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.

Mudskipper interleukin-34 modulates the functions of monocytes/macrophages via the colony-stimulating factor-1 receptor 1

Interleukin-34(IL-34)is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colonystimulating factor-1 receptor(CSF-1R).However,information on the function of IL-34 in fish remains limited.In the present study,we identified an IL-34 homolog from mudskippers(Boleophthalmus pectinirostris).In silico analysis showed that the mudskipper IL-34(BpIL-34)was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper(Epinephelus coioides)homolog.BpIL-34 transcripts were constitutively expressed in various tissues,with the highest level of expression found in the brain.Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues.The recombinant mature BpIL-34 peptide(rBpIL-34)was purified and used to produce anti-rBpIL-34 IgG.Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages(MOs/MФs)was N-glycosylated.In vitro,rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs,as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factorα(BpTNF-α)and BpIL-1βin these cells.Furthermore,the knockdown of mudskipper CSF-1R1(BpCSF-1R1),but not mudskipper BpCSF-1R2,significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФfunction.In conclusion,our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.

Stromal cell-derived factor-1α regulates chondrogenic differentiation via activation of the Wnt/β-catenin pathway in mesenchymal stem cells

BACKGROUND Mesenchymal stem cells(MSCs)have been applied to treat degenerative articular diseases,and stromal cell-derived factor-1α(SDF-1α)may enhance their therapeutic efficacy.However,the regulatory effects of SDF-1αon cartilage differentiation remain largely unknown.Identifying the specific regulatory effects of SDF-1αon MSCs will provide a useful target for the treatment of degenerative articular diseases.AIM To explore the role and mechanism of SDF-1αin cartilage differentiation of MSCs and primary chondrocytes.METHODS The expression level of C-X-C chemokine receptor 4(CXCR4)in MSCs was assessed by immunofluorescence.MSCs treated with SDF-1αwere stained for alkaline phosphatase(ALP)and with Alcian blue to observe differentiation.Western blot analysis was used to examine the expression of SRY-box transcription factor 9,aggrecan,collagen II,runt-related transcription factor 2,collagen X,and matrix metalloproteinase(MMP)13 in untreated MSCs,of aggrecan,collagen II,collagen X,and MMP13 in SDF-1α-treated primary chondrocytes,of glycogen synthase kinase 3β(GSK3β)p-GSK3βandβ-catenin expression in SDF-1α-treated MSCs,and of aggrecan,collagen X,and MMP13 in SDF-1α-treated MSCs in the presence or absence of ICG-001(SDF-1αinhibitor).RESULTS Immunofluorescence showed CXCR4 expression in the membranes of MSCs.ALP stain was intensified in MSCs treated with SDF-1αfor 14 d.The SDF-1αtreatment promoted expression of collagen X and MMP13 during cartilage differentiation,whereas it had no effect on the expression of collagen II or aggrecan nor on the formation of cartilage matrix in MSCs.Further,those SDF-1α-mediated effects on MSCs were validated in primary chondrocytes.SDF-1αpromoted the expression of p-GSK3βandβ-catenin in MSCs.And,finally,inhibition of this pathway by ICG-001(5μmol/L)neutralized the SDF-1α-mediated up-regulation of collagen X and MMP13 expression in MSCs.CONCLUSION SDF-1αmay promote hypertrophic cartilage differentiation in MSCs by activating the Wnt/β-catenin pathway.These findings provide further evidence for the use of MSCs and SDF-1αin the treatment of cartilage degeneration and osteoarthritis.

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.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats

AIM:To observe the effects of N-acetylserotonin(NAS)administration on retinal ischemia-reperfusion(RIR)injury in rats and explore the underlying mechanisms involving the high mobility group box 1(HMGB1)/receptor for advanced glycation end-products(RAGE)/nuclear factor-kappa B(NF-κB)signaling pathway.METHODS:A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye.Eighty male Sprague Dawley were randomly divided into five groups:sham group(n=8),RIR group(n=28),RIR+NAS group(n=28),RIR+FPS-ZM1 group(n=8)and RIR+NAS+FPS-ZM1 group(n=8).The therapeutic effects of NAS were examined by hematoxylin-eosin(H&E)staining,and retinal ganglion cells(RGCs)counting.The expression of interleukin 1 beta(IL-1β),HMGB1,RAGE,and nod-like receptor 3(NLRP3)proteins and the phosphorylation of nuclear factorkappa B(p-NF-κB)were analyzed by immunohistochemistry staining and Western blot analysis.The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats.With NAS therapy,the HMGB1 and RAGE expression decreased significantly,and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression.Additionally,NAS exhibited an anti-inflammatory effect by reducing IL-1βexpression.The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression,so as to the IL-1βexpression and retinal edema,accompanied by an increase of RGCs in RIR rats.CONCLUSION:NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway,which may be a useful therapeutic target for retinal disease.

