Exosome-transported IncRNA H19 regulates insulin-like growth factor-1 via the H19/let-7a/insulin-like growth factor-1 receptor axis in ischemic stroke

LncRNA(long non-coding RNA) H19 is a transcript of the H19 gene that is expressed during embryogenesis.We previously discove red a role for circular lncRNA H19 in the onset and prognosis of cerebral ischemic stroke.In this study,we used serum from patients with ischemic stroke,and mouse and cell culture models to elucidate the roles of plasma and neuronal exosomes in the regulatory effect of lncRNA H19 on insulin-like growth factor-1 and its mechanism in ischemic stroke,using western blotting,quantitative real-time polymerase chain reaction,and enzyme-linked immunosorbent assays.Plasma exosomal IncRNA H19 was negatively associated with blood levels of insulin-like growth factor-1 in samples from patients with cerebral ischemic stroke.In a mouse model,levels of exosomal IncRNA H19 were positively correlated with plasma and cerebral lncRNA H19.In a cell co-culture model,we confirmed that IncRNA H19 was transported from neuro ns to astrocytes by exosomes to induce downregulation of insulin-like growth factor-1 through the H19/let-7 a/insulin-like growth factor-1 receptor axis.This study provides the first evidence for the transpo rtation of IncRNA H19 by exosomes and the relationship between IncRNA H19 and insulinlike growth factor-1.

Correlation between expression of two transforming growth factor-beta 1 receptors and microvascular density in a rat model of cerebral ischemia and reperfusion injury

The effects of transforming growth factor-β1 （TGF-β1） are currently controversial. Whether TGF-β1 promotes or inhibits revascularization under different conditions remains poorly understood. Based on previous studies, the current experiment established rat models of cerebral ischemia and reperfusion injury （IRI）, and demonstrated that pathological and functional damage was also increased after IRI. The most serious damage was observed at 3 days after reperfusion, at which time microvascular density fell to its lowest level. Soon afterwards, microvascular density increased, new collateral circulation was gradually established at 4 to 7 days after reperfusion, and pathological damage and neurological deficits were improved. TGF-β1, activin receptor-like kinase 5 （ALK5） mRNA and protein expression levels increased gradually over time. In contrast, ALK1 mRNA and protein expression decreased over the same period. A significant negative correlation was detected between microvascular density and expression of the ALK5 gene transcript. There was no correlation between microvascular density and ALK1 gene transcriptional expression following cerebral IRI in a rat model. These findings suggest that ALK5, rather than ALK1, is the critical receptor in the TGF-β1 signal pathways after cerebral IRI.

Expression of transforming growth factor-β_1 and its typeⅠ receptor in different phases of post-burn hypertrophic scars

Objective: To analyze and compare the expression pattern of the transforming growth factor-β1(TGF-β1) and its type I receptor (TGF-β RI ) in nounal human skin and various phases of post-burn hypertrophic scars (HTS). Method: The immunohistochemical ABC method was employed. Results: In nounal human skin, no evident immunoreactivity of TGF-β1 and TGF-β R I was observed. In activation phase of post-burn HTS, TGF-β R I and TGF-β1 were highly expressed in most dermal fibroblasts which seemed to be the same subset. However, in remission phase, no staining was seen in der mal fibroblasts. Conclusion: The formation of all may involve the increase of TGF-β responsiveness in fibroblasts The ac cumulation at the wound site and failure of apoptosis of over-resposive fibroblasts may contribute to the formation of HTS.

