Research progress in correlation between insulin-like growth factor 2 mRNA binding protein 2 gene and type 2 diabetes mellitus / 中国生物制品学杂志

@#Type 2 diabetes mellitus(T2DM)was a chronic,non-communicable disease with a combination of multiple genetic and environmental factors,of which the main characteristics included insufficient insulin secretion and insulin resistance.Insulin-like growth factor 2 mRNA binding protein 2(IGF2BP2/IMP2),an important insulin secretion-related protein in human body,is mainly expressed in tissues and cells such as pancreas,fat and intestine.It has been confirmed that IGF2BP2 can down-regulate the expression of IGF2 and the function damage of the related islet β cell is an important cause of T2DM and vascular complications.Therefore,IGF2BP2 gene can be used as an important predictor for diabetes mellitus risk.This paper reviews the correlation between IGF2BP2 gene and T2DM.

Effects of chronic stress during pregnancy on depression-like behavior and methylation of imprinted gene IGF-2/H19 in female offspring rats / 中华行为医学与脑科学杂志

Objective:To investigate the effects of chronic stress during pregnancy on depressive behavior and DNA methylation of insulin-like growth factor-2 ( IGF-2 )/long non-coding RNA ( lncRNA ) H19 in hippocampus of female offspring rats.Methods:A total of 32 SPF female SD rats were divided into model group and control group according to the random number table. The rats in the model group were treated with chronic unpredictable mild stress (CUMS) to establish the depression model, and the rats in the control group were fed normally.On the 7th day of stress stimulation, all female rats mated with male rats. One day before stress stimulation and 1, 7, 14, 21, 28 days after stress stimulation, blood samples were collected from the inner canthus vein of the rats to determine the plasma corticosterone concentration. Eight female pups were randomly selected from each group on postnatal day 28(PND28) and postnatal day 42 (PND42). Plasma corticosterone concentration was measured after angular vein blood collection. At PND42, the depression-like behavior of female pups in the two groups was measured by sucrose preference test, tail suspension test and forced swimming test. The expression of IGF-2/H19 and related transferases in hippocampus of offspring rats was detected by RT-PCR and Western blot. Methyl target technology was used to capture and sequence 19 CpG sites of IGF-2 differentially methylated region(DMR) fragment 2, 8 CpG sites in H19 imprinting control region (ICR) fragment 1 and 15 CpG sites in H19-ICR fragment 2, and calculate the methylation level of each CpG site. SPSS 26.0 was used for statistical analysis of relevant data by repeated measurement ANOVA, t test and non-parametric test. Results:(1) The data of plasma corticosterone content of the two groups of female rats at different times were analyzed by repeated measurement variance.The results showed that the the interaction effect between time and group was not significant ( F=2.997, P=0.066), and the main effect of time was significant ( F=4.44, P=0.010). The main effect of group was significant ( F=41.40, P=0.001). According to the independent effect analysis of factors between groups, on the 14th, 21st, and 28th days of stress, the plasma corticosterone concentration of the model group was higher than that of the control group (all P<0.001). (2) In the sucrose preference test, the total liquid consumption (11.10(10.38, 11.58) mL, 13.55(12.00, 15.77) mL, Z=-3.055, P=0.002), 1% sucrose water consumption ((5.50±1.30) mL, (8.56±2.04) mL, t=-3.582, P=0.003) and 1% sucrose preference percentage ( (51.35±8.69) %, (62.11±8.05) %, t=-2.576, P=0.022) of female pups in the model group were significantly lower than those in the control group. (3) The duration of immobility in tail suspension test ((126.95±39.89) s, (54.30±25.00) s, t=4.375, P=0.001) and forced swimming test ((7.97±6.66) s, (1.85±2.12) s, t=2.478, P=0.037) of female offspring in the model group were longer than those in the control group. (4) The expression of IGF-2 mRNA ((0.46±0.24), (1.00±0.00), t=3.821, P=0.019) and H19 mRNA ((0.60±0.25), (1.00±0.00), t=3.574, P=0.007) in hippocampus of female pups in the model group were lower than those of control group. The relative expression of IGF-2 protein in female offspring of model group was lower than that in control group ((0.77±0.04), (1.00±0.00), t=9.876, P=0.01). The relative expression of CCTC-binding factor (CTCF) mRNA ((1.29±0.12), (1.00±0.00), t=-4.850, P=0.003) and protein ((1.90±0.28), (1.00±0.00), t=-5.513, P=0.005) were higher than those in the control group. (5) The methylation levels of three CpG sites in the IGF-2 DMR region of female offspring in the model group were lower than those in the control group ( t=-3.21, -3.00, -3.34, all P<0.05), located at chr1215831028, chr1215831055 and chr1215831205, respectively. The methylation level of IGF-2 DMR fragment was lower than that of the control group ( t=-3.453, P=0.048). The relative expression levels of DNMT3A mRNA ( t=5.102, P=0.002), DNMT3A ( t=10.213, P<0.001) and DNMT3B ( t=4.169, P=0.014) in female offspring of the model group were lower than those in the control group. Conclusion:Chronic stress during pregnancy causes depression and despair in female offspring mice, and the mechanism may be related to the decrease of methylation level of imprinted gene IGF-2 DMR caused by the decrease of methyltransferase expression.