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.

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.

Insulin-like growth factor-1 induces lymphangiogenesis and facilitates lymphatic metastasis in colorectal cancer

AIM:To investigate the expression of insulin-like growth factor-1(IGF-1)/insulin-like growth factor-1 receptor(IGF-1R)in colorectal cancer(CRC)tissues and to analyze their correlation with lymphangiogenesis and lymphatic metastasis.METHODS:Immunohistochemistry was used to evaluate IGF-1 and IGF-1R expression and lymphatic vessel density(LVD)in 40 CRC specimens.The correlation between IGF-1/IGF-1R and LVD was investigated.Effects of IGF-1 on migration and invasion of CRC cells were examined using transwell chamber assays.A LoVo cell xenograft model was established to further detect the role of IGF-1 in CRC lymphangiogenesis in vivo. RESULTS:Elevated IGF-1 and IGF-1R expression in CRC tissues was correlated with lymph node metastasis(r=0.715 and 0.569,respectively,P<0.05)and tumor TNM stage(r=0.731 and 0.609,P<0.05).A higher LVD was also found in CRC tissues and was correlated with lymphatic metastasis(r=0.405,P<0.05).A positive correlation was found between LVD and IGF-1R expression(r=0.437,P<0.05).Transwell assays revealed that IGF-1 increased the migration and invasion of CRC cells.In vivo mouse studies showed that IGF-1 also increased LVD in LoVo cell xenografts.CONCLUSION:IGF-1/IGF-1R signaling induces tumorassociated lymphangiogenesis and contributes to lymphatic metastasis of CRC.

Insulin-like growth factor receptor-1 overexpression is associated with poor response of rectal cancers to radiotherapy

AIM:To explore the potential correlation between insulin-like growth factor receptor-1(IGF-1R)expression and rectal cancer radiosensitivity.METHODS:Eighty-seven rectal cancer patients(c TNMⅠ-Ⅲ)treated in our department between January 2011and December 2012 were enrolled.All subjects were treated with preoperative radiotherapy and radical resection of rectal carcinoma.Immunohistochemistry and reverse transcription polymerase chain reaction(RT-PCR)were performed to detect IGF-1R expression in pre-treatment and postoperative colorectal cancer specimens.Radiosensitivity for rectal cancer specimens was evaluated by observing rectal carcinoma mass regression combined with fibrosis on HE staining,degree of necrosis and quantity of remaining tumor cells.The relative IGF-1R expression was evaluated for association with tumor radiosensitivity.RESULTS:Immunohistochemistry showed diffuse IGF-1R staining on rectal cancer cells with various degrees of signal density.IGF-1R expression was significantly correlated with c TNM staging(P=0.012)while no significant association was observed with age,sex,tumor size and degree of differentiation(P=0.424,0.969,0.604,0.642).According to the Rectal Cancer Regression Grades(RCRG),there were 31 cases of RCRG1(radiation sensitive),28 cases of RCRG2 and 28 cases of RCRG3(radiation resistance)in 87 rectal cancer subjects.IGF-1R protein hyper-expression was significantly correlated with a poor response to radiotherapy(P<0.001,r=0.401).RT-PCR results from pre-radiation biopsy specimens also showed that IGF-1R m RNA negative group exhibited a higher radiation sensitivity(P<0.001,r=0.497).Compared with the pre-radiation biopsy specimens,the paired post-operative specimens showed a significantly increased IGF-1R protein and m RNA expression in the residual cancer cells(P<0.001,respectively).CONCLUSION:IGF-1R expression level may serve as a predictive biomarker for radiosensitivity of rectal cancer before preoperative radiotherapy.

