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.

Interaction between insulin-like growth factor binding protein-related protein 1 and transforming growth factor beta 1 in primary hepatic stellate cells

BACKGROUND: We previously showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel mediator in liver fibrosis. Transforming growth factor beta 1 (TGF beta 1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGF beta 1 in primary hepatic stellate cells (HSCs). METHODS: We overexpressed TGF beta 1 or IGFBPrP1 and inhibited TGF beta 1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of a-smooth muscle actin (alpha-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3). RESULTS: We found that the adenovirus vector encoding the TGF beta 1 gene (AdTGF beta 1) induced IGFBPrP1 expression while that of alpha-SMA, collagen I, fibronectin, and TGF beta 1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGF beta 1, alpha-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGF beta 1 expression reduced the IGFBPrP1-stimulated expression of alpha-SMA, collagen I, fibronectin, and p-Smad2/3. CONCLUSIONS: These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGF beta 1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGF beta 1-depedent manner, and may act as an upstream regulatory factor of TGF beta 1 in the Smad pathway.

Significance of highly phosphorylated insulin-like growth factor binding protein-1 and cervical length for prediction of preterm delivery in twin pregnancies

BACKGROUND A twin pregnancy can carry greater risks than singleton pregnancies.About 60 in 100 twin pregnancies result in spontaneous birth before 37 wk,which is associated with several complications in the premature babies.Clinical detection of biomarkers may help to predict the possibility of premature birth so that corresponding interventions can be given to the pregnant women in a timely manner,in order to reduce the risk of preterm birth and improve the outcomes of the newborn infants.AIM To explore the clinical value of transvaginal ultrasound measurement of cervical length combined with insulin-like growth factor binding protein-1(IGFBP-1)hyperphosphorylation in cervical secretions as predictors of preterm delivery in twin pregnancies.METHODS A total of 254 pregnant women with twin pregnancies,who were admitted to Hainan General Hospital and underwent maternity examination,were selected as the study subjects from January 2015 to December 2018.All participants received transvaginal ultrasound measurement of cervical length and phosphorylated IGFBP-1(phIGFBP-1)test between 24 and 34 wk gestation.The pregnancy outcomes were analyzed.RESULTS Of the women with a positive phIGFBP-1 test result,preterm birth rate was higher in those with a cervical length≤25 mm than those with a cervical length>25 mm(all P<0.05).Similarly,in women with a negative phIGFBP-1 test result,preterm birth rate was higher in those with a cervical length≤25 mm than those with a cervical length>25 mm(all P<0.05).The sensitivity,specificity,and positive and negative predictive values of the phIGFBP-1 test combined with the cervical length test were 95.71%,91.21%,95.12%and 92.22%,respectively,for the prediction of preterm birth.CONCLUSION Cervical length combined with phIGFBP-1 tests is of value for the prediction of outcomes of preterm delivery in twin pregnancies.

Insulin-like growth factor-binding protein-3 inhibits IGF-1-induced proliferation of human hepatocellular carcinoma cells

OBJECTIVE Basic fibroblast growth factor(b FGF)and platelet-derived growth factor(PDGF)produced by hepatocellular carcinoma(HCC)cells are responsible for the cell growth.Accumulating evidence shows that insulin-like growth factor-binding protein-3(IGFBP-3)suppresses HCC cell proliferation in both IGF-dependent and independent manners.The present study is to investigate whether treatment with exogenous IGFBP-3 inhibits bF GF and PDGF production and the cell proliferation of HCC cells.METHODS Cell Counting Kit 8 assay were designed to detect HCC cell proliferation,transcription factor early growth response-1(EGR1)involving in IGFBP-3 regulation of b FGF and PDGF were detected by RT-PCR and Western blot assays.Western blot assay was adopted to detect the IGFBP-3 regulating insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS The present study demonstrates that IGFBP-3 suppressed IGF-1-induced b FGF and PDGF expression while it does not affect their expression in the absence of IGF-1.To delineate the underlying mechanism,Western-blot and RT-PCR assays confirmed that the transcription factor early growth response protein 1(EGR1)is involved in IGFBP-3 regulation of b FGF and PDGF.IGFBP-3 inhibition of type 1 insulin-like growth factor receptor(IGF1R),ERK and AKT activation is IGF-1-dependent.Furthermore,transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1,b FGF and PDGF expression.CONCLUSION In conclusion,these findings suggest that IGFBP-3suppresses transcription of EGR1 and its target genes b FGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation.It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation,suggesting that IGFBP-3 could be a target for the treatment of 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.