Effects of p,p'-DDE on H19 DMR methylation and insulin secretion of INS-1 cells / 环境与职业医学

Background Diabetes is a major threat to public health across the world. Studies have shown that exposure to p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) is closely related to the occurrence of type 2 diabetes mellitus. However, the relevant molecular mechanism is not clear. Objective To investigate the effects of p,p'-DDE on H19 differentially methylated region (DMR) methylation and insulin secretion of rat insulinoma cells (INS-1 cells). Methods INS-1 cells were cultured with different concentrations (0, 3.125, 6.25, 12.5, 25, 50, and 75 µmol·L−1) of p,p'-DDE for 24 h, and the viability of INS-1 cells was detected by CCK-8 method. INS-1 cells were exposed to 0, 12.5, 25, and 50 µmol·L−1 p,p'-DDE for 24 h in subsequent experiments. The methylation levels of 24 CpG sites in H19 DMR were analyzed by bisulfite genomic sequencing. The expression levels of insulin-like growth factor 2 (IGF2) mRNA were detected by real-time quantitative PCR. The expression levels of IGF2 and insulin-like growth factor-1 receptor (IGF1R) proteins were detected by Western blotting. The insulin secretion function of INS-1 cells was determined by glucose-stimulatedinsulin secretion test (5 and 25 mmol·L−1 glucose, respectively). Results Compared with the control group, the viability of INS-1 cells increased significantly after treatment with 12.5 µmol·L−1 p,p'-DDE; however, it was significantly inhibited after treatment with 50 or 75 µmol·L−1 p,p'-DDE (P<0.01); therefore, 50 µmol·L−1 was chosen as the maximum concentration of exposure for subsequent experiments. The 25 µmol·L−1 p,p'-DDE treatment decreased the methylation levels of CpG18 and CpG22-CpG24 sites in H19 DMR, and the 50 µmol·L−1 p,p'-DDE treatment decreased the methylation levels of CpG10-CpG24 sites (P<0.05 or P<0.05). Multiple concentrations (12.5, 25, and 50 µmol·L−1) of p,p'-DDE down-regulated the mRNA and protein relative expression levels of IGF2 and the protein relative expression levels of IGF1R. The transcription level of IGF2 decreased to 67.8%, 68.6%, and 62.5% of the control group, the protein level of IGF2 decreased to 73.3%, 79.5%, and 80.9% of the control group, and the protein level of IGF1R decreased to 54.8%, 25.6%, and 12.9% of the control group, respectively (P<0.01). In the high glucose context, p,p'-DDE at selected concentrations inhibited the insulin secretion levels to 85.0%, 58.6%, and 49.5% of the control group, respectively (P<0.01). Conclusion p,p'-DDE could down-regulate methylation level of H19 DMR, interfere the IGF2/IGF1R signaling pathway, and inhibit insulin secretion of islet cells.

The mechanism of enriched environment repairing the learning and memory impairment in offspring of prenatal stress by regulating the expression of activity-regulated cytoskeletal-associated and insulin-like growth factor-2 in hippocampus

BACKGROUND@#Prenatal stress can cause neurobiological and behavioral defects in offspring; environmental factors play a crucial role in regulating the development of brain and behavioral; this study was designed to test and verify whether an enriched environment can repair learning and memory impairment in offspring rats induced by prenatal stress and to explore its mechanism involving the expression of insulin-like growth factor-2 (IGF-2) and activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus of the offspring.@*METHODS@#Rats were selected to establish a chronic unpredictable mild stress (CUMS) model during pregnancy. Offspring were weaned on 21st day and housed under either standard or an enriched environment. The learning and memory ability were tested using Morris water maze and Y-maze. The expression of IGF-2 and Arc mRNA and protein were respectively measured by using RT-PCR and Western blotting.@*RESULTS@#There was an elevation in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy. Maternal stress's offspring exposed to an enriched environment could decrease their plasma corticosterone level and improve their weight. The offspring of maternal stress during pregnancy exhibited abnormalities in Morris water maze and Y-maze, which were improved in an enriched environment. The expression of IGF-2, Arc mRNA, and protein in offspring of maternal stress during pregnancy was boosted and some relationships existed between these parameters after being exposed enriched environment.@*CONCLUSIONS@#The learning and memory impairment in offspring of prenatal stress can be rectified by the enriched environment, the mechanism of which is related to the decreasing plasma corticosterone and increasing hippocampal IGF-2 and Arc of offspring rats following maternal chronic stress during pregnancy.