Vascular endothelial growth factor A, secreted in response to transforming growth factor-β1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells

Hypoxia and transforming growth factor-β1 （TGF-β1） increase vascular endothelial growth factor A （VEGFA） expression in a number of malignancies. This effect of hypoxia and TGF-β1 might be responsible for tumor progression and metastasis of advanced prostate cancer. In the present study, TGF-β1 was shown to induce VEGFA165 secretion from both normal cell lines （HPV7 and RWPE1） and prostate cancer cell lines （DU 145 and PC3）. Conversely, hypoxia-stimulated VEGFA165 secretion was observed only in prostate cancer cell lines. Hypoxia induced TGF-β1 expression in PC3 prostate cancer cells, and the TGF-β1 type I receptor （ALK5） kinase inhibitor partially blocked hypoxia-mediated VEGFA16s secretion. This effect of hypoxia provides a novel mechanism to increase VEGFA expression in prostate cancer cells. Although autocrine signaling of VEGFA has been implicated in prostate cancer progression and metastasis, the associated mechanism is poorly characterized. VEGFA activity is mediated via VEGF receptor （VEGFR） 1 （Fit-l） and 2 （FIk-I/KDR）. Whereas VEGFR-1 mRNA was detected in normal prostate epithelial cells, VEGFR-2 mRNA and VEGFR protein were expressed only in PC3 cells. VEGFA165 treatment induced phosphorylation of extracellular signal-regulated kinase 1/2 （ERKI/2） in PC3 cells but not in HPV7 cells, suggesting that the autocrine function of VEGFA may be uniquely associated with prostate cancer. Activation of VEGFR-2 by VEGFA165 was shown to enhance migration of PC3 cells. A similar effect was also observed with endogenous VEGFA induced by TGF-β1 and hypoxia. These findings illustrate that an autocrine loop of VEGFA via VEGFR-2 is critical for the tumorigenic effects of TGF-β1 and hypoxia on metastatic prostate cancers.

Total flavone of Abelmoschus manihot suppresses epithelial-mesenchymal transition via interfering transforming growth factor-β1 signaling in Crohn's disease intestinal fibrosis

AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1,Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates liver fibrosis by suppressing the transforming growth factor-β1 signal pathway

Background: Histone deacetylases(HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid(SAHA) by suppressing transforming growth factor-β1(TGF-β1) signaling. Methods: Male Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride(CCl 4) and LX2 cell(human hepatic stellate cell line) was stimulated by TGF-β1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor(CTGF) mRNA levels were detected by real-time polymerase chain reaction(PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3(H3), Smad7, Smad2/3, Acetyl-Histone H3(AH3), HDAC2, α-smooth muscle actin( α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-β1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin(HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. Results: Compared with control group, the TGF-β1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats( P < 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats( P < 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-β1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced( P < 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats( P < 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver( P < 0.01). Conclusion: The HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-β1 signaling.

Dab2 attenuates brain injury in APP/PS1 mice via targeting transforming growth factor-beta/SMAD signaling

Transforming growth factor-beta （TGF-β） type II receptor （TβRⅡ） levels are extremely low in the brain tissue of patients with Alzheimer＇s disease. This receptor inhibits TGF-β1/SMAD signaling and thereby aggravates amyolid-beta deposition and neuronal injury. Dab2, a specific adapter protein, protects T RII from degradation and ensures the effective conduction of TGF-β 1/SMAD signaling. In this study, we used an adenoviral vector to overexpress the Dab2 gene in the mouse hippocampus and investigated the regulatory effect of Dab2 protein on TGF-β1/SMAD signaling in a mouse model of Alzheimer＇s disease, and the potential neuroprotective effect. The results showed that the TβRⅡ level was lower.in APP/PS1 mouse hippocampus than in normal mouse hippocampus. After Dab2 expression, hippocampal TβRⅡ and p-SMAD2/3 levels were signifi- cantly increased, while amyloid-beta deposition, microglia activation, tumor necrosis factor- and interleulin-6 levels and neuronal loss were significantly attenuated in APP/PS1 mouse brain tissue. These results suggest that Dab2 can exhibit neuroprotective effects in Alzheimer＇s disease by regulating TGF-β1/SMAD signaling.

