Insulin-like growth factor 2 targets IGF1R signaling transduction to facilitate metastasis and imatinib resistance in gastrointestinal stromal tumors

BACKGROUND Gastrointestinal stromal tumors(GISTs)are typical gastrointestinal tract neoplasms.Imatinib is the first-line therapy for GIST patients.Drug resistance limits the long-term effectiveness of imatinib.The regulatory effect of insulin-like growth factor 2(IGF2)has been confirmed in various cancers and is related to resistance to chemotherapy and a worse prognosis.AIM To further investigate the mechanism of IGF2 specific to GISTs.METHODS IGF2 was screened and analyzed using Gene Expression Omnibus(GEO:GSE225819)data.After IGF2 knockdown or overexpression by transfection,the phenotypes(proliferation,migration,invasion,apoptosis)of GIST cells were characterized by cell counting kit 8,Transwell,and flow cytometry assays.We used western blotting to evaluate pathway-associated and epithelial-mesenchymal transition(EMT)-associated proteins.We injected transfected cells into nude mice to establish a tumor xenograft model and observed the occurrence and metastasis of GIST.RESULTS Data from the GEO indicated that IGF2 expression is high in GISTs,associated with liver metastasis,and closely related to drug resistance.GIST cells with high expression of IGF2 had increased proliferation and migration,invasiveness and EMT.Knockdown of IGF2 significantly inhibited those activities.In addition,OEIGF2 promoted GIST metastasis in vivo in nude mice.IGF2 activated IGF1R signaling in GIST cells,and IGF2/IGF1R-mediated glycolysis was required for GIST with liver metastasis.GIST cells with IGF2 knockdown were sensitive to imatinib treatment when IGF2 overexpression significantly raised imatinib resistance.Moreover,2-deoxy-D-glucose(a glycolysis inhibitor)treatment reversed IGF2 overexpressionmediated imatinib resistance in GISTs.CONCLUSION IGF2 targeting of IGF1R signaling inhibited metastasis and decreased imatinib resistance by driving glycolysis in GISTs.

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.

Neuroprotective effects of insulin-like growth factor-2 in 6-hydroxydopamine-induced cellular and mouse models of Parkinson’s disease

Skin-derived precursor Schwann cells have been reported to play a protective role in the central nervous system. The neuroprotective effects of skin-derived precursor Schwann cells may be attributable to the release of growth factors that nourish host cells. In this study, we first established a cellular model of Parkinson’s disease using 6-hydroxydopamine. When SH-SY5 Y cells were pretreated with conditioned medium from skin-derived precursor Schwann cells, their activity was greatly increased. The addition of insulin-like growth factor-2 neutralizing antibody markedly attenuated the neuroprotective effects of skin-derived precursor Schwann cells. We also found that insulin-like growth factor-2 levels in the peripheral blood were greatly increased in patients with Parkinson’s disease and in a mouse model of Parkinson’s disease. Next, we pretreated cell models of Parkinson’s disease with insulin-like growth factor-2 and administered insulin-like growth factor-2 intranasally to a mouse model of Parkinson’s disease induced by 6-hydroxydopamine and found that the level of tyrosine hydroxylase, a marker of dopamine neurons, was markedly restored, α-synuclein aggregation decreased, and insulin-like growth factor-2 receptor downregulation was alleviated. Finally, in vitro experiments showed that insulin-like growth factor-2 activated the phosphatidylinositol 3 kinase(PI3 K)/AKT pathway. These findings suggest that the neuroprotective effects of skin-derived precursor Schwann cells on the central nervous system were achieved through insulinlike growth factor-2, and that insulin-like growth factor-2 may play a neuroprotective role through the insulin-like growth factor-2 receptor/PI3 K/AKT pathway. Therefore, insulin-like growth factor-2 may be an useful target for Parkinson’s disease treatment.