Mechanism of insulin-like growth factor 2 regulating human ovarian granulosa cell proliferation through the phosphatidylinositol 3-kinase/ protein kinase B signaling pathway / 解剖学报

Objective To explore the effect and mechanism of insulin-like growth factor 2 ( IGF2 ) on the proliferation of human ovarian granulosa cells ( KGN ). Methods KGN cells cultured in vitro and treated with different concentrations of IGF2 were divided into control group and IGF2 group (25 μg/L, 50 μg/L, 100 μg/L), and then cells were divided into control group, 100 μg/L IGF2 group, LY294002 group, and IGF2 +LY294002 group after intervened the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway with LY294002. MTS and 5-ethynyl-2'- deoxyuridine (EdU) method was used to detect the effect of IGF2 on KGN cell proliferation, and enzyme linked immunosorbent assay was used to detect the contents of estrogen and progesterone in cell culture supernatant. The expressions of insulin like growth factor 1 receptor (IGF1R), protein kinase B (Akt), phosphorylated protein kinase B(p-Akt) and CYP19A1 protein in each group were detected by Western blotting. Results With the concentration gradient of IGF2, the proliferation rate of KGN cells and the secretion of estrogen and progesterone gradually increased. The cell proliferation rate and hormone level in the group treated with lOOfig/L IGF2 were the highest (P<0.01), while the PI3K/Akt signaling pathway was inhibited, and the cell proliferation rate and hormone secretion decreased significantly (P<0.01). The protein expression levels of IGF1R, p-Akt and CYP19A1 in different concentration groups increased significantly (P<0.05). While the expression of the above proteins were affected by intervened the PI3K/Akt signaling pathway. Compared with the control group, the protein expression of IGF1R and p-Akt increased significantly in IGF2 group and IGF2 +LY294002 group(P<0.01), CYP19A1 increased significantly in IGF2 group(P<0.01), the protein expression of p-Akt and CYP19A1 decreased significantly in LY294002 group (P<0.05), there was no significant difference in the protein expression of IGF 1R. Compared with the IGF2 group, the protein expression of p-Akt and CYP19A1 decreased in IGF2 +LY294002 group (P<0.01), there was no statistically significant difference in the protein expression of IGF1R, and the expression levels of IGF1R, p-Akt and CYP19A1 were significantly reduced in LY294002 group (P<0.01). Conclusion IGF2 may promote the proliferation and secretion of human ovarian granulosa cells through the PI3K/Akt signaling pathway mediated by IGF1R.

Síndrome de Doege Potter / Doege-Potter syndrome

Resumen El síndrome de Doege Potter es un síndrome caracterizado por hipoglucemia severa asociada a tumor fibroso de la pleura. Presentamos el caso de una paciente de 67 años con antecedente por biopsia de tumor fibroso de pleura, quien seis meses después de este diagnóstico presenta episodios de alteración del estado de conciencia con desorientación y documentación de hipoglucemia con triada de Whipple presente. Se realiza test de ayuno el cual es positivo para hipoglucemia no hiperinsulinémica y dado sus antecedentes, se hace el diagnóstico de un síndrome de Doege Potter. Se realiza manejo quirúrgico con resección total de masa tumoral con posterior resolución de la hipoglucemia.(Acta Med Colomb 2020; 45. DOI:https://doi.org/10.36104/amc.2020.1503).

Abstract Doege-Potter syndrome is characterized by severe hypoglycemia associated with a fibrous tumor of the pleura. We present the case of a 67-year-old patient with a history of a fibrous tumor of the pleura, diagnosed through biopsy, who six months after this diagnosis experienced episodes of altered consciousness with disorientation, and documented hypoglycemia with Whipple's triad. A fasting test was positive for non-hyperinsulinemic hypoglycemia and, given his history, he was diagnosed with Doege-Potter syndrome. He was treated surgically through total removal of the tumor mass, with subsequent resolution of the symptoms.(Acta Med Colomb 2020; 45. DOI:https://doi.org/10.36104/amc.2020.1503).