DIAGNOSTIC VALUE OF SERUM INSULIN- LIKE GROWTH FACTOR BINDING PROTEIN- 3 IN CHILDREN WITH OR WITHOUT GROWTH HORMONE DEFICIENCY

OBJECTIVE: To study the value of serum insulin-like growth factor binding protein-3 (IGFBP-3) levels in differential diagnosis of growth hormone deficiency (GHD). METHODS: To measure serum IGFBP-3 levels by RIA in normal children and adolescents, GHD children and short-stature children without GHD. RESULTS: Serum level of IGFBP-3 in 129 children with untreated GHD and with no pubertal development was 1.6 +/- 0.9 mg/L, which was less than that in normal group of the same age, but overlapped with the normal children in Tanner stage I. After six-month treatment with recombinant human growth hormone (rhGH), serum level of IGFBP-3 in 59 GHD significantly increased from 1.3 +/- 0.7 mg/L to 2.7 +/- 0.9 mg/L, accompanied by an increase of body heights, growth velocities and serum level of IGF-1. Serum level of IGFBP-3 in 55 short-stature children without GHD was 3.3 +/- 2.2 mg/L, which was not significantly different from that in normal group. CONCLUSION: Serum IGFBP-3 level can reflect the status of GH secretion in children with GHD and is a useful marker for differential diagnosis of GHD.

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.

Increased expression of insulin-like growth factor-binding protein-3 is implicated in erectile dysfunction in two-kidney one-clip hypertensive rats after propranolol treatment

This study aimed to investigate the role of insulin-like growth factor-binding protein-3 （IGFBP-3） in erectUe dysfunction （ED） in two-kidney one-clip （2K-1C） hypertensive rats treated with the β-blocking agent propranolol. Adult male Wistar rats were randomly divided into three groups： a normal control group, a hypertensive control group and a propranolol treatment group （n=9）. After 4 weeks of propranolol treatment, intracavemous pressure （ICP） responses to electrical stimulation of the cavernous nerves were evaluated. The expression of IGFBP-3 and insulin-like growth factor-1 （IGF-1） mRNA and protein in the rat cavernous tissue were detected by quantitative real-time PCR and Western blot, respectively. The concentration of cyclic guanosine monophosphate （cGMP） in the cavernous tissue was determined by enzyme-linked immunosorbent assay （ELISA）. Cavernosal pressure in response to cavernous nerve stimulation was decreased 4 weeks after propranolol treatment （P〈0.01, compared to the hypertensive control group）. IGFBP-3 mRNA and protein expression was increased in the propranolol treatment group compared to the hypertensive control group （P〈O.01）, whereas IGF-1 expression was decreased in the propranolol treatment group compared to the hypertensive control group （P〈0.01）. In addition, cavernous cGMP concentration was decreased in the prepranolol treatment group compared to the hypertensive control group （P〈0.01）. Taken together, these results suggest that the upregulation of IGFBP-3 may play a role in the development of ED in hypertensive rats.

Modified insulin-like growth factor 1 containing collagen-binding domain for nerve regeneration

Insulin-like growth factor 1 （IGF-I） is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve targeted and long-lasting treatment, we investigated the addition of a binding structure by fusing a collagen-binding domain to IGF- 1. After confirming its affinity for collagen, the biological activity of this construct was examined by measuring cell proliferation after transfection into PC12 and Schwann cells using a 3-（4,5-dimethyl-2-thiazolyl）-2,5-di- phenyl-2-H-tetrazolium bromide assay. Immunofluorescence staining was conducted to detect neurofilament and microtubule-associated protein 2 expression, while real time-polymerase chain reaction was utilized to determine IGF-1 receptor and nerve growth/actor mRNA expression. Our results demonstrate a significant increase in collagen-binding activity of the recombinant protein compared with IGF-1. Moreover, the recombinant protein promoted proliferation of PC12 and Schwann cells, and increased the expression of neurofilament and microtubule-associated protein 2. Importantly, the recombinant protein also stimulated sustained expression of IGF-1 receptor and nerve growth factor mRNA for days. These results show that the recombinant protein achieved the goal of targeting and long-lasting treatment, and thus could become a clinically used factor for promoting nerve regeneration with a prolonged therapeutic effect.