Mendelian randomization provides evidence for a causal effect of serum insulin-like growth factor family concentration on risk of atrial fibrillation

BACKGROUND Atrial fibrillation(AF)is one of the most common persistent arrhythmias among adult cardiovascular diseases.It is important to identify potential risk factors for AF.Members of the insulin-like growth factor(IGF)family exert a variety of effects on various cell types in the context of the pathogenesis of cardiovascular diseases,and previous population-based studies indicate associations between IGF family members and AF.However,the causal effects of IGF family members in AF have not been evaluated.assess genetic relationships between IGF family members and AF.METHODS MR was performed based on genome-wide association study(GWAS)datasets,and concentration levels of 14 IGF family members were retrieved.An initial MR analysis was conducted to identify single nucleotide polymorphisms potentially associated with IGF serum concentrations.A GWAS meta-analysis including 60620 AF cases and 970216 control participants of European ancestry was then conducted to identify AF causal effects.Two-sample MR packages were used to perform MR analysis in R.MR-Egger,weighted median(WM),and inverse va-riance weighted(IVW)methods were used.RESULTS Core Tip:Due to the high prevalence of atrial fibrillation(AF),and adverse outcomes related to it,it is important to identify risk factors associated with development of the condition.Insulin-like growth factor(IGF)family members exert a variety of effects on various cell types in the context of the pathogenesis of cardiovascular diseases,and previous population-based studies indicate associations between IGF family members and AF.However,the causal effects of IGF family members in AF have not been evaluated.The results of the current study provide novel insights on the pathogenesis of AF,and implic-ations of serum IGF family member concentrations when assessing the risk of AF.The study generated evidence on the potential roles of developmental pathological effects in the pathogenesis of AF.Further observational and experimental studies are critically needed.

Single Nucleotide Polymorphism Analysis on 5' Regulatory Region of Goose Insulin-like Growth FactorⅠGene

[ Objedive] This study was aimed to determine the single nucleotide polymorphisms （SNPs） of IGF-I gene in two breeds, Wanxi white goose and Langde goose. [ Method] Two pair of primers was designed based on chicken and porcine genomic sequence to amplify the 5＇ regulatory region of IGF-I, and the sequence was determined and analyzed. [ Result] A total of four SNPs were identified in this region by PCR-SSCP meth- od, that is, A to T at 26 nt, A to G at 215 nt, A to G at 314 nt, and A to T at 325 nt. [ Conclusioa] The two breeds ,wanxi white geese and Langde geese, agree with Hardy-weinberg equilibrium with respect to these SNPS.

Reactivation of the insulin-like growth factor-Ⅱsignaling pathway in human hepatocellular carcinoma

Constitutive activation of the insulin-like growth factor (IGF)-signaling axis is frequently observed in human hepatocellular carcinoma(HCC).Especially the over- expression of the fetal growth factor IGF-Ⅱ,IGF-Ⅰ receptor(IGF-IR),and cytoplasmic downstream effectors such as insulin-receptor substrates(IRS)contribute to proliferation,anti-apoptosis,and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱsignaling during HCC development and progression.Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies,various experimental strategies have been developed,including neutralizing antibodies and selective receptor kinase inhibitors,with respect to the specific and efficient reduction of oncogenic IGF-Ⅱ/IGF-IR-signaling.

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.

Abnormal expression of insulin-like growth factor-Ⅱ and its dynamic quantitative analysis at different stages of hepatocellular carcinoma development

BACKGROUND:Hepatocellular carcinoma(HCC)is characterized by multiple causes,clear multiple stages and a multifocal process of tumor progression related intimately to the overexpression of many cellular factors. The aim of this study was to investigate the dynamic expression of insulin-like growth factor-Ⅱ(IGF-Ⅱ) and its abnormal alteration in the early stages of HCC development. METHODS:Hepatoma models were induced by 2-fluorenylacetamide(2-FAA)in male Sprague-Dawley rats.Morphological changes of rat livers were assessed by pathological examination(HE staining).The levels of IGF-Ⅱexpression in the livers and sera of rats were quantitatively detected by an enzyme-linked immunosorbent assay(ELISA).Simultaneously,the expression and cellular distribution of liver IGF-Ⅱwere analyzed by immunohistochemistry. RESULTS:Histological examination confirmed that rat hepatocytes showed changes from granule-like degeneration to atypical hyperplasia to HCC,and progressively increasing hepatic IGF-Ⅱlevels during HCC development.The levels of hepatic or serum IGF- Ⅱin HCC tissues and sera were significantly higher than those in normals and rats with degeneration.The immunohistochemical evidence indicated the positiveexpression and hepatocyte distribution of IGF-Ⅱin rat hepatoma.A positive relationship of IGF-Ⅱlevels was found between liver tissues and sera of experimental rats (P【0.01). CONCLUSION:Hepatic IGF-Ⅱmay participate in hepatocyte cancer development and detection of IGF-Ⅱ expression during HCC development could be a useful molecular marker for its early diagnosis.