The relation between of insulin like growth factor II and endometrial carcinoma

Background: Endometrial cancer is the most common gynecologic malignancy in the United States and the fourth most common cancer in women. The need of a soft marker that can be used with CA-125 tumor marker for early detection of endometrial cancer and to predict late stages and advanced histopathological grades and to specify the cases who will be managed by complete surgical staging including para aortic and pelvic lymphadenectomy is of great importance. The aim of the study was to evaluate the role of insulin like growth factor 2 in endometrial carcinoma and to correlate it with different histopathological grades of the disease.Methods: This study was applied on sixty patients with abnormal uterine bleeding and were divided into two groups, Group A included 30 cases of endometrial carcinoma, while Group B included 30 cases complaining of abnormal vaginal bleeding due to other causes as a control group. Serum samples were taken from all patients and estimation of IGF-2 serum levels using ElISA technique was done. Comparison of IGF-2 serum level between both groups and correlation of its levels with different histopathological grades of endometrial cancer group were done.Results: As regard comparison between both groups and ILGF2 serum level, study results demonstrated that ILGF2 levels ranged between 600.0-1440.0 ng/ml and 40.0-560.0 ng/ml with the mean of 781.33 ng/ml±196.45 and 336.0 ng/ml±212.86 for cases Group A and control Group B respectively. There was a statistically significant difference between the two studied groups regarding ILGF2 serum level (p<0.001). As regards correlation between histopathological grades and ILGF-2 serum level in cases Group A, the study revealed a strong positive correlation.Conclusions: ILGF-2 can be used as a serum marker for endometroid adenocarcinoma of the body of the uterus and to predict its higher histopathological grades.

Dynamic expression of insulin￣like growth factor 2 during critical period of visual development in lateral geniculate body of mice / 解剖学报

Objective To detect the dynamic expression of insulin-like growth factor 2 (IGF2) in lateral geniculate body (LGB) during the critical period of visual development. Methods Three groups of Kunming mice of different ages were selected for testing, which were 3 weeks old, 5 weeks old and 7 weeks old, twelve in each group. The forepaw-reaching reflex test was used to detect whether the visual function of the mice was normal in each group. Immunohistochemical technique was used to detect the expression of IGF2 protein and its receptor in the lateral geniculate body of normal mice at week 3, 5 and 7 postnatal, and to analyze the expression of the protein of IGF2 and its receptor in each part of the lateral geniculate body. Results The expression of IGF2 protein in the dorsal lateral geniculate nucleus decreased significantly at week 5 postnatal and increased significantly at week 7 postnatal, and increased gradually over time at week 5 and week 7 postnatal in the ventral geniculate nucleus. The expression of IGF2 receptor protein in the dorsal lateral geniculate nucleus and ventral nucleus increased significantly at week 5 postnatal, and at week 7 postnatal, the expression of IGF2 receptor decreased to week 3 level in lateral geniculate body of mice. Conclusion The expression of IGF2 and its receptor in lateral geniculate body of mice during critical period of visual development changed dynamically, and the expression patterns of IGF2 and its receptor in different parts of LGB were not completely consistent. The expression of IGF2 and its receptors may be related to the plasticity of visual development in mice.

In vitro immune response of insulin-like growth factor 2 mRNA-binding protein 3 restricted cytotoxic T lymphocyte epitope from lung cancer immunotherapy target / 解剖学报

Objective Cancer immunotherapy is attractive for antigen-specific T cell-mediated anti-tumor therapy, especially in induction of cytotoxic T lymphocytes(CTL). In this report, we evaluated insulin-like growth factor 2 mRNA-binding protein 3(IGF2P3) restricted epitope-induced cytotoxic T lymphocytes effects in human lung cancer cells. Methods The human leukocyte antigen A2(HLA-A2) restricted epitope peptides of IGF2P3 were selected by NetCTL 1. 2, SYFPEITHI and IEDB software prediction. The binding affinity of the peptides to HLA-A02 molecules was evaluated by T2A2 cells binding assay. enzyme-linked immunosport(ELISPOT) assay was used to investigate the ability of the peptide to induce specific restricted cytotoxic T lymphocytes (CTL) and release of interferon γ(IFN-γ). The ability of the peptides to induce T cell response was investigated by carboxyfluorescein succinimidyl amino ester (CFSE) fluorescent staining. Results The candidate peptide P143, P199, P280, P409 and P515 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P409, P515 were able to induce specific CTL and higher levels of IFN-γ were released. The CTL induced by P409, P515 lysed target cells. Conclusion P409 and P515 have the potential for adoptive immunotherapy and can be used as candidate epitopes for new anti-tumor polypeptide immunotherapy vaccine. P409 and P515 can be used in peptide-based immunotherapy for lung cancer.

