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.

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 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.

Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells

Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.

Effects of ethanol on insulin-like growth factor-Ⅰ system in primary cultured rat hepatocytes: Implications of JNK1/2 and alcoholdehydrogenase

AIM: To evaluate the effects of ethanol on the insulin- like growth factor-Ⅰ (IGF-Ⅰ) system involved in c-Jun N-terminal kinase (JNK1/2) and alcoholdehydrogenase (ADH) activity in primary cultured rat hepatocytes. METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol for different durations of time. The cells were pretreated with SP600125 (10 μmol/L) and 4-MP (200 μmol/L), and then treated with ethanol (200 mmol/L). We then measured IGF-Ⅰ secretion, IGF-Ⅰ mRNA expression, cell viability and JNK1/2 activity by radioimmunoassay, RT-PCR, MTT assay and Western blot, respectively (n = 6). RESULTS: Ethanol induced the activity of phospho (p)-JNK1/2, reaching a maximum at 60 min and then decreasing at 180 min. The effects of ethanol on the IGF-Ⅰ system were increased at 60 min (secretion: 7.11 ± 0.59 ng/mg protein vs 4.91 ± 0.51 ng/mg, mRNA expression: 150.2% ± 10.2% vs 101.5% ± 11.3%, P = 0.045) and then decreased at 180 min (secretion: 3.89 ± 0.25 ng/mg vs 5.4 ± 0.54 ng/mg protein; mRNA expression: 41.5% ± 10.4% vs 84.7% ± 12.1%, P = 0.04), however cell viability was decreased in a dose- and time-dependent manner. SP600125 blocked the ethanol-induced changes (at 60 min). Additionally, 4-methylpyrazole prevented the ethanol-induced decreases in the IGF-Ⅰ system, cell viability and p-JNK1/2 activity (at 180 min). CONCLUSION: This study suggests that ethanol- induced p-JNK1/2 activation is associated with the IGF-Ⅰ system and cell viability in hepatocytes. Furthermore, alcohol dehydrogenase is involved in the relationship between ethanol-induced inactivation of p-JNK1/2 and the changes of the IGF-Ⅰ system and cell viability.

The effect of polymorphism in gene of insulin-like growth factor-Ⅰ on the serum periparturient concentration in Holstein dairy cows

Objective:To investigate the relationship between polymorphism within the 5'-untranslated region(5'-UTR)of IGF-I gene and its periparturient concentration in Iranian Holstein dairy cows.Methods:Blood samples(5 mL,n=37)were collected by caudal venipuncture from each animal into sample lubes containing the EDTA and DNA was extracted from blood.In order to measure ICF-I concentration the collection of blood samples(n=111)was also done at 14 d before calving(prepartum),25 and 45 d postpartum.Results:We found evidence for a significant effect of C to T mutation in position 512 of IGF-I gene on its serum concentration in dairy cows in Iran.Cows with CC genotype had significantly higher concentration(Mean-SD)of IGF-I at 14 d prepartum(91.8±18.1)μg/L compared to those with TT genotype(73.3±14.4)μg/L(P=0.04).A significant trend(quadratic)was found for IGF-I concentration,as higher in CC cows compared to ones with TT genotype,during the 14 d before calving to 45 d postpartum(P=0.01).Conclusions:We concluded that C/T transition in the promoter region of IGF-I gene can influence the serum concentration of ICF-I in periparturient dairy cows.

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.

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.

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.

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.

Relationship between Insuline-like Growth Factor-I and Progesterone Secretion of Cultured Human Trophoblast Cells in Vitro

Objective To investigate the effect of insuline-like growth factor-Ⅰ （IGF-Ⅰ） on progesterone genesis and regulation. Methods Cytotrophoblast cells were collected by trypsin-collagenase digestion and percoll gradient centrifugation for primary culture. After stimulated with different concentrations（100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml） of IGF-Ⅰ at the same time and with different duration（12 h,24 h,48 h, 72 h） of IGF-Ⅰ with the same concentration, progesterone levels in the media were measured by radioimmunoassay. Simultaneously, semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） was applied to determine the expression of low density lipoprotein receptor （LDLR） mRNA. Results Progesterone levels correlated positively with IGF-Ⅰ along with the IGF-Ⅰ concentration increasing, progesterone level began to increase at 12 h, and reached the climax at 48 h when cultured with 100 μg/L IGF-Ⅰ. The expression of LDLR mRNA was detectable in every group and accordant with variation of progesterone level. Conclusion Progesterone secretion has time- and dose-dependent effect on IGF-Ⅰ, and IGF-1 can up-regulate the expression of LDLR mRNA. IGF-Ⅰ may play an important role in promoting secretion of progesterone in trophoblast cells.