Effect of endogenous insulin-like growth factor and stem cell factor on diabetic colonic dysmotility

AIM: To investigate whether the reduction of stem cell factor (SCF) is mediated by decreased endogenous insulin-like growth factor (IGF)-1 in diabetic rat colon smooth muscle. METHODS: Sixteen Sprague-Dawley rats were randomly divided into two groups: control group and streptozotocin-induced diabetic group. After 8 wk of streptozotocin administration, colonic motility function and contractility of circular muscle strips were measured. The expression of endogenous IGF-1 and SCF was tested in colonic tissues. Colonic smooth muscle cells were cultured from normal adult rats. IGF-1 siRNA transfection was used to investigate whether SCF expression was affected by endogenous IGF-1 expression in smooth muscle cells, and IGF-1 induced SCF expression effects were studied. The effect of high glucose on the expression of endogenous IGF-1 and SCF was also investigated. RESULTS: Diabetic rats showed prolonged colonic transit time (252 ± 16 min vs 168 ± 9 min, P < 0.01) and weakness of circular muscle contraction (0.81 ± 0.09 g vs 2.48 ± 0.23 g, P < 0.01) compared with the control group. Endogenous IGF-1 and SCF protein expression was significantly reduced in the diabetic colonic muscle tissues. IGF-1 and SCF mRNA expression also showed a paralleled reduction in diabetic rats. In the IGF-1 siRNA transfected smooth muscle cells, SCF mRNA and protein expression was significantly decreased. IGF-1 could induce SCF expression in a concentration and time-dependent manner, mainly through the extracellular-signal-regulated kinase 1/2 signal pathway. High glucose inhibited endogenous IGF-1 and SCF expression and the addition of IGF-1 to the medium reversed the SCF expression. CONCLUSION: Myopathy may resolve in colonic motility dysfunction in diabetic rats. Deficiency of endogenous IGF-1 in colonic smooth muscle cells leads to reduction of SCF expression.

Overexpression of insulin-like growth factor-Ⅰ receptor as a pertinent biomarker for hepatocytes malignant transformation

AIM: To investigate the dynamic features of insulin-like growth factor-I receptor (IGF-IR) expression in rat hepatocarcinogenesis, and the relationship between IGF-IR and hepatocytes malignant transformation at mRNA or protein level.METHODS: Hepatoma models were made by inducing with 2-fluorenylacetamide (2-FAA) on male Sprague-Dawley rats. Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining, the dynamic expressions of liver and serum IGF-IR were quantitatively analyzed by an enzyme-linked immunosorbent assay. The distribution of hepatic IGF-IR was located by immunohistochemistry. The fragments of IGF-IR gene were amplified by reverse transcription-polymerase chain reaction, and confirmed by sequencing.RESULTS: Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration, precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-IR expression. The incidences of liver IGF-IR, IGF-IR mRNA, specific IGF-IR concentration (ng/mg wet liver), and serum IGF-IR level (ng/mL) were 0.0%, 0.0%, 0.63 ± 0.17, and 1.33 ± 0.47 in the control; 50.0%, 61.1%, 0.65 ± 0.2, and 1.51 ± 0.46 in the degeneration; 88.9%, 100%, 0.66 ± 0.14, and 1.92 ± 0.29 in the precancerosis; and 100%, 100%, 0.96 ± 0.09, and 2.43 ± 0.57 in the cancerous group, respectively. IGF-IR expression in the cancerous group was significantly higher (P < 0.01) than that in any of other groups at mRNA or protein level. The closely positive IGF-IR relationship was found between livers and sera (r = 0.91, t = 14.222, P < 0.01), respectively.CONCLUSION: IGF-IR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.