A preliminary study on abnormal expression of long noncoding RNA CTD-2012K14.6 in placentas and its role in inducing macrosomia / 中华内分泌代谢杂志

Objective To explore the role of long noncoding RNA ( lncRNA) CTD-2012K14. 6 in the development of gestational diabetes mellitus (GDM) related macrosomia. Methods The quantitative real-time PCR ( qRT-PCR) was performed to measure the expression of CTD-2012K14.6 in placentas of women with or without GDM, and the quantity of CTD-2012K14. 6 expression and its association with fetal weights were analyzed; Bioinformatic analysis was performed to predict the downstream molecules. CTD-2012K14. 6 over-expressing lentiviral and siRNA was constructed in human trophoblastic cell line HTR-8/SVneo cells, qRT-PCR and Western blot (WB) were used to invest its effect in modulating the expression of downstream molecules. Results The expression of CTD-2012K14.6 in GDM placentas was significantly higher than that in normal controls (1.70 ± 0.63 vs 1.00 ± 0.56,t=3.68,P＜0.01), and positively correlated with fetal weight (r=0.8501, P＜0.01); on-line analysis showed that CTD-2012K14.6 was located at chr16:67,549,214-67,563,958, which was located in the intron of CCCTC-binding factor( CTCF); Up-regulating CTD-2012K14.6 could significantly reduce the expression of CTCF mRNA and protein, and increase the expression of insulin-like growth factor-Ⅱ( IGF-Ⅱ) mRNA and protein, while down-regulating CTD-2012K14.6 could significantly increase the expression of CTCF mRNA and protein, and reduce the expression of IGF-ⅡmRNA and protein. Conclusion The CTD-2012K14. 6 may play an important role in the pathogenesis of GDM related macrosomia by upregulating the expression of CTCF and IGF-Ⅱ.

Effects of down-regulation of IGF2 gene on the biological characteristics of HCT116 colon cancer stem cells / 中华肿瘤杂志

Objective@#To investigate the effect of down-regulation of insulin-like growth factor 2 (IGF2) gene on the biological characteristics of HCT116 colon cancer stem cells (CSCs).@*Methods@#Flow cytometry sorting technology was used to isolate CSCs from colon cancer cell line HCT116 by a monoclonal antibody against CD133; serum free floating culture assay was used for the enrichment of CSCs. The proportion of CD133+ cells was analyzed by flow cytometry; CSCs were identified by sphere culturing, immunofluorescence analysis and soft agar clone formation. RT-qPCR method was used to examine transcriptional level of IGF2 gene in CSCs. Western blotting was used to examine IGF2 protein expression in CSCs. siRNA was used to establish IGF2 transient knock down model in CSCs. Cell proliferation array, cell cycle and apoptosis analysis, cell invasion array and colony forming assay were used to further examine the role of IGF2 on the biological characteristics of colon CSCs.@*Results@#CSCs were successfully isolated from HCT116 cell lines, which were cultured to form cell spheres in serum-free stem cell culture medium. We found that the morphology of sphere-forming-like cells after several passages maintained the same characteristics as that of the first passage. The results of immunofluorescence showed that CSC markers including CD133 and ALDH continued positively expressing on the cell surface of CSCs, and flow cytometry analysis showed that more than 90% of the spheroid cells remained CD133 positive. The clone formation rate of non-CSCs group and CSCs group were (28.10±2.66)% and (43.73±2.30)% respectively, with significant difference (P<0.01). The RT-qPCR results showed that the transcriptional level IGF2 gene in non-CSCs group and CSCs group were (1.06±0.24) and (2.17±0.51) respectively, with significant difference (P<0.05). The western blot results showed that the protein expression of IGF2 in CSCs group and non-CSCs group were (1.10±0.55) and (2.14±0.23) respectively, with significant difference (P<0.05). Knockdown of IGF2 significantly decreased the percentage of CD133+ cells in CSCs and cell proliferation (P<0.01). Knockdown of IGF2 increased the percentage of G2/M phase (23.46% of siNC group vs 60.14% of siIGF2 group) and cell apoptosis (2.80% of siNC group vs 40.70% of siIGF2 group), while decreased the percentage of G0/G1 phase (40.77% of siNC group vs 17.73% of siIGF2 group). The invasion results showed that the number of cells penetrating into the basement surface in siNC group and siIGF2 group was (109.00±16.37) and (54.00±8.19) respectively, with significant difference (P<0.01). The rate of sphere-forming of colon CSCs in siNC group and siIGF2 group were (51.70±7.42)% and (21.27±2.35)% respectively, with significant difference (P<0.01). The clone formation rate of siNC group and siIGF2 group were (37.20±3.87)% and (18.23±2.25)% respectively, with significant difference (P<0.01).@*Conclusion@#IGF2 gene plays an important role in maintaining the biological characteristics of colon cancer stem cells and promoting self-renewal and stemness of colon CSCs.