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis

AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied,with all subjects having liver biopsy data and DNA available for testing.This study assessed the association of eight single nucleotide polymorphisms(SNPs)in a total of six genes including toll-like receptor 4(TLR4),transforming growth factor-beta(TGF-β),oxoguanine DNA glycosylase,monocyte chemoattractant protein 1,chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity.Genotyping was performed using high resolution melt analysis and sequencing.The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration.RESULTS:There were significant associations between the cofactors of male gender(P=0.0001),increasing age(P=0.006),alcohol consumption(P=0.0001),steatosis(P=0.03),hepatic iron concentration(P<0.0001)and the presence of hepatic fibrosis.Of the candidate gene polymorphisms studied,none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors.We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied.Importantly,in this large,well characterised cohort of patients there was no association between SNPs for TGF-βor TLR4and the presence of fibrosis,cirrhosis or increasing fibrosis stage in multivariate analysis.CONCLUSION:In our large,well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.

Hsa_circRNA_102610 upregulation in Crohn’s disease promotes transforming growth factor-β1-induced epithelial-mesenchymal transition via sponging of hsa-miR-130a-3p

BACKGROUND The incidence of inflammatory bowel disease,a chronic intestinal inflammatory disorder that includes Crohn’s disease(CD)and ulcerative colitis,is rising.Circular RNAs are considered valuable diagnostic biomarkers for CD.Current evidence supports the views that epithelial-mesenchymal transition(EMT)plays an important role in CD pathogenesis,and that hsa-miR-130a-3p can inhibit transforming growth factor-β1(TGF-β1)-induced EMT.Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients.Moreover,we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p.Thus,we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD.AIM To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD.METHODS The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction.The proliferation of human intestinal epithelial cells(HIECs)and normal-derived colon mucosa cell line 460(NCM460)cells was detected by cell counting kit-8,5-ethynyl-2’-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610.Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics.The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays.The relative expression levels of CyclinD1,mothers against decapentaplegic homolog 4(SMAD4),E-cadherin,N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression,TGF-β1-induced EMT or hsa-miR-130a-3p mimic transfection(in rescue experiments).RESULTS Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD(r=0.359,P=0.007)by Pearson correlation analysis.Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells,while hsa-miR-130a-3p reversed the cell proliferationpromoting effects of hsa_circRNA_102610.Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells.An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed(r=-0.290,P=0.024)by Pearson correlation analysis.Moreover,overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting.Furthermore,overexpression of hsa_circRNA_102610 promoted TGF-β1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p,with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin.CONCLUSION Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.

Alendronate disturbs femoral growth due to changes during immunolocalization of transforming growth factor-β1 and bone morphogenetic protein-2 in epiphyseal plate

BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone.Despite the abovementioned atractive reasons for alendronate’s use,few data on the effect of alendronate during epiphyseal growth exist.AIM Verify the effect of alendronate on the growth epiphyseal plate,and compare its effect with the size of the femur during the double-staining of the immunolocalization of transforming growth factor-β1(TGF-β1)and bone morphogenetic protein-2(BMP2)in endochondral ossifing in specimens that have received alendronate.METHODS Forty newborn rats were randomly divided into two groups:a control group(were given applications of 1 mg/kg physiologic saline)and a group that received Alendronate(a dose of 2.5 mg/kg).These groups were then divided into two subgroups for euthanasia in two and 12 d of life.After euthanasia,the femurs were removed,and the femoral bones were measured linearly between the apex of the greater trochanter until the lower intercondylar midlle face to verify the probable bone growth between 3 and 12 d in control and alednroanto treated rats.Posteriorly,the surgical pieces were also sent to the histopathology laboratory to produce histological slides.The obtained slides were stained with hematoxylin and eosin to measure each of the cartilage zones in endochondral development.and other slides were immunohistochemically tested for anti-TGF-β1 and BMP-2 antibodies to investigate the immunolocalization of these proteins in the epiphyseal plaque area.RESULTS On the third day,some diferences between the control group and specimens treated with alendronate were verified.Macroscopiccaly,we found similarities in size between the femoral bones when we compared the control group with the specimens that received alendronate.On the 12^th day,the bone size of the mice receiving the drug was significantly smaller than those of the control group.These results coincide with changes in the TGF-β1 and BMP-2 expression.In the specimens that received alendronate,the TGF-β1 was expressed in some sites of trabecular bone that was neoformed,peripherally to the bone marrow area.The BMP-2 was also positive in proliferative chondrocytes and hypertrofic chondrocytes.On the 12^th day,all layers of chondrocytes exhibited positivity for BMP-2 in the specimens that received alendronate.In the interface between the trabecular bone and cartilage,an area of disorganized bone deposition was evident.Neoformed bone also appeared to be different at 12 d.In the control group,BMP-2 was positive in an intense area of bone trabeculae,whereas the alendronate-treated group showed TGF-β1 positive trabeculae and a greater bone area.CONCLUSION Alendronate alters the immunolocalization of TGF-β1 and BMP-2 simultaneously,a condition that changes the usual histological aspects of the cartilage zone and impairs epiphysis growth and femur growth.