Mitochondrial protection by low doses of insulin-like growth factor-Ⅰin experimental cirrhosis

AIM： To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor-Ⅰ （IGF-Ⅰ ） therapy （4 wk） is able to induce beneficial effects on damaged mitochondria leading to cellular protection. METHODS： Wistar rats were divided into three groups： Control group, untreated cirrhotic rats and cirrhotic rats treated with IGF-Ⅰ treatment （2 μg/1O0 g bw/d）. Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria, caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three expedmental groups. RESULTS： Untreated cirrhotic rats showed a mitochondrial dysfunction characterized by a significant reduction of mitochondrial membrane potential （in status 4 and 3）; an increase of intramitochondrial reactive oxigen species （ROS） generation and a significant reduction of ATPase activity. IGF-Ⅰ therapy normalized mitochondrial function by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production. Activity of the electron transport complexes Ⅰ and Ⅲ was increased in both cirrhotic groups. In addition, untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis. IGF- Ⅰ therapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis. CONCLUSION： These results show that IGF- Ⅰ exerts a mitochondrial protection in experimental cirrhosis leading to reduced apoptosis and increased ATP production.

Insulin-like growth factor-1, IGF binding protein-3, and the risk of esophageal cancer in a nested case-control study

To assess the relationship between serum levels of insulin-like growth factor-1 (IGF1)/IGF-binding protein-3 (IGFBP3) and the risk of esophageal carcinoma.METHODSWe assessed the relationship between the serum levels of these molecules and the risk of esophageal cancer in a prospective, nested case-control study of participants from the Japan Collaborative Cohort Study. A baseline survey was conducted from 1988 to 1990. Of the 110585 enrolled participants, 35% donated blood samples. Those who had been diagnosed with esophageal cancer were considered cases for nested case-control studies. A conditional logistic model was used to estimate odds ratios for the incidence of esophageal cancer associated with serum IGF1 and IGFBP3 levels.RESULTSThirty-one cases and 86 controls were eligible for the present assessment. The molar ratio of IGF1/IGFBP3, which represents the free and active form of IGF1, was not correlated with the risk of esophageal carcinoma. A higher molar difference between IGFBP3 and IGF1, which estimates the free form of IGFBP3, was associated with a decreased risk of esophageal carcinoma (P = 0.0146), and people in the highest tertile had the lowest risk (OR = 0.107, 95%CI: 0.017-0.669). After adjustment for body mass index, tobacco use, and alcohol intake, the molar difference of IGFBP3-IGF1 was inversely correlated with the risk of esophageal carcinoma (P = 0.0150).CONCLUSIONThe free form of IGFBP3, which is estimated by this molar difference, may be inversely associated with esophageal cancer incidence.

Effects of Bifidobacterium infantis on cytokine-induced neutrophil chemoattractant and insulin-like growth factor-1 in the ileum of rats with endotoxin injury