miR-483-5p promotes growth, migration and invasion of hepatocellular carcinoma cells by up-regulating IGF2 gene transcription / 中国病理生理杂志

AIM:To investigate the effect of miR-483-5p on P3 promoter-driven mRNA(P3 mRNA)expres-sion of human insulin-like growth factor 2(IGF2)gene and its role in the development of hepatocellular carcinoma (HCC).METHODS: The expression levels of miR-483-5p and P3 mRNA were analyzed by real-time PCR in human HCC cell lines Huh7,Hep3B,Bel-7402,HepG2 and SMMC-7721,normal human liver cell line HL-7702,83 cases of hu-man HCC tissues and their matched adjacent nontumorous tissues(MANT), and 22 cases of normal adult liver tissues (NALT).The association between P3 mRNA level and miR-483-5p level was evaluated by Pearson correlation analysis. The full-length sequences of 5'UTR of P3 mRNA containing wild-type and mutant miR-483-5p-binding sequences were cloned into pGL3 promoter vector, which were termed pGL3-P3-5'UTR-WT and pGL3-P3-5'UTR-MUT, respectively. These luciferase reporter constructs were transfected into HeLa,293T and Huh7 cells together with miR-483-5p mimic, miR-483-5p inhibitor or scrambled control,and the luciferase activity was measured using dual-luciferase reporter system. The miR-483-5p mimic,miR-483 inhibitor and scrambled control were also transfected into Huh 7 cells and Hep3B cells, and P3 mRNA level was detected by real-time PCR.The expression levels of miR-483-5p in the nuclear and cytoplasmic fractions of Hep3B cells and Huh7 cells were detected by real-time PCR.The effect of miR-483-5p on P3 mRNA transcrip-tion was evaluated by nuclear run-on assay.The effect of miR-483-5p on the stability of P3 mRNA was analyzed by RNA stability assay.Furthermore,the effects of miR-483-5p on the viability, apoptosis, migration and invasion of Huh7 cells were investigated.RESULTS:Significamtly high levels of miR-483-5p and P3 mRNA were detected in the 5 human HCC cell lines and the human HCC tissues as compared with the human normal liver cell line HL-7702, and the MANT and NALT,respectively.Linear correlation analysis revealed that P 3 mRNA level was positively correlated to miR-483-5p level in the 5 human HCC cell lines and the human HCC tissues.miR-483-5p directly recognized the P3 mRNA 5'UTR to pro-mote gene expression.Overexpression of miR-483-5p resulted in a significant increase in P3 mRNA expression in a dose-dependent manner in the Huh7 cells and Hep3B cells.The mature miR-483-5p was present in both cytoplasm and nucleus of Hep3B cells and Huh7 cells.miR-483-5p induced nascent P3 mRNA transcription in the nucleus of Huh7 cells.miR-483-5p did not alter P3 mRNA stability in Huh7 cells.Furthermore,miR-483-5p led to increased viability,apoptosis inhi-bition,and enhanced migration and invasion abilities in the Huh 7 cells.CONCLUSION:High expression of miR-483-5p promotes the growth,migration and invasion of HCC cells in part through up-regulating P3 mRNA transcription,and is con-sequently involved in the development of HCC.

Neuroprotective effects and mechanisms of Cerebroprotein Hydrolysate for Injection (Ⅰ) on vascular dementia in rats / 药物评价研究

