibrosis

AIM: To investigate the relationship between 90-kuD ribosomal S6 kinase (p90RSK) and collagen type Ⅰ expression during the development of hepatic fibrosis in vivo and in vitro. METHODS: Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type Ⅰ were determined by co-immunofluoresence and confocal microscopy. Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell (HSC) line. The expression of collagen type Ⅰ in HSC-T6 cells was assessed by Western blotting and real-time polymerase chain reaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, then collagen type Ⅰ promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated with or without platelet-derived growth factor (PDGF)-BB at a final concentration of 20 μg/L and the cell growth was determined by MTS conversion. RESULTS: In fibrotic liver tissues, p90RSK was overexpressed in activated HSCs and had a significant positive correlation with collagen type Ⅰ levels. In HSC-T6 cells transfected with RNAi targeted to p90RSK, the expression of collagen type Ⅰ was downregulated (61.8% in mRNA, P < 0.01, 89.1% in protein, P < 0.01). However, collagen type Ⅰ promoter activity was not increased with over-expression of p90RSK and not decreased with low expression either, compared with controls in the same cell line ( P = 0.076). Furthermore, p90RSK siRNA exerted the inhibition of HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation. CONCLUSION: p90RSK is over-expressed in activated HSCs and involved in regulating the abnormal expression of collagen type Ⅰ through initiating the proliferation of HSCs.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on the expression of PPARγ in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs. METHODS: The activated HSCs were divided into three groups: control group, 3 μmol/L rosiglitazone group, and 10 μmol/L rosiglitazone group. The expression of PPARγ, α-smooth muscle actin (α-SMA), and type Ⅰ and Ⅲ collagen was detected by RT-PCR, Western blot and immunocytochemiccal staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry. RESULTS: The expression of PPARy at mRNA and protein level markedly increased in HSCs of 10 umol/L rosiglitazone group (t value was 10.870 and 4.627 respectively, P<0.01 in both). The proliferation of HSCs in 10 μmol/L rosiglitazone group decreased significantly (t=5.542, P<0.01), α-SMA expression level and type I collagen synthesis ability were also reduced vs controls (t value = 10.256 and 14.627 respectively, P<0,01 in both). The apoptotic rate of HSCs significantly increased in 10 μmol/L rosiglitazone group vs control (X2=16.682, P<0,01). CONCLUSION: By increasing expression of PPARγ in activated HSCs, rosiglitazone, an agonist of PPARγ, decreases α-SMA expression and type I collagen synthesis, inhibits cell proliferation, and induces cell apoptosis.     

TGF-β1 promotes differentiation of hiPSC into functional smooth-muscle-like cells

Background Cell source is one of the most important constructions for tissue engineered blood vessels(TEBV). As human adult vascular cells are limited by the replicative life spans and poor collagen secretion, stem cell has become a promising cell source. Hence, we investigated the differentiation of human induced pluripotent stem cells(hiPSC) into functional smooth-muscle-like cells(SMLCs) by embryoid bodies method and explored whether transforming growth factor-β1(TGF-β1) can promote the differentiation. Methods HiPSCs were cultured in smooth muscle cell medium with or without TGF-β1 after forming embryoid bodies. The cell morphology, cell characteristics and contractility were compared after 7 days of differentiation. Real-time PCR and Western blot were used to assess the mRNA and protein expression levels of α-SMA, Calponin, SM22α, Collagen I and Collagen III. Functional contraction study was performed using carbachol. Results HiPSC could successfully differentiate into cells that were similar to typical smooth muscle cells in morphology. The expression of α-SMA, Calponin and SM22α up-regulated after induction. TGF-β1 could further up-regulated α-SMA expression.Immunofluorescence images showed that more than 80% of the hiPSC-derived SMLCs by TGF-β1 stained with smooth muscle cell markers α-SMA, SMMHC, SM22α and Calponin. Analyses of expression in collagen showed that hiPSC-derived SMLCs exhibited higher levels of Collagen I and Collagen III after induction by TGF-β1. Conclusion The hiPSC could successfully differentiate into smooth-muscle-like cells using embryoid bodies method. TGF-β1 can promote the differentiation and enhance collagen synthesis[.S Chin J Cardiol 2019;20(1):44-53]     

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like...     

Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

AIM: To investigate the potential mechanism of Arg-Gly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl₄-induced hepatic fibrosis in rats.METHODS: We constructed a rat model of CCl₄-induced hepatic fibrosis and treated the rats with different formulations of OM. To evaluate the antifibrotic effect of OM, we detected levels of alkaline phosphatase, hepatic histopathology (hematoxylin and eosin stain and Masson staining) and fibrosis-related gene expression of matrix metallopeptidase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-1 as well as type I procollagen via quantitative real-time polymerase chain reaction. To detect cell viability and apoptosis of hepatic stellate cells (HSCs), we performed 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and flow cytometry. To reinforce the combination of oxymatrine with HSCs, we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM, and its targeting of HSCs was examined by fluorescent microscopy.RESULTS: OM attenuated CCl₄-induced hepatic fibrosis, as defined by reducing serum alkaline phosphatase (344.47 ± 27.52 U/L vs 550.69 ± 43.78 U/L, P < 0.05), attenuating liver injury and improving collagen deposits (2.36% ± 0.09% vs 7.70% ± 0.60%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). OM inhibited cell viability and induced apoptosis of HSCs in vitro. RGD promoted OM targeting of HSCs and enhanced the therapeutic effect of OM in terms of serum alkaline phosphatase (272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L, P < 0.05), liver injury, collagen deposits (0.26% ± 0.09% vs 2.36% ± 0.09%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). Moreover, in vitro assay demonstrated that RGD enhanced the effect of OM on HSC viability and apoptosis.CONCLUSION: OM attenuated hepatic fibrosis by inhibiting viability and inducing apoptosis of HSCs. The RGD-labeled formulation enhanced the targeting efficiency for HSCs and the therapeutic effect.     

Notch3 regulates the activation of hepatic stellate cells

AIM: To investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells (HSCs).METHODS: Immunohistochemistry was used to detect the expression of Notch3 in fibrotic liver tissues of patients with chronic active hepatitis. The expression of Notch3 in HSC-T6 cells treated or not with transforming growth factor (TGF)-β1 was analyzed by immunofluorescence staining. The expression of Notch3 and myofibroblastic marker α-smooth muscle actin (α-SMA) and collagen I in HSC-T6 cells transfected with pcDNA3.1-N3ICD or control vector were detected by Western blotting and immunofluorescence staining. Moreover, effects of Notch3 knockdown in HSC-T6 by Notch3 siRNA were investigated by Western blotting and immunofluorescence staining.RESULTS: The expression of Notch3 was significantly up-regulated in fibrotic liver tissues of patients with chronic active hepatitis, but not detected in normal liver tissues. Active Notch signaling was found in HSC-T6 cells. TGF-β1 treatment led to up-regulation of Notch3 expression in HSC-T6 cells, and over-expression of Notch3 increased the expression of α-SMA and collagen I in HSC-T6 without TGF-β1 treatment. Interestingly, transient knockdown of Notch3 decreased the expression of myofibroblastic marker and antagonized TGF-β1-induced expression of α-SMA and collagen I in HSC-T6.CONCLUSION: Notch3 may regulate the activation of HSCs, and the selective interruption of Notch3 may provide an anti-fibrotic strategy in hepatic fibrosis.     