Effects of Heparin on Transforming Growth Factor-β_1 and Extracellular Matrix Components in the Glomeruli of Diabetic Rats

The effects of heparin on the expression of transforming growth factor-β 1 (TGF-β 1) and two extracellular matrix components laminin (LN) and fibronectin (FN) in diabetic rat glomeruli were investigated. Twenty-six rats were randomly divided into control group (C, n=8), diabetic group (D, n=9), and diabetes+heparin group (DH, n=9). After 8-week therapy of heparin (200 U once daily by abdominal injection), TGF-β 1, LN and FN expression in glomeruli was detected by immunohistochemical method. The results showed that the expression levels of TGF-β 1, LN and FN were higher in group D than in group C. It was found that heparin could reduce 24-h urinary albumin excretion and inhibit overexpression of TGF-β 1, LN and FN in glomeruli of diabetic rats. It suggested that the inhibitory effect of heparin on diabetic glomerular sclerosis was at least partly related with the inhibition of TGF-β 1 expression.

Expression of Mesenger RNA for Transforming Growth Factor-β_1 in Bovine Trabecular Meshwork

Purpose: To investigate the relationship between transforming growth factor-β1(TGF-β1) and primary open-angle glaucoma, we have determined whether trabec-ular tissues have the expression of messenger RNA for TGF-β1.Methods: Total RNA of 24 newborn bovine trabecular tissue were extracted byGuanidine isothiocyanate method. The TGF-β33 plasmid was brought into E. col-ibacillius HB101 and amplificated. After Bam HI endolase degradation and labelwith a-32p-dATP the RNA was hybridized with the cDNA (complementary DNA)probe and examined by autoradiography.Results: The presence of mRNA for TGF-β1 in bovine trabecular meshwork wasconfirmed.Conclusions: The TGF-β1 present in normal aqueous humor must be at least partlyderived from the trabecular meshwork. It offered a basis for understanding therelationship between abnormal synthesis, activation and clearance of TGF-β1 andthe pathogenesis of primary open-angle glaucoma (POAG) in molecular biology.Eye Science 1996; 12:1-4.

Effects of Lingguizhugan Decoction on α-SMA and collagen synthesis in rat myocardial fibroblasts induced by transforming growth factor-β_(1)