A peptide containing the receptor binding site of insulin-like growth factor binding protein-2 enhances bone mass in ovariectomized rats

Male Igfbp2-/-mice have a significant reduction in bone mass and administration of a peptide that contains the insulin-like growth factor binding protein-2(IGFBP-2) receptor-binding domain stimulates bone formation in these animals. Female Igfbp2-/-mice do not have this phenotype but following ovariectomy(OVX) lose more bone than OVX wild-type mice. This suggests that in the absence of estrogen, IGFBP-2 is required to maintain bone mass. Therefore these studies were undertaken to determine if this peptide could stimulate bone acquisition in OVX rats. OVX rats were divided into seven treatment groups: sham animals, OVX animals, OVX animals receiving a control scrambled peptide, or one of three doses of the active peptide termed PEG-HBD-1(0.7, 2,and 6 mg·kg^(-1)) and an OVX group receiving parathyroid hormone(PTH)(50 μg·kg-1 per day). The peptides were administered for8 weeks. DXA revealed a significant reduction in femoral and tibial areal bone mineral density(aBMD) after OVX, whereas treatment with the high-dose peptide increased aBMD by 6.2% ± 2.4%(P < 0.01) compared to control peptide; similar to the increase noted with PTH(5.6% ± 3.0%, P < 0.01). Similar increases were noted with two lower doses of the peptide(3.8% ± 1.5%, P < 0.05 for low dose; 3.1% ± 1.6%, P = 0.07 for middle dose). Micro CT showed that the OVX control peptide animals had reductions of 41% and64% in femoral trabecular BV/TV and trabecular number, respectively. All three doses of the peptide increased bone volume/total volume(BV/TV) significantly, while the low and middle doses increased trabecular number. Cortical BV/TV and thickness at the midshaft increased significantly with each dose of peptide(18.9% ± 9.8%, P < 0.01 and 14.2% ± 7.9%, P < 0.01 for low dose; 23.7% ±10.7%, P < 0.001 and 15.8% ± 6.1%, P < 0.001 for middle dose; 19.0% ± 6.9%, P < 0.01 and 16.2% ± 9.7%, P < 0.001 for high dose)and with PTH(25.8% ± 9.2%, P < 0.001 and 19.4% ± 8.8%, P < 0.001). Histomorphometry showed that the lowest dose of peptide stimulated BV/TV, trabecular thickness, mineral apposition rate(MAR), bone formation rate/bone surface(BFR/BS), number of osteoblasts/bone perimeter(N.ob/B.pm), and decreased osteoclast surface/bone perimeter(Oc.S/B.Pm). The highest dose stimulated each of these parameters except MAR and BFR/BS. Thus, the heparin-binding domain receptor region of IGFBP-2 accounts for its anabolic activity in bone. Importantly, this peptide enhances bone mass in estrogen-deficient animals.

Changes of insulin-like growth factor-Ⅱ and insulin-like growth factor binding protein-3 in cerebrospinal fluid of children with tuberculous meningitis