The expression of insulin-like growth factor-Ⅰ mRNA and polypeptide in rat osteoblasts with exposure to parathyroid hormone

Objective To investigate the insulin-like growth factor-Ⅰ (IGF-Ⅰ) gene and polypeptide expression in cultured rat osteoblast (ROB) and the role of IGF-Ⅰ in mediating the cell-to-cell communication by mimicking the pharmacokinetics of parathyroid hormone (PTH). Methods The ROB was cultured with three kinds of treatment: (1) Control (Ctr), the cells were cultured without PTH during the first 6 hours and the subsequent 42 hours in a 48-hour cycle; (2) Intermittent exposure to PTH (Itm), the cells were cultured with PTH during the first 6 hours, but without PTH in the subsequent 42 hours; and (3) Continuous exposure to PTH (Ctu), the cells were cultured with PTH during the first 6 hours and the subsequent 42 hours. Results The bone-forming activities of ROB were increased in Itm and inhibited in Ctu. The IGF-Ⅰ mRNA content in Itm cells was elevated only during the first 6 hours and that in Ctu cells was elevated at any time during an incubation cycle. The free IGF-Ⅰ concentration in the medium of Itm cells was generally higher and that of the Ctu cells was generally lower compared with those of the Ctr cells. The IGF-Ⅰ antibody significantly reduced the alkaline phosphatase activity within the cells of Ctr and Itm. Conclusions PTH rapidly and constantly stimulates the IGF-Ⅰ gene transcription of osteoblast. There was an obvious discrepancy between the IGF-Ⅰ mRNA content within the osteoblast and the free IGF-Ⅰ level around the osteoblast in either mode of PTH action. The IGF-Ⅰ might be important for osteoblast-osteoblast communication and bone-forming activity, not only in intermittent PTH administration, but also in the physiological functioning of osteoblasts.

Effects of insulin, insulin-like growth factor-I and -II on proliferation and intracellular signaling in endometrial carcinoma cells with different expression levels of insulin receptor isoform A

Background Hyperinsulinemia, insulin-like growth factor （IGF）-I and -Ⅱ （IGF-Ⅱ） are associated with increased risk of endometrial carcinoma. Insulin receptor isoform A （IR-A） is more frequently expressed in endombtrial carcinoma than in normal endometrial tissues. To better understand their roles in endometrial carcinoma, we investigated the effects of insulin, IGF-I, and IGF-II in endometrial carcinomas cells with different IR-A expression levels. Methods To explore the role of IR-A in mediating the activity of IGF-I, IGF-II, and insulin, we investigate the cellular proliferation of endometrial carcinoma cell lines RL95-2 and RL95-2-1R-A by MTS assays. Then we examined the protein kinase Akt phosphorylation and extracellular signal-regulated kinase （ERK） 1/2 phosphorylation in both cell lines by Western blotting. The effect of IGF-II and AG1024 on cell cycle progression and apoptosis was assessed by fiowcytometry. To examine whether the effects of IGFs were mediated by IR-A, we blocked IGF-I receptor （IGF-IR） in both cell lines using AG1024, an IGF-IR-specific inhibitor. Results IGF-I and IGF-II significantly enhanced proliferation of both cell lines （P 〈0.05）. By contrast, insulin significantly increased proliferation of RL95-2-1R-A cells only （P 〈0.05）. IGF-I and IGF-II significantly increased pAkt levels in RL95-2 cells and pERK1/2 levels in RL95-2-1R-A cells （all, P 〈0.05）. Insulin increased pERK1/2 levels in RL95-2-1R-A cells only （P 〈0.05）. LY294002 and PD98059 inhibited the specific signaling activities and cellular proliferation. After AG1024 pretreatment, neither IGF-I nor IGF-II affected pAkt levels in RL95-2 cells. IGF-II, but not IGF-I, increased pERK1/2 levels in RL95-2-1R-A cells. After AG1024 pretreatment, the proliferation rate and DNA content corresponding to the S phase increased and apoptosis decreased significantly in IGF-II-treated RL95-2-1R-A cells only （P 〈0.05）. Conclusions The proliferation effect of insulin is mediated by IR-A. When IR-A dominates in a cell line, IGF-II activated cell proliferation mainly through the ERKI/2 pathway. On the other hand, IGF-II activated cell proliferation mainly through the Akt pathway. IR-A can at least partly mediate the proliferative and anti-apoptotic effects of IGF-II through the ERKI/2 pathway.