Objective:To explore the protective effect of Linggui Zhugan Decoction(LGZGD)medicated serum on myocardial fibrosis induced by transforming growth factor-β1(TGF-β1).Methods:Using enzyme digestion method,combined with differential adherence to isolate and culture Sprague-Dawley(SD)suckling mouse Cardiac fibroblasys(CFB)in vitro.Divided into:blank group,blank rat serum group,model group,and LGZGD medicated serum group(5%、10%、20%).Except for blank group and blank rat serum group,they were stimulated with 5 ng/ml TGF-β1 for 12 hours,and then then intervene with LGZGD medicated serum(5%、10%、20%)and continue to culture for 24 hours.Use immunofluorescence and Western blot(WB)to detect the expression ofα-smooth muscle actin(α-SMA),Enzyme-linked immunosorbent assay(ELISA)and WB to detect type Ⅰ collagen(Collagen Ⅰ),type Ⅰ collagen(Collagen Ⅲ)and fibronectin(FN)expression.Results:Compared with the blank group,the expressions of Collagen Ⅰ,Collagen Ⅲ,α-SMA and FN in the model group were significantly increased(P<0.01);Compared with the model group,the expressions of Collagen Ⅰ and Collagen Ⅲ in each concentration group of the experiment were significantly reduced(P<0.01);the expression ofα-SMA and FN were significantly reduced(P<0.01).Conclusions:LGZGD has an inhibitory effect on collagen synthesis and the expression ofα-SMA and FN,indicating that the anti-fibrosis effect of LGZGD is related to it.

Transforming growth factor beta-1 upregulates glucose transporter 1 and glycolysis through canonical and noncanonical pathways in hepatic stellate cells

BACKGROUND Hepatic stellate cells(HSCs)are the key effector cells mediating the occurrence and development of liver fibrosis,while aerobic glycolysis is an important metabolic characteristic of HSC activation.Transforming growth factor-β1(TGF-β1)induces aerobic glycolysis and is a driving factor for metabolic reprogramming.The occurrence of glycolysis depends on a high glucose uptake level.Glucose transporter 1(GLUT1)is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism,thus affecting cell proliferation and growth.However,little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs.AIM To investigate the mechanisms of action of GLUT1,TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis.METHODS Immunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue.A Seahorse extracellular flux(XF)analyzer was used to examine changes in aerobic glycolytic flux,lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation.The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothersagainst-decapentaplegic-homolog 2/3(Smad2/3)signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase(PI3K)/AKT signaling pathways.In addition,GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs.Finally,a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis.RESULTS GLUT1 protein expression was increased in both mouse and human fibrotic liver tissues.In addition,immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins,indicating that GLUT1 expression was related to the development of liver fibrosis.TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad,p38 MAPK and P13K/AKT signaling pathways.The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression.GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration.A GLUT1 inhibitor was administered in a mouse model of liver fibrosis,and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis.CONCLUSION TGF-β1 induces GLUT1 expression in HSCs,a process related to liver fibrosis progression.In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs.In addition,in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.

Insulin-like growth factor-1 mRNA isoforms and insulinlike growth factor-1 receptor mRNA expression in chronic hepatitis C

AIM: to evaluate the expression of different insulinlike growth factor(IGF)-1 mRNA isoforms and IGF-1 receptor(IGF-1R) mRNA in hepatitis C virus(HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from chronic hepatitis C(CH-C) patients were obtained before anti-viral therapy. Inflammatory activity(grading) and advancement of fibrosis(staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instruction. Expression levels of IGF-1 mRNA isoforms(IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS: The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms(P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2(r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA(r = 0.891; r = 0.821, respectively), with IGF-1B mRNA(r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA(r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA(r = 0.956; r = 0.869, respectively), and B with C isoforms(r = 0.868)(P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading(G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data(e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION: Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.

Targeting Transforming Growth Factor-<i>β</i>(TGF-<i>β</i>) in Cancer and Non-Neoplastic Diseases