BACKGROUND： Recent studies have found that insulin-like growth factors （IGFs） and insulin-like growth factor binding protein-3 （IGFBP-3） have stronger neurotrophic and neuroprotective effects. But whether their levels in cerebrospinal fluid could be used as an auxiliary indicator in differentially diagnosing tuberculous meningitis and viral encephalitis is not yet clear. OBJECTIVE： To explore the changes of insulin-like growth factor-Ⅱ （IGF-Ⅱ ） and IGFBP-3 in cerebrospinal fluid （CSF） of children with tuberculous meningitis and the significance of the changes. DESIGN： A non-randomized concurrent controlled study. SETTING： Department of Pediatric Internal Medicine, the First Affiliated Hospital of Xinxiang Medical College. PARTICIPANTS： Thirty children with tuberculous meningitis （14 males and 16 females） were selected from the Department of Pediatric Internal Medicine, the First Affiliated Hospital of Xinxiang Medical College from January 2005 to December 2006. Tuberculous meningitis was diagnosed according to their clinical manifestations, the history of close contact with tuberculosis, typical cerebrospinal fluid changes of tuberculous meningitis, positive tuberculosis antibody and effective antituberculosis treatment. There were 30 children （13 males and 17 females） with viral encephalitis, and viral encephalitis was diagnosed according to epidemiological history, clinical manifestations, conventional and biochemical changes of cerebrospinal fluid, and negative bacteriology judgment. Meanwhile, 30 children （13 males and 17 females） without infectious and central nervous system disease were selected as the control group. Informed consent was obtained from the parents of all the enrolled children. METHODS： ①The lumbar puncture operation was implemented immediately to obtain cerebrospinal fluid （3 mL）. The contents of IGF-Ⅱ and IGFBP-3 were detected with immunoradiometric assay. The concentrations of glucose and protein in cerebrospinal fluid were determined with a dry-chemical method. The number of white blood cells was counted by Fushi Method. ②The Pearson correlation analysis was used to analyze the correlation of the contents of IGF-Ⅱ and IGFBP-3 in cerebrospinal fluid with the leucocyte counting and the concentrations of glucose and protein in cerebrospinal fluid. MAIN OUTCOME MEASURES： The contents of IGF- Ⅱ and IGFBP-3 in cerebrospinal fluid, and their correlation with the leucocyte counting and the concentrations of glucose and protein in cerebrospinal fluid. RESULTS： ①Contents of IGF-Ⅱ and IGFBP-3 in cerebrospinal fluid： The contents of IGF-Ⅱ and IGFBP-3 in cerebrospinal fluid in the tuberculous meningitis group were significantly higher than those in the encephalitis virus group and control group （P 〈 0.05）. There was no significant difference in the contents of IGF- Ⅱ and IGFBP-3 in cerebrospinal fluid between the viral encephalitis group and control group （P 〉 0.05）. ②Correlation： The IGF- Ⅱ and IGFBP-3 contents in cerebrospinal fluid were positively correlated with the protein concentration in cerebrospinal fluid （r =0.821, 0.855, P 〈 0.01）, but negatively with the glucose （r =0.742, - 0.605, P 〈 0.01）. CONCLUSION- ①IGFs and IGVBPs are involved in the pathophysiological process of tuberculous meningitis, as well as the glucose and protein metabolism in cerebrospinal fluid. ②The IGF-Ⅱ and IGFBP-3 contents in cerebrospinal fluid can be used as the auxiliary indicators to differentially diagnose tuberculous meningitis and viral enceohalitis.

Preparatory training attenuates drastic response of the insulin-like growth factor binding protein 1 at the point of maximal oxygen consumption in handball players

Background:Intensive exercise changes physiological need for glucose and several biochemical pathways responsible for its metabolism response.Among them are those which involve insulin,insulin-like growth factor(IGF-1),and IGF-binding proteins(IGFBPs).Different types and degrees of exercise,as well as an athlete's fitness,may induce a range of responses regarding concentrations and time needed for the alteration.The idea of the work was to find out whether and how insulin/IGF axis responds to additional physical activity in the already trained subjects and if so,is the adaptation potentially beneficial from the aspect of metabolic control.Methods:The effect of 4-week intensive training on campus(preparatory training) on the levels of insulin,IGF-1,and IGFBPs during maximal progressive exercise test(MPET) on a treadmill was compared to the results obtained during MPET conducted after a regular training season of a female elite handball team(n = 17,age:17 ± 1 years,height:171 ± 8 cm,weight:65 ± 8 kg,body mass index:22 ± 1 kg/m^2 at the beginning of the study;there were no significant changes at the end).Serum samples were obtained from players immediately before the test(basal),at the end of the test after reaching the point of maximal oxygen consumption(VO_(2max)),and after recovery.Results:The concentration of insulin decreased at VO_(2max),but remained higher in players after preparatory training(12.2 ± 2.5 m U/L vs.8.9 ± 4.4 m U/L,p = 0.049).The level of IGFBP-1 decreased in players at VO_(2max) in either case of training,but it remained much higher in tests performed after the preparatory regime than before(p = 0.029).Concentrations of IGF-1,IGFBP-2,-3,and-4 did not change significantly.Conclusion:The inverse relation between insulin and IGFBP-1 was lost during MPET,as these 2 molecules changed in the same direction.The results obtained suggest less severe stress-induced depression of insulin and IGFBP-1 after preparatory training.But another metabolic mechanism cannot be excluded,and that is potentially impaired insulin sensitivity resulting in higher level of IGFBP-1.