Objective To investigate the neuroprotective mechanisms of Cerebroprotein Hydrolysate for Injection (Ⅰ) on vascular dementia in rats.Method The rat vascular dementia model was prepared using an improved two-vessel occlusion method,and the common carotid artery was only isolated but not blocked in sham group.Rats were randomly divided into sham group,model group,Cerebroprotein Hydrolysate for Injection (Ⅰ) groups with low,medium and high dose (5,10,20 mg/kg) and Cerebroprotein Hydrolysate Injection group (Cerebrolysin,Positive drug,10 mg/kg).The drug was administered by iv injection of rat tail vein once a day for two weeks,while the same volume of saline was administered in sham and model group.At the end of administration,the plasma was collected through abdominal aorta to separate serum,and rat cortex was isolated to prepare homogenate.The levels of nerve growth factor (NGF) and insulin-like growth factor 2 (IGF-2) in serum and level of gamma-aminobutyric acid (GABA) in cortex were detected by ELISA.Level of glutamate (Glu) in cortex of VaD rats was detected by colorimetry.Results Compared with model group,levels of NGF and IGF-2 in the serum of VaD rats and level of GABA in cortex were significantly increased,while level of Glu in cortex was significantly decreased after administration of Cerebroprotein Hydrolysate for Injection (Ⅰ).The increased IGF-2 and GABA levels by Cerebroprotein Hydrolysate for Injection (Ⅰ) were significantly higher than that of Cerebrolysin at same dose.Conclusion The mechanisms underlying the increased leaming and memory ability of VaD rats by Cerebroprotein Hydrolysate for Injection (Ⅰ),are possibly related to the increased levels of NGF and IGF-2 in body and a regulation of the balance between excitatory and inhibitory amino acid neurotransmitters.

Neuroprotective effects and mechanisms of Cerebroprotein Hydrolysate for Injection (Ⅰ) on vascular dementia in rats / 药物评价研究

Objective To investigate the neuroprotective mechanisms of Cerebroprotein Hydrolysate for Injection (Ⅰ) on vascular dementia in rats.Method The rat vascular dementia model was prepared using an improved two-vessel occlusion method,and the common carotid artery was only isolated but not blocked in sham group.Rats were randomly divided into sham group,model group,Cerebroprotein Hydrolysate for Injection (Ⅰ) groups with low,medium and high dose (5,10,20 mg/kg) and Cerebroprotein Hydrolysate Injection group (Cerebrolysin,Positive drug,10 mg/kg).The drug was administered by iv injection of rat tail vein once a day for two weeks,while the same volume of saline was administered in sham and model group.At the end of administration,the plasma was collected through abdominal aorta to separate serum,and rat cortex was isolated to prepare homogenate.The levels of nerve growth factor (NGF) and insulin-like growth factor 2 (IGF-2) in serum and level of gamma-aminobutyric acid (GABA) in cortex were detected by ELISA.Level of glutamate (Glu) in cortex of VaD rats was detected by colorimetry.Results Compared with model group,levels of NGF and IGF-2 in the serum of VaD rats and level of GABA in cortex were significantly increased,while level of Glu in cortex was significantly decreased after administration of Cerebroprotein Hydrolysate for Injection (Ⅰ).The increased IGF-2 and GABA levels by Cerebroprotein Hydrolysate for Injection (Ⅰ) were significantly higher than that of Cerebrolysin at same dose.Conclusion The mechanisms underlying the increased leaming and memory ability of VaD rats by Cerebroprotein Hydrolysate for Injection (Ⅰ),are possibly related to the increased levels of NGF and IGF-2 in body and a regulation of the balance between excitatory and inhibitory amino acid neurotransmitters.

The relationship between serum insulin-like growth factor-2 levels and clinical characteristics in patients with schizophrenia / 中国神经精神疾病杂志

Objective To explore the change of serum insulin-like growth factor-2 (IGF-2) and its relationship with clinical characteristics in patients with schizophrenia. Methods Fifty-one schizophrenic patients were recruited in the present study and 50 healthy volunteers served as controls. The serum IGF-2 level was measured using enzyme linked immunosorbent assay (ELISA). Positive and Negative Syndrome Scale (PANSS) was used to evaluate the psychotic symp?toms of patients. Trail Making Test-A (TMTA), Digit-Symbol Coding Test (DSCT), Continuous Performance Test (CPT) and Stroop Color-Word Test (SCWT) were used to evaluate the cognitive function of both groups. Results There were sig?nificant differences in the results of TMTA, DSCT, CPT and SCWT between patient and control groups. The serum levels of IGF-2 were significantly lower in patients than that in controls [(202.7±40.7) ng/mL vs. (365.9±65.5) ng/mL, P0.05). Furthermore, significant correlations were found between the serum IGF-2 level and the negative symptom sub?scale of PANSS (r=-0.397, P=0.004), CPT score (r=0.378, P=0.006), SCWT-word number (r=0.289, P=0.040), SC? WT-color number (r=0.327, P=0.019) and SCWT-word/color number (r=0.386, P=0.005) in schizophrenic patients. Con?clusion The serum IGF-2 levels of patients with schizophrenia are significantly lower than that of healthy controls, and the IGF-2 level is associated with the severity of negative symptoms and cognitive impairments in patients, indicating that serum IGF-2 might be an indicator of the severity of schizophrenia.