BACKGROUND The digestive tract is the maximal immunizing tissue in the body, and mucosal integrity and functional status of the gut is very important to maintain a healthy organism. Severe infection is one of the most common causes of gastrointestinal dysfunction, and the pathogenesis is closely related to endotoxemia and intestinal barrier injury. Bifidobacterium is one of the main probiotics in the human body that is involved in digestion, absorption, metabolism, nutrition, and immunity.Bifidobacterium plays an important role in maintaining the intestinal mucosal barrier integrity. This study investigated the protective mechanism of Bifidobacterium during ileal injury in rats.AIM To investigate the effects of Bifidobacterium on cytokine-induced neutrophil chemoattractant(CINC) and insulin-like growth factor 1(IGF-1) in the ileum of rats with endotoxin injury.METHODS Preweaning rats were randomly divided into three groups: Control(group C),model(group E) and treatment(group T). Group E was intraperitoneally injected with lipopolysaccharide(LPS) to create an animal model of intestinal injury.Group T was intragastrically administered Bifidobacterium suspension 7 d before LPS. Group C was intraperitoneally injected with normal saline. The rats were killed at 2, 6 or 12 h after LPS or physiological saline injection to collect ilealtissue samples. The expression of ileal CINC mRNA was evaluated by reverse transcription-polymerase chain reaction(RT-PCR), and expression of ileal IGF-1 protein and mRNA was detected by immunohistochemistry and RT-PCR,respectively.RESULTS The ileum of rats in Group C did not express CINC mRNA, ileums from Group E expressed high levels, which was then significantly decreased in Group T(F =23.947, P < 0.05). There was no significant difference in CINC mRNA expression at different times(F = 0.665, P > 0.05). There was a high level of IGF-1 brown granules in ileal crypts and epithelial cells in Group C, sparse staining in Group E, and dark, dense brown staining in Group T. There was a significant difference between Groups C and E and Groups E and T(P < 0.05). There was no significant difference in IGF-1 protein expression at different times(F = 1.269, P > 0.05). IGF-1 mRNA expression was significantly different among the three groups(P < 0.05),though not at different times(F = 0.086, P > 0.05).CONCLUSION Expression of CINC mRNA increased in the ileum of preweaning rats with endotoxin injury, and exogenous administration of Bifidobacterium reduced CINC m RNA expression. IGF-1 protein and mRNA expression decreased in the ileum of preweaning rats with endotoxin injury, and exogenous administration of Bifidobacterium prevented the decrease in IGF-1 expression. Bifidobacterium may increase IGF-1 expression and enhance intestinal immune barrier function in rats with endotoxin injury.

Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer

AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol3 kinase (PI3K), and PD98059, a specific inhibitor of mitogen-activated protein kinase, were added to the media. After 72 h, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed to analyze cell proliferation. A wound assay was performed to analyze cell motility with hematoxylin and eosin (HE) staining 48 h after addition of each inhibitor. RESULTS: All cell lines clearly expressed not only IGF-IR but also phosphorylated IGF-IR. PPP significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 36.9% ± 2.4% (mean ± SD), 30.9% ± 5.5%, 23.8% ± 3.9%, 37.1% ± 5.3%, 10.4% ± 4.5%, 52.5% ± 4.5% and 22.6% ± 0.4%, at 2 μmol/L, respectively (P < 0.05). LY294002 significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 44.4% ± 7.6%, 32.9% ± 8.2%, 53.9% ± 8.0%, 52.8% ± 4.0%, 32.3% ± 4.2%, 51.8% ± 4.5%, and 30.6% ± 9.4%, at 50 μmol/L, respectively (P < 0.05). PD98059 did not significantly suppress cell proliferation. PPP at 2 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 3.0% ± 0.2%, 0%, 0%, 2.0% ± 0.1%, 5.0% ± 0.2%, 3.0% ± 0.1%, and 5.0% ± 0.2%, respectively (P < 0.05). LY294002 at 50 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 to 3.0% ± 0.2%, 0%, 3.0% ± 0.2%, 0%, 0%, 0% and 3% ± 0.1%, respectively (P < 0.05). PD980509 at 20 μmol/L did not suppress motility. Cells were observed by microscopy to analyze the morphological changes induced by the inhibitors. Cells in medium treated with 2 μmol/L PPP or 50 μmol/L LY294002 had pyknotic nuclei, whereas those in medium with 20 μmol/L PD98059 did not show apoptosis.CONCLUSION: IGF-IR and PI3K are good candidates for molecular therapy of pancreatic cancer.

Insulin-like growth factor 2 mRNA-binding protein 1 promotes cell proliferation via activation of AKT and is directly targeted by microRNA-494 in pancreatic cancer