Effects of transfected adenovirus-mediated transcription factor X-box binding protein 1 on hippocampal-derived neural stem cell proliferation and apoptosis under hypoxia

BACKGROUND： Neural stem cell （NSC） survival is closely associated with cell apoptosis in ischemic-hypoxic regions following transplantation. Numerous studies have revealed that X-box binding protein 1 （XBP1） is a transcription factor during endoplasmic reticulum unfolded protein response and is essential for cell survival, differentiation, and anti-apoptotic effects. OBJECTIVE： To determine the effects of the XBP1 gene on NSC proliferation and apoptosis under hypoxic conditions following XBP1 gene transfection into rat embryonic hippocampal NSCs using recombinant adenovirus vector. DESIGN, TIME AND SETTING： In vitro experiments were performed at the Laboratory of Cell Biology of Jilin University and Laboratory of Proteomics, Department of Neurology, Jilin University China from September 2008 to November 2009. MATERIALS： Recombinant adenovirus package XBP1 gene and Ad-XBPl-enhanced green fluorescent protein plasmid （Guangzhou Easywin BioMed Technology, China）, rabbit anti-XBP1 and its target gene estrogen receptor degradation-enhancing a-mannosidase-like protein （EDEM） glucose-regulated protein 78 （GRP78）, anti-apoptotic molecule Bcl-2 and proapoptotic molecule Bax polyclonal antibody （Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA）, and COCI2 （Sigma, St. Louis, MO, USA） were used in the present study. METHODS： Hippocampi from embryonic, Sprague Dawley rats on gestational day 16 were harvested for NSC isolation and cloning, followed by immunofluorescence for Nestin and sub-culturing. The recombinant adenovirus Ad-XBPl-enhanced green fluorescent protein plasmid was transfected into rat embryonic hippocampal NSCs, and then CoCl2 was applied to induce hypoxia. MAIN OUTCOME MEASURES： Cell quantification and 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide colorimetric assay were utilized to detect proliferation in XBPl-transfected NSCs for 7 consecutive days. Western blot assay was utilized to quantify XBP1 GRP78, EDEM, Bcl-2, and Bax expression. Flow cytometry was used to measure apoptosis. RESULTS： NSC proliferation was significantly enhanced following XBP1 gene transfection （P 〈 0.05）. Under hypoxic conditions, GRP78, EDEM, and Bcl-2 levels increased, but Bax levels decreased. In addition, NSC apoptosis decreased following transfection （P 〈 0.05）. CONCLUSION： The XBP1 gene was successfully transfected into rat embryonic hippocampal NSCs using a recombinant adenovirus vector. NSC proliferation following transfection, as well as anti-apoptotic effects under hypoxia, was significantly increased.

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.

Tribulus terrestris extracts alleviate muscle damage and promote anaerobic performance of trained male boxers and its mechanisms： Roles of androgen, IGF-1, and IGF binding protein-3