Transforming growth factor-β?(TGF-β) superfamily is a key player in the regulation of a wide variety of physiological processes from development to pathogenesis. Since the discovery of the prototypic member, TGF-β, almost three decades ago, there have been tremendous advances in our understanding of its complex biology. TGF-β?misregulation has been implicated in the pathogenesis of a variety of diseases, including cancer with a direct role in facilitating metastasis, fibrosis and inflammation. Consequently, TGF-β?is currently explored as a prognostic candidate biomarker of tumor invasiveness and metastasis;and it offers an attractive target for cancer therapy. Several anti-TGF-β?approaches, such as TGF-β?antibodies, antisense oligonucleotides and small molecules inhibitors of TGF-β?type 1 receptor kinase, have shown great promise in the preclinical studies. Here, we consider why the TGF-βsignaling pathway is a drug target, the potential clinical applications of TGF-β?inhibition, the issues arising with anti-TGF-β?therapy and how these might be adopted using personalized approaches with a special care for patient selection and timing of therapy so that we may bring forward all the potentials of targeting this pathway for therapeutic uses in both cancer, preferentially in combination therapy, and non-neoplastic diseases.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Effects of targeted-edited oncogenic insulin-like growth factor-1 receptor with specific-sgRNA on biological behaviors of Hep G2 cells

BACKGROUND Insulin-like growth factor-1 receptor(IGF-1R)is over-expressed in hepatocellular carcinoma(HCC).However,the relationship between IGF-1R activation and HCC progression remains unidentified.AIM To investigate the effects of editing IGF-1R on the biological features of HCC cells.METHODS Immunohistochemistry analyzed the expressions of IGF-1R and P-glyco protein(P-gp)in HCC tissues and their distal non-cancerous tissues(non-Ca).IGF-1R was edited with Crispr/Cas9 system,screened specific sg RNAs,and then transfected into Hep G2 cells.CCK-8,scratch wound test detected cell proliferation,migration,invasion and transwell assays,respectively.Alterations of IGF-1R and P-gp were confirmed by Western blotting.Alterations of anti-cancer drug IC_(50)values were analyzed at the cell level.RESULTS The positive rates of IGF-1R(93.6%,χ~2=63.947)or P-gp(88.2%,χ~2=58.448)were significantly higher(P<0.001)in the HCC group than those(36.6%in IGF-1R or 26.9%in P-gp)in the non-Ca group.They were positively correlated between high IGF-1R and P-gp expression,and they were associated with hepatitis B virus infection and vascular invasion of HCC.Abnormal expressions of circulating IGF-1R and P-gp were confirmed and associated with HCC progression.Biological feature alterations of HCC cells transfected with specific sg RNA showed IGF-1R expression down-regulation,cell proliferation inhibition,cell invasion or migration potential decreasing,and enhancing susceptibility of Hep G2 cells to anti-cancer drugs.CONCLUSION Edited oncogenic IGF-1R was useful to inhibit biological behaviors of Hep G2 cells.

Inhibition of Ubiquitin-specific Protease 4 Attenuates Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via Transforming Growth Factor Beta Receptor Type Ⅰ

Objective Ubiquitin-specific protease 4(USP4)facilitates the development of transforming growth factor-beta 1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in various cancer cells.Moreover,EMT of renal tubular epithelial cells(RTECs)is required for the progression of renal interstitial fibrosis.However,the role of USP4 in EMT of RTECs remains unknown.The present study aimed to explore the effect of USP4 on the EMT of RTECs as well as the involved mechanism.Methods In established unilateral ureteral obstruction(UUO)rats and NRK-52E cells,immunohistochemistry and Western blot assays were performed.Results USP4 expression was increased significantly with obstruction time.In NRK-52E cells stimulated by TGF-β1,USP4 expression was increased in a time-dependent manner.In addition,USP4 silencing with specific siRNA indicated that USP4 protein was suppressed effectively.Meanwhile,USP4 siRNA treatment restored E-cadherin and weakened alpha smooth muscle actin(α-SMA)expression,indicating that USP4 may promote EMT.After treatment with USP4 siRNA and TGF-β1 for 24 h,the expression of TGF-β1 receptor type I(TβRI)was decreased.Conclusion USP4 promotes the EMT of RTECs through upregulating TβRI,thereby facilitating renal interstitial fibrosis.These findings may provide a potential target of USP4 in the treatment of renal fibrosis.