Effects of Insulin-like Growth Factor 1 Receptor and Its Inhibitor AG1024 on the Progress of Lung Cancer

Summary： The type 1 insulin-like growth factor receptor （IGF-1R） and its downstream signaling com- ponents have been increasingly recognized to drive the development of malignancies, including non-small cell lung cancer （NSCLC）. This study aimed to investigate the effects of IGF-1R and its in- hibitor, AG1024, on the progression of lung cancer. Tissue microarray and immunohistochemistry were employed to detect the expressions of IGF-1 and IGF-1R in NSCLC tissues （n=198）. Western blotting was used to determine the expressions oflGF-1 and phosphorylated IGF-1R （p-IGF-1R） in A549 human lung carcinoma cells, and MTT assay to measure cell proliferation. Additionally, the expressions of IGF-1, p-IGF-1R and IGF-1R in a mouse model of lung cancer were detected by Western blotting and real-time fluorescence quantitative polymerase chain reaction （FQ-PCR）, respectively. The results showed that IGF-1 and IGF-1R were overexpressed in NSCLC tissues. The expression levels of IGF-1 and p-IGF-1R were significantly increased in A549 cells treated with IGF-1 as compared to those treated with IGF-1 ＋AG 1024 or untreated cells. In the presence of IGF-1, the proliferation of A549 cells was significantly increased. The progression of lung cancer in mice treated with IGF-1 was significantly increased as compared to the group treated with IGF-l＋AG1024 or the control group, with the same trend mirrored in IGF-1/p-IGF-1R/IGF-1R at the protein and/or mRNA levels. It was concluded that IGF- 1 and IGF inhibitor AG 1024 promotes lung cancer progression.

Expression of c-erbB-2 oncogene protein, epidermal growth factor receptor, and TGF-β1 in human pancreatic ductal adenocarcinoma

Objective: To detect the relations of c-erbB-2 onco-gene protein, epidermal growth factor receptor (EG-FR) and transforming growth factor-β1 (TGF-β1)to the progression or metastasis of pancreatic carci-noma.Methods: Using streptavidinbiotin complex (SABC)method, c-erbB-2 oncongene protein, we examinedimmunohistochemically EGFR and TGF-β1 expres-sions in wax-tissue sections from 10 individuals withnormal pancreas (NP), 13 patients with chronic pan-creatitis (CP) and 36 patients with pancreatic ductaladenocarcinoma (PC).Results: The positive expression rates of c-cerbB-2oncogene protein, EGFR and TGF-β1 in the NP, CPand PC groups were 0, 0, 10%; 7.7%, 7.7%,7.7%; and 41.7%, 50.0%, 44.4%, respectively.The positive expression rates of the three specific pro-teins increased more significantly in the PC groupthan in the NP and CP groups (P【0.05). The indi-vidual expression of c-erbB-2, EGFR and TGF-β1was not related to the age and sex of the patients aswell as the site, size and histopathological grade oftumors (P】0.05), but to the clinical stage of tumors(P【0.01). The coexpression rate of the three pro-teins was 27.8 % (10/36). This coexpression in thePC group was correlated with the histopathologicalgrades and clinical stages of tumors (P【0.01).Conclusion: Detection of c-erbB-2 oncogene protein,EGFR, and TGF-β1 expressions in pancreatic tissueis helpful to judge the malignancy, progression, andmetastasis of PC.

Clinicopathological and Prognostic Significance of Hypoxia-inducible Factor-1 alpha in Lung Cancer: a Systematic Review with Meta-analysis