Purpose: To investigate the effects of Tribulus terrestris(TT) extracts on muscle mass, muscle damage, and anaerobic performances of trained male boxers and its mechanisms: roles of plasma androgen, insulin growth factor 1(IGF-1), and IGF-1 binding protein-3(IGFBP-3).Methods: Fifteen male boxers were divided into exercise group(E, n = 7) and exercise plus TT group(E + TT, n = 8). The 2 groups both undertook3-week high-intensity and 3-week high-volume trainings separated by a 4-week rest. TT extracts(1250 mg/day) were orally administered by boxers in E + TT group. TT extract compositions were detected by UHPLC–Q-TOF/MS. Before and at the end of the 2 trainings, muscle mass, anaerobic performance, and blood indicators were explored.Results: Compared with E group, decreases of plasma CK(1591.5 ± 909.6 U/L vs. 2719.9 ± 832.5 U/L) and IGFBP-3(3075.5 ± 1072.5 ng/m L vs. 3950.8 ± 479.3 ng/m L) as well as increases of mean power(MP, 459.4 ± 122.3 W vs. 434.6 ± 69.5 W) and MP/body weight(MP/BW, 7.5 ± 0.9 W/kg vs. 7.1 ± 1.1 W/kg) were detected in E + TT group after a high-intensity training. For high-volume training, reduction of IGFBP-3(2946.4 ± 974.1 ng/m L vs. 3632.7 ± 470.1 ng/m L) and increases of MP(508.7 ± 103.2 W vs. 477.8 ± 49.9 W) and MP/BW(8.2 ± 0.3 W/kg vs.7.5 ± 0.9 W/kg) were detected in E + TT group, compared with E group. Muscle mass, blood levels of testosterone, dihydrotestosterone(DHT),and IGF-1 were not signifiantly changed between the 2 groups.Conclusion: Taking 1250 mg capsules containing TT extracts did not change muscle mass and plasma levels of testosterone, DHT, and IGF-1 but significantly alleviated muscle damage and promoted anaerobic performance of trained male boxers, which may be related to the decrease of plasma IGFBP-3 rather than androgen in plasma.

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.

Improvement in erectile dysfunction after insulin-like growth factor-1 gene therapy in diabetic rats

Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic rats were transfected with AdCMV-βgal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction （RT-PCR）, Western blot and histology. The penis β-galactosidase activity and localization of the STZ diabetic rats were also determined. Results： One to two days after transfection, the β-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-βgal. One to 2 days after administration of AdCMV- IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure （ICP/MAP） and total intracavernous pressure （ICP）, was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. Conclusion： Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF- 1 might be a new therapeutic intervention for the treatment of erectile dysfunction （ED） in the STZ diabetic rats.

Brain and spinal cord trauma:what we know about the therapeutic potential of insulin growth factor 1 gene therapy

Although little attention has been paid to cognitive and emotional dysfunctions observed in patients after spinal co rd injury,several reports have described impairments in cognitive abilities.Our group also has contributed significantly to the study of cognitive impairments in a rat model of spinal co rd injury.These findings are very significant because they demonstrate that cognitive and mood deficits are not induced by lifestyle changes,drugs of abuse,and combined medication.They are related to changes in brain structures involved in cognition and emotion,such as the hippocampus.Chronic spinal cord injury decreases neurogenesis,enhances glial reactivity leading to hippocampal neuroinflammation,and trigge rs cognitive deficits.These brain distal abnormalities are recently called te rtiary damage.Given that there is no treatment for Tertiary Damage,insulin growth factor 1 gene therapy emerges as a good candidate.Insulin growth factor 1 gene thera py recove rs neurogenesis and induces the polarization from pro-inflammato ry towards anti-inflammatory microglial phenotypes,which represents a potential strategy to treat the neuroinflammation that supports te rtiary damage.Insulin growth factor 1 gene therapy can be extended to other central nervous system pathologies such as traumatic brain injury where the neuroinflammatory component is crucial.Insulin growth factor 1 gene therapy could emerge as a new therapeutic strategy for treating traumatic brain injury and spinal cord injury.

Role of insulin/insulin-like growth factor 1 signaling pathway in longevity

The insulin/insulin-like growth factor 1 (IGF-1) signaling pathway is evolutionary conserved in diverse speciesincluding C.elegans, saccharomyces cerevisiae, Drosophila melanogaster, rodents and humans, which is involved in many interrelated functions that are necessary for metabolism, growth and reproduction. Interestingly, more and more research has revealed that insulin/IGF-1 signaling pathway plays a pivotal role in the regulation of longevity. Generally, disruption of the power of this pathway will extend longevity in species ranging from C.elegansto humans. The role of insulin/IGF-1 in longevit yis probably related to stress resistance. Although the underlying mechanisms of longevity are not fully understood, the Insulin/IGF-1 signaling pathway has attracted substantial attention and it will be a novel target to prevent or postpone age-related diseases and extend life span. In this review, we mainly focus on the similar constitution and role of insulin/IGF-1 signaling pathway in C.elegans, saccharomyces cerevisiae, rodents and humans.

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.