Metabolic shift in liver: Correlation between perfusion temperature and hypoxia inducible factor-1α

AIM: To study at what temperature the oxygen carried by the perfusate meets liver requirements in a model of organ perfusion. METHODS: in this study, we correlated hypoxia induciblefactor(Hi F)-1α expression to the perfusion temperature and the hepatic oxygen uptake in a model of isolated perfused rat liver. Livers from Wistar rats were perfused for 6 h with an oxygenated medium at 10, 20, 30 and 37 ℃. Oxygen uptake was measured by an oxygen probe; lactate dehydrogenase activity, lactate release and glycogen were measured spectrophotometrically; bile flow was gravitationally determined; p H of the perfusate was also evaluated; Hi F-1α m RNA and protein expression were analyzed by real time-polymerase chain reaction and ELi SA, respectively. RESULTS: Livers perfused at 10 and 20 ℃ showed no difference in lactate dehydrogenase release after 6 h of perfusion(0.96 ± 0.23 vs 0.93 ± 0.09 m U/min per g) and had lower hepatic damage as compared to 30 and 37 ℃(5.63 ± 0.76 vs 527.69 ± 45.27 m U/min per g, respectively, P s 0.01). After 6 h, tissue ATP was significantly higher in livers perfused at 10 and 20 ℃than in livers perfused at 30 and 37 ℃(0.89 ± 0.06 and 1.16 ± 0.05 vs 0.57 ± 0.09 and 0.33 ± 0.08 nmol/mg, respectively, P s 0.01). No sign of hypoxia was observed at 10 and 20 ℃, as highlighted by low lactate release respect to livers perfused at 30 and 37 ℃(121.4 ± 12.6 and 146.3 ± 7.3 vs 281.8 ± 45.3 and 1094.5 ± 71.7 nmol/m L, respectively, P s 0.02), and low relative Hi F-1α m RNA(0.40 ± 0.08 and 0.20 ± 0.03 vs 0.60 ± 0.20 and 1.47 ± 0.30, respectively, P s 0.05) and protein(3.72 ± 0.16 and 3.65 ± 0.06 vs 4.43 ± 0.41 and 6.44 ± 0.82, respectively, P s 0.05) expression.CONCLUSION: Livers perfused at 10 and 20 ℃ show no sign of liver injury or anaerobiosis, in contrast to livers perfused at 30 and 37 ℃.     

Antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline on bile duct ligation induced liver fibrosis in rats

AIM:To investigate the preventive effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on bile duct ligation (BDL)induced liver fibrosis in rats. METHODS:Liver fibrosis in rats was induced by BDL and AcSDKP was infused subcutaneously for 2 wkvia a osmotic minipump (Alzet 2ML4) immediately after BDL operation. After scarifying, serum and liver specimens were collected. Hematoxylin and eosin staining, Sirius red staining, enzyme linked immunosorbent assay, Western blot or real-time polymerase chain reaction were used to determinate liver functions, histological alterations, collagen deposition, mRNA expression of markers for fibroblasts, transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). RESULTS:When compared to model rats, chronic exogenous AcSDKP infusion suppressed profibrogenicTGF-β1 signaling, α-smooth muscle actin positivity (α-SMA), fibroblast specific protein-1 (FSP-1) staining and collagen gene expression. Col Ⅰ, Col Ⅲ, matrix metalloproteinase-2, tissue inhibitors of metallopro-teinase-1 and tissue inhibitors of metalloproteinase-2 mRNA expressions were all significantly downregulated by AcSDKP infusion (2.02 ± 1.10vs 14.16 ± 6.50, 2.02 ± 0.45vs 10.00 ± 3.35, 2.91 ± 0.30vs 7.83 ± 1.10, 4.64 ± 1.25 vs 18.52 ± 7.61, 0.46 ± 0.16 vs 0.34 ± 0.12, respectively, P 0.05). Chronic exogenous AcSDKP infusion attenuated BDL-induced liver injury, inflammation and fibrosis. BDL caused a remarkable increase in alanine transaminase, aspartate transaminase, total bilirubin, and prothrombin time, all of which were reduced by AcSDKP infusion. Mast cells, collagen accumulation, α-SMA, TGF-β1, FSP-1 and BMP-7 increased. The histological appearance of liver specimens was also improved. CONCLUSION:Infusion of exogenous AcSDKP attenu-ated BDL-induced fibrosis in the rat liver. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.     