Hypoxia-inducible factor-1 alpha（HIF-1α） plays a vital role in the initiation, evaluation and prognosis in lung cancer. The prognostic value of HIF-1α reported in diverse study remains disputable. Accordingly, a meta-analysis was implemented to further understand the prognostic role of HIF-1α in lung cancer. The relationship between HIF-1α and the clinicopathological characteristics and prognosis of lung cancer were investigated by a meta-analysis. Pub Med and Embase were searched from their inception to January 2015 for observational studies. Fixed-effects or random-effects meta-analyses were used to calculate odds ratios and 95% confidence intervals of different comparisons. A total of 20 studies met the criteria. The results showed that HIF-1α expression in lung cancer tissues was significantly higher than that in normal lung tissues. Expression of HIF-1α in patients with squamous cell carcinoma was significantly higher than that of patients with adenocarcinomas. Similarly, non-small cell lung cancer（NSCLC） patients had higher HIF-1α expression than small cell lung cancer（SCLC） patients. Moreover, lymph node metastasized tissues had higher HIF-1α expression than non-lymph node metastasized tissues. A high level HIF-1α expression was well correlated with the expression of vascular endothelial growth factor and epidermal growth factor receptor in the NSCLC. Notably, NSCLC or SCLC patients with positive HIF-1α expression in tumor tissues had lower overall survival rate than patients with negative HIF-1α expression. It was suggested that HIF-1α expression may be a prognostic biomarker and a potential therapeutic target for lung cancer.

GENE EXPRESSION OF TRANSFORMING GROWTH FACTOR β_1 TYPEII RECEPTOR IN HCC AND ITS CLINICAL SIGNIFICANCE

Objective: Transforming Growth Factor-β1 (TGF-β1)plays a central role in the process of . growth suppressionof the hepatocytes, and its type II receptor (TGF-β1R II)transfers the signal of growth suppression. In this study,the gene expression of TGF-β1R II in HCC and itsclinical significance was investigated. Methods: Theexpression of TGF-β1R II mRNA in 30 cases Of HCCtissue and the surrounding liver tissue was separatelydetected using reverse transcription-PCR. Results:The positive expression rate of TGF-β1R II mRNA wassignificantly lower in HCC tissue (11/30) than that in thesurrounding liver tissue (23/30) (P<0.01). Further, theless the cancer tissue expressed TGF-β1R II mRNA, themore poorly the tumoral hepatocyte differentiated(P<0.01) and the more portal vein cancer embolusexisted (p=0.0465). Conclusion: The decreaseexpression of TGF-β1 R II mRNA by tumoral hepatocyteresults in the defect of its negative growth regulation,and this may be one of the most important reasons forits carcinogenesis and uncontrolled growth.

Allograft Inflammatory Factor-1 in Cardiac Ischemia Re-perfusion Injury: Release of Molecular Markers in an <i>in Vitro</i>Setting

Initial ischemia/reperfusion injury (IRI) may have an impact on recipient immune responses after transplantation. Allograft inflammatory factor-1 (AIF-1) has been implicated in the regulation of inflammation associated with organ rejection. We hypothesized that it is either passively released from injured tissues during organ procurement, or actively secreted by allograft infiltrating cells contributing to allograft dysfunction. We investigated the impact of IRI in an in vitro study of human heart tissue during the process of transplantation. The mRNA expression levels for both isoforms of the AIF-1, I2 and I3 were significantly increased after 30 minutes reperfusion (AIF-1 I2: p 0.01 vs. AIF-1 I3: p 0.005). Expression levels for IL-18 and the TLRs were increased after 30 minutes of reperfusion. Only IL-18 and TLR-2 were statistically significant (IL-18: p 0.0001 vs. TLR-2: p 0.01). The mRNA expression levels for AIF-1 I2 and IL-18 were decreased from the original levels of ischemia after 60 and 90 minutes reperfusion. The TLR-2 and -4 were presented with minimal levels of reduction after 60 minutes. However, mRNA expression levels for all were decreased to the original levels of ischemia after 90 minutes, except for AIF-1 I3, but the difference was not statistically significant. AIF-1 and IL-18 were specifically detected in myocytes and interstitial tissues by immunohistochemistry (IHC) stain after IRI. TLR-4 was non-specific, and TLR2 was minimally expressed. The study discusses the evidence supporting that the AIF-1 may have therapeutic potential for strategies in the control of innate immune responses early on, after transplantation.