BACKGROUND Studies have shown that insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1)plays critical roles in the genesis and development of human cancers.AIM To investigate the clinical significance and role of IGF2BP1 in pancreatic cancer.METHODS Expression levels of IGF2BP1 and microRNA-494(miR-494)were mined based on Gene Expression Omnibus datasets and validated in both clinical samples and cell lines by quantitative real-time polymerase chain reaction and Western blot.The relationship between IGF2BP1 expression and clinicopathological factors of pancreatic cancer patients was analyzed.The effect and mechanism of IGF2BP1 on pancreatic cancer cell proliferation were investigated in vitro and in vivo.Analyses were performed to explore underlying mechanisms of IGF2BP1 upregulation in pancreatic cancer and assays were carried out to verify the posttranscriptional regulation of IGF2BP1 by miR-494.RESULTS We found that IGF2BP1 was upregulated and associated with a poor prognosis in pancreatic cancer patients.We showed that downregulation of IGF2BP1 inhibited pancreatic cancer cell growth in vitro and in vivo via the AKT signaling pathway.Mechanistically,we showed that the frequent upregulation of IGF2BP1 was attributed to the downregulation of miR-494 expression in pancreatic cancer.Furthermore,we discovered that reexpression of miR-494 could partially abrogate the oncogenic role of IGF2BP1.CONCLUSION Our results revealed that upregulated IGF2BP1 promotes the proliferation of pancreatic cancer cells via the AKT signaling pathway and confirmed that the activation of IGF2BP1 is partly due to the silencing of miR-494.

Tyrosine kinase of insulin-like growth factor receptor as target for novel treatment and prevention strategies of colorectal cancer

AIM： To investigate the antineoplastic potency of the novel insulin-like growth factor 1 receptor （IGF-1R） tyrosine kinase inhibitor （TKI） NVP-AEW541 in cell lines and primary cell cultures of human colorectal cancer （CRC）. METHODS： Cells of primary colorectal carcinomas were from 8 patients. Immunostaining and crystal violet staining were used for analysis of growth factor receptor protein expression and detection of cell number changes, respectively. Cytotoxicity was determined by measuring the release of the cytoplasmic enzyme lactate dehydrogenase （LDH）. The proportion of apoptotic cells was determined by quantifying the percentage of sub-G1 （hypodiploid） cells. Cell cycle status reflected by the DNA content of the nuclei was detected by flow cytometry. RESULTS： NVP-AEW541 dose-dependently inhibited the proliferation of colorectal carcinoma cell lines and primary cell cultures by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by caspase-3 activation and nuclear degradation. Cell cycle was arrested at the G1/S checkpoint. The NVP-AEW541-mediated cell cycle-related signaling involved the inactivation of Akt and extracellular signal-regulated kinase （ERK） 1/2, the upregulation of the cyclin-dependent kinase inhibitors p21^waf1/CIP1 and p27^kjp1, and the downregulation of the cell cycle promoter cyclin D1. Moreover, BAX was upregulated during NVP-AEW541-induced apoptosis, whereas Bcl-2 was downregulated. Measurement of LDH release showed that the antineoplastic effect of NVP-AEW541 was not due to general cytotoxicity of the compound. However, augmented antineoplastic effects were observed in combination treatments of NVP-AEW541 with either 5-FU, or the EGFR-antibody cetuximab, or the HMG-CoA-reductase inhibitor fluvastatin. CONCLUSION： IGF-1R-TK inhibition is a promising novel approach for either monoor combination treatment strategies of colorectal carcinoma and even for CRC chemoprevention.

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM:To investigate the role of insulin-like growth factor binding protein-7 (IGFBP-7) in the activation and transdifferentiation of hepatic stellate cells (HSC) in vitro.METHODS:Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor (TGF)-β1,IGFBP-7 or antiIGFBP-7 antibody for 24 h.The supernatant or a cytoplasm suspension was obtained from cultured HSC,followed by transfer of cells to form cell-coated dishes.Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin,collagen and α-smooth muscle actin (SMA).The pro-apoptotic effect of antiIGFBP-7 antibody was determined by flow cytometry.RESULTS:Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group.In addition,fibronectin,collagen and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent.Moreover,flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC,which is responsible for the development of liver fibrosis,and may represent a novel pathway and target for therapeutic intervention.CONCLUSION:IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC.AntiIGFBP-7 antibody may ameliorate liver fibrogenesis.