Liver cold preservation induce lung surfactant changes and acute lung injury in rat liver transplantation

AIM:To investigate the relationship between donor liver cold preservation,lung surfactant (LS) changes and acute lung injury (ALI) after liver transplantation.METHODS:Liver transplantation models were estab-lished using male Wistar rats.Donor livers were pre-served in University of Wisconsin solution at 4 ℃ for different lengths of time.The effect of ammonium pyr-rolidinedithiocarbamate (PDTC) on ALI was also detect-ed.All samples were harvested after 3 h reperfusion.The severity of ALI was evaluated by lung weight/body weight ratio,lung histopathological score,serum nitric oxide (NO) and endothelin (ET)-1 levels,lung tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels.Lung surfactants (LSs) were determined by micellar electrokinetic capillary chromatography.RESULTS:With extended donor liver cold preservation time (CPT),lung histopathological scores,serum ET-1 levels,lung weight/body weight ratio and the level of TNF-α and IL-1β in lung were increased significantly in the 180-min group compared with the sham group (3.16 ± 0.28 vs 1.12 ± 0.21,P 0.001;343.59 ± 53.97 vs 141.53 ± 48.48,P 0.001;0.00687 ± 0.00037 vs 0.00557 ± 0.00056,P 0.001;17.5 ± 3.0 vs 1.3 ± 0.3,P 0.001;10.8 ± 2.3 vs 1.8 ± 0.4,P 0.001),but se-rum NO levels decreased remarkably (74.67 ± 10.01 vs 24.97 ± 3.18,P 0.001).The expression of lung phos-phatidylcholine (PC),phosphatidylethanolamine (PE),phosphatidylinositol (PI) and phosphatidylserine (PS) increased when CPT was 120 min,and decreased when CPT was 180 min (PC:1318.89 ± 54.79 vs 1011.18 ± 59.99,P 0.001;PE:1504.45 ± 119.96 vs 1340.80 ± 76.39,P=0.0019;PI:201.23 ± 34.82 vs 185.88 ± 17.04,P=0.2265;PS:300.43 ± 32.95 vs 286.55 ± 55.55,P=0.5054).All these ALI-associated indexes could be partially reversed by PDTC treatment.CONCLUSION:Prolonged CPT could induce or inhibit the expression of LSs at the compensation or decom-pensation stage,and some antioxidants (e.g.,PDTC) may reverse the pathological process partially.     

Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice

AIM: To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice.METHODS: We employed a recently developed novel insulin gene therapy strategy using a synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes. We intravenously administered a recombinant adenovirus expressing furin-cleavable rat insulin under the control of the synthetic promoter (rAd-SP-rINSfur) into diabetic Lepr^db/db mice. A recombinant adenovirus expressing β-galactosidase under the cytomegalovirus promoter was used as a control (rAd-CMV-βgal). Blood glucose levels and body weights were monitored for 50 d. Glucose and insulin tolerance tests were performed. Immunohistochemical staining was performed to investigate islet morphology and insulin content.RESULTS: Administration of rAd-SP-rINSfur lowered blood glucose levels and normoglycemia was maintained for 50 d, whereas the rAd-CMV-βgal control virus-injected mice remained hyperglycemic. Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice cleared exogenous glucose from the blood more efficiently than control virus-injected mice at 4 wk [area under the curve (AUC): 21  508.80 ± 2248.18 vs 62  640.00 ± 5014.28, P < 0.01] and at 6 wk (AUC: 29  956.60 ± 1757.33 vs 60  016.60 ± 3794.47, P < 0.01). In addition, insulin sensitivity was also significantly improved in mice treated with rAd-SP-rINSfur compared with rAd-CMV-βgal-treated mice (AUC: 9150.17 ± 1007.78 vs 11  994.20 ± 474.40, P < 0.05). The islets from rAd-SP-rINSfur-injected mice appeared to be smaller and to contain a higher concentration of insulin than those from rAd-CMV-βgal-injected mice.CONCLUSION: Based on these results, we suggest that insulin gene therapy might be one therapeutic option for remission of type 2 diabetes.