肝癌和癌旁肝组织中IGF-Ⅰ,IGF-Ⅰ受体mRNA的表达

目的 探讨胰岛素样生长因子Ⅰ（ＩＧＦⅠ） 及其受体（ＩＧＦⅠＲ）在肝细胞癌变过程中的作用及意义．方法 采用ＤＮＡＲＮＡ 原位杂交方法观察肝癌（ ｎ ＝ ３０） 和癌旁肝组织中ＩＧＦⅠ，ＩＧＦⅠ受体ｍ ＲＮＡ 的表达．结果 在肝癌和癌旁肝组织中ＩＧＦⅠ的表达率分别为４０－０ ％和５０－０ ％ ，ＩＧＦⅠ受体的表达率分别为４６－７ ％ 和５３－３ ％ ；ＩＧＦⅠ，ＩＧＦⅠ受体在分化较差的癌细胞和不典型增生肝细胞中表达最为明显．结论 ＩＧＦⅠ和ＩＧＦⅠ受体可能在肝细胞癌变过程中发挥重要作用．

RNA干扰沉默IGF-Ⅰ R表达对非小细胞肺癌细胞生物学特性及化疗敏感性的影响

目的:评价RNA干扰(RNA interference,RNAi)技术,对人肺癌A549细胞系中胰岛素样生长因子类受体(IGF-Ⅰ R)表达的阻断效应,和IGF-Ⅰ R基因沉默后细胞增殖、凋亡等生物学特性及肿瘤细胞对化疗药物敏感性的改变。方法:应用U6启动子,介导DNA模板转录生成短发夹样RNA(shRNA),并转染人肺癌A549细胞株,从而产生IGF-Ⅰ R特异性小干扰RNA(siRNA),经RT-PCR和Western blot检测IGF-Ⅰ R表达的改变;联合应用化疗药物顺铂(DDP),通过MTT法和流式细胞技术等,观察细胞生长、细胞周期、细胞凋亡及DDP半数致死量(IC50)的变化。结果:IGF-Ⅰ R表达水平明显下降(抑制率高达89.8%),肿瘤细胞增殖能力明显减弱,细胞滞留于G0期的比例上升;DDP对肿瘤细胞24h、48h、72h的IC50均明显减少,IGF-Ⅰ R siRNA1组DDP作用72h的IC50为0.92 mg/L,明显低于control-siRNA组的3.77mg/L,0.5mg/L DDP联合IGF-Ⅰ R siRNA1作用48h后,A549细胞的生长抑制率达32.1%,明显高于control-siRNA组的18.9%,细胞凋亡率从27.8%上升至44.2%。结论:运用RNAi技术能有效抑制A549细胞IGF-Ⅰ R的表达,使细胞增殖能力减弱,化疗敏感性增加。

草原红牛胰岛素样生长因子Ⅰ受体基因3′UTR序列多态性分析

以58头草原红牛为试验材料,采用PCR-SSCP技术对胰岛素样生长因子Ⅰ受体(IGF-ⅠR)基因3′UTR序列多态性进行了检测。结果表明,在IGF-ⅠR基因3′UTR中发现PCR-SSCP的多态位点,有2种等位基因和3种基因型。对所得基因型与生长和胴体性状的相关性分析结果发现,在3′UTR中AB型的胴体产肉率、肉骨比显著高于AA、BB型(P<0.05);AA型的脂肪覆盖率显著高于BB型(P<0.05),与AB型差异不显著(P>0.05)。

胰岛素样生长因子Ⅰ、胰岛素样生长因子Ⅰ受体在大肠癌中的表达及其意义

目的检测胰岛素样生长因子Ⅰ(IGF-Ⅰ)、胰岛素样生长因子Ⅰ受体(IGF-ⅠR)蛋白在大肠癌中的表达,并分析与临床病理学因素的关系,探讨两者在大肠癌发病机制中的相互作用及与患者预后的关系。方法选取不同类型的大肠癌组织48例(试验组),其中男性25例,女性23例;年龄34～78岁,平均年龄53岁。任意选癌旁5cm以上的正常组织24例(正常对照组)。采用SABC法检测IGF-Ⅰ、IGF-ⅠR的表达。结果在48例大肠癌组织中,IGF-Ⅰ、IGF-ⅠR的阳性表达率分别为64.58%、58.33%,均显著高于癌旁正常组织中的16.67%、16.67%。IGF-Ⅰ、IGF-ⅠR在大肠癌组织中的表达与肿瘤的浸润程度、淋巴结转移、Dukes’分期有关,而与其他的病理因素及患者5年生存情况无关。IGF-Ⅰ和IGF-ⅠR在大肠癌组织中的表达呈明显相关性。结论IGF-Ⅰ、IGF-ⅠR可能相互协同作用参与大肠癌的浸润、转移,可作为反映大肠癌进展的重要生物学指标,但IGF-Ⅰ、IGF-ⅠR阳性表达与大肠癌患者的预后无明显相关性。