Interplay between micro RNA-17-5p, insulin-like growth factor-Ⅱ through binding protein-3 in hepatocellular carcinoma

AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mir Vana mi RNA Isolation Kit. microR NA-17-5p(miR-17-5p) expression was mimicked and antagonized in Hu H-7 cell lines using Hi Per Fect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cD NA followed by quantification of mi R-17-5p and IGFBP-3 expression using Taq Man real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-Ⅱ protein was measured in transfected Hu H-7 cells using IGF-Ⅱ ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where mi R-17-5p was extensively underexpressed in HCC tissues(P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients(P = 0.0041) compared to healthy donors. Forcing mi R-17-5p expression in Hu H-7 cell lines showed a significant downregulation of IGFBP-3 mR NA expression(P = 0.0267) and a significant increase in free IGF-Ⅱ protein(P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of mi R-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone(P = 0.0474).CONCLUSION: These data suggest that regulating IGF-Ⅱ bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miR NAs.

Epigenetic regulation of insulin-like growth factor axis in hepatocellular carcinoma

The insulin-like growth factor(IGF) signaling path-way is an important pathway in the process of hepa-tocarcinogenesis,and the IGF network is clearly dysregulated in many cancers and developmental abnormalities.In hepatocellular carcinoma(HCC),only a minority of patients are eligible for curative treatments,such as tumor resection or liver transplant.Unfortunately,there is a high recurrence of HCC after surgical tumor removal.Recent research efforts have focused on targeting IGF axis members in an attempt to find therapeutic options for many health problems.In this review,we shed lights on the regulation of members of the IGF axis,mainly by micro RNAs in HCC.Micro RNAs in HCC attempt to halt the aberrant expression of the IGF network,and a single micro RNA can have multiple downstream targets in one or more signaling pathways.Targeting micro RNAs is a relatively new approach for identifying an efficient radical cure for HCC.

Insulin-like growth factor binding protein related protein 1 knockdown attenuates hepatic ?brosis via the regulation of MMPs/TIMPs in mice

Background: Previous research suggested that insulin-like growth factor binding protein related protein 1(IGFBPrP1), as a novel mediator, contributes to hepatic fibrogenesis. Matrix metalloproteinases(MMP) and tissue inhibitors of metalloproteinases(TIMP) play an essential role in hepatic fibrogenesis by regulating homeostasis and remodeling of the extracellular matrix(ECM). However, the interaction between IGFBPrP1 and MMP/TIMP is not clear. The present study was to knockdown IGFBPrP1 to investigate the correlation between IGFBPrP1 and MMP/TIMP in hepatic fibrosis. Methods: Hepatic fibrosis was induced by thioacetamide(TAA) in mice. Knockdown of IGFBPrP1 expression by ultrasound-targeted microbubble destruction-mediated CMB-shRNA-IGFBPrP1 delivery, or inhibition of the Hedgehog(Hh) pathway by cyclopamine treatment, was performed in TAA-induced liver fibrosis mice. Hepatic fibrosis was determined by hematoxylin and eosin and Sirius red staining. Hepatic expression of IGFBPrP1, α-smooth muscle actin( α-SMA), transforming growth factor β 1(TGF β1), collagen I, MMPs/TIMPs, Sonic Hedgehog(Shh), and glioblastoma family transcription factors(Gli1) were investigated by immunohistochemical staining and Western blotting analysis. Results: We found that hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I were increased longitudinally in mice with TAA-induced hepatic fibrosis, concomitant with MMP2/TIMP2 and MMP9/TIMP1 imbalance and Hh pathway activation. Knockdown of IGFBPrP1 expression, or inhibition of the Hh pathway, reduced the hepatic expression of IGFBPrP1, TGF β1, α-SMA, and collagen I and re-established MMP2/TIMP2 and MMP9/TIMP1 balance. Conclusions: Our findings suggest that IGFBPrP1 knockdown attenuates liver fibrosis by re-establishing MMP2/TIMP2 and MMP9/TIMP1 balance, concomitant with the inhibition of hepatic stellate cell activation, down-regulation of TGF β1 expression, and degradation of the ECM. Furthermore, the Hh pathway mediates IGFBPrP1 knockdown-induced attenuation of hepatic fibrosis through the regulation of MMPs/TIMPs balance.