Anisodine hydrobromide alleviates oxidative stress caused by hypoxia/reoxygenation in human cerebral microvascular endothelial cells predominantly via inhibition of muscarinic acetylcholine receptor 4

Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.Endothelial dysfunction can be caused by hypoxia/reoxygenation(H/R)via oxidative stress and metabolic alterations.The present study investigated whether AT3 regulates the production of nitric oxide(NO)and reactive oxygen species(ROS),and the HIF-1αpathway via regulation of muscarinic acetylcholine receptors(mAChRs)in brain microvascular endothelial cells after H/R exposure.Methods:Under H/R conditions,hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3.Specific inhibitors of M2-and M4-mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells.Then,mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction.The intracellular ROS level was measured using DCFH-DA probes.The expression of hypoxia-inducible transcription factor 1α(HIF-1α)was also detected.Results:While H/R induced the expression of M2-and M4-mAChRs,AT3 suppressed the H/R-upregulated M2-and M4-mAChRs.H/R also induced the production of NO,ROS,and apoptosis.AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis.HIF-1αwas induced by H/R,but was suppressed by AT3.Conclusion:Thus,the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α,NO and ROS,predominantly through the downregulation of M4-mAChR.The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/reperfusion injury.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

C-X-C chemokine receptor type 7 antibody enhances neural plasticity after ischemic stroke

Stromal cell-derived factor-1 and its receptor C-X-C chemokine receptor 4(CXCR4) have been shown to regulate neural regeneration after stroke.Howeve r,whether stromal cell-derived factor-1 receptor CXCR7,which is widely distributed in the develo ping and adult central nervous system,participates in neural regeneration remains poorly unde rstood.In this study,we established rat models of focal cerebral ischemia by injecting endothelin-1 into the cerebral co rtex and striatum.Starting on day 7 after injury,CXCR7-neutralizing antibody was injected into the lateral ventricle using a micro drug delivery system for 6 consecutive days.Our results showed that CXCR7-neutralizing antibody increased the total length and number of sprouting co rticospinal tra ct fibers in rats with cerebral ischemia,increased the expression of vesicular glutamate transporter 1 and growth-related protein 43,marke rs of the denervated spinal cord synapses,and promoted the differentiation and maturation of oligodendrocyte progenitor cells in the striatum.In addition,CXCR7 antibody increased the expression of CXCR4 in the striatum,increased the protein expression of RAS and ERK1/2 associated with the RAS/ERK signaling pathway,and im proved rat motor function.These findings suggest that CXCR7 improved neural functional recovery after ischemic stroke by promoting axonal regeneration,synaptogenesis,and myelin regeneration,which may be achieved by activation of CXCR4 and the RAS/ERK1/2 signaling pathway.

Expression and signifi cance of TLR4 and HIF-1α in pancreatic ductal adenocarcinoma

AIM:To investigate the expression of toll-like receptor(TLR) 4,nuclear factor-κB(NF-κB) p65 and hypoxiainducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma and their clinical significance.METHODS:The mRNA of TLR4 and HIF-1α were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues,and expression of TLR4,NF-κB p65 and HIF-1α protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent tissues.The relationship between TLR4 or HIF-1α and pathologic features,as well as the association between TLR4 and HIF-1α,were also analyzed.Kaplan-Meier method was used to assess the impact of expression of TLR4 and HIF-1α on survival of patients with pancreatic cancer.RESULTS:The relative quantif ication of TLR4 and HIF-1α mRNA in tumor tissues was 0.81±0.10 and 0.87±0.11,respectively,signif icantly higher than that in adjacent tissues(0.81±0.10 vs 0.70±0.16,P=0.002;0.87±0.11 vs 0.68±0.13,P=0.000).The protein expression of TLR4,NF-κB p65 and HIF-1α in tumor tissues was 69.20%,66.15% and 70.80%,respectively,being signif icantly higher than that in adjacent normal tissues(69.20% vs 39.50%,P=0.003;66.15% vs 31.58%,P=0.001;70.80% vs 36.80%,P=0.001).There was no signif icant correlation between TLR4 or HIF-1α expression and the age,gender,tumor location,the degree of tumor differentiation in the patients(P>0.05).However,there was signif icant correlation between the expression of TLR4 or HIF-1α and tumor size,lymph node metastasis,venous invasion and clinical staging(P<0.05).The expression of TLR4 and HIF-1α had a signif icant impact on survival of patients with pancreatic adenocarcinoma.CONCLUSION:TLR4,NF-κB p65 and HIF-1α are overexpressed in pancreatic adenocarcinoma,TLR4 may be partly involved in up-regulating HIF-1α,and both synergestically promote development of pancreatic adenocarcinoma.