Inhibitory Effect of siRNA Expression Vector Inhibiting IGF2 Gene on the Proliferation of Hepatoma Cell Line Huh-7 / 中国药房

OBJECTIVE:To investigate the inhibitory effect of siRNA expression vector inhibiting human insulin-like growth factor 2(IGF2)gene on the proliferation of hepatoma cell line Huh-7. METHODS:siRNA expression vector pGL3-hAFP-hTERT-siRNA3(“siRNA3”)which inhibited IGF2 gene by dual promoter regulation of recombinant human alpha-foetoprotein(hAFP)and human telomerase reverse transcriptase(hTERT)was transfected into the Huh-7 cell and normal hepatocyte L-02,and then a nega-tive control group(vector pGL3-hAFP-hTERT)and a blank control group were set up. IGF2 mRNA expression was detected by re-al-time fluorescent quantitative polymerase chain reaction 48 h after transfection into the cells in all groups;the activity of the cells by the microplate reader 0,24,48 and 72 h thereafter;and the cell cycle and apoptosis by the flow cytometer 48 h thereafter,and the changes in the protein levels of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in the cell were detected by Western blot. RESULTS:Compared with the negative control group and blank control group,IGF2 mRNA expression in the Huh-7 cell transfected with siRNA3 was obviously weaker;at 48 and 72 h after transfection,the activity of Huh-7 cell signigicantly reduced, Huh-7 cells at G1 phase obviously increased and those at S phase markedly decreased;the occurrence of early,late and total apopto-sis in Huh-7 cells apparently increased,and the protein expression of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in cells significantly weakened,with statistically significance(P0.05). CONCLUSIONS:siRNA which inhibited IGF2 gene by dual promoter regulation of recombinant hAFP and hTERT can specially inhibit IGF2 gene expression and the prolifer-ation of Huh-7 cells,which may be involved with down-regulated protein expression of cell proliferation-associated gene PCNA, cell cycle control-associated genes Cyclin E2,Cyclin D2 and Cdc2 and apoptosis regulation-associated gene Bcl-2 as a result of down-regulated IGF2 mRNA expression and protein expres-sion.

Serum IGFBP4 concentration decreased in dairy heifers towards day 18 of pregnancy

This study was conducted to determine if the main components of the somatotropic axis change during the early phase of pregnancy in the maternal blood system and whether differences exist on day 18 after pregnancy recognition by the maternal organism. Blood samples of pregnant heifers (Holstein Friesian; n = 10 after embryo transfer) were obtained on the day of ovulation (day 0), as well as on days 7, 14, 16 and 18 and during pregnant, non-pregnant and negative control cycles. The oncentrations of progesterone (P4), oestrogen, growth hormone (GH), insulin-like growth factor-1 and -2 (IGF1, -2) and IGF-binding protein-2, -3 and -4 (IGFBP2, -3, -4) were measured. The mRNA expressions of growth hormone receptor 1A, IGF1, IGF2, IGFBP2, IGFBP3 and IGFBP4 were detected using RT-qPCR in liver biopsy specimens (day 18). In all groups, total serum IGF1 decreased from day 0 to 16. Notably, IGFBP4 maternal blood concentrations were lower during pregnancy than during non-pregnant cycles and synchronized control cycles. It can be speculated that the lower IGFBP4 in maternal blood may result in an increase of free IGF1 for local action. Further studies regarding IGFBP4 concentration and healthy early pregnancy are warranted.

Bioinformatic analysis of the polymorphism and function of IGF2R gene 3′untranslated region / 医学研究生学报

Objective Genetic variants in microRNA (miRNA) binding regions of the gene 3′untranslated region (3′UTR) can affect the regulation of gene expression .The aim of this study was to predict insulin like growth factor 2 receptor (IGF2R) 3′UTR variants and to test their effects on IGF2R gene expression by bioinformatic analysis . Methods Single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) in the IGF2R gene 3′UTR were obtained from online databases .The frequency distribution of all the selected IGF2R 3′UTR variants genotypes among the different populations and the linkage disequilibrium ( LD) values of all SNPs were calculated .Additionally , the potential miRNA binding sites were also predicted with the help of online bioinformatic tool . Finally, correlation analysis of the mRNA expression of IGF 2R genotype and different variant genotypes in the lymphoblastoid cell lines was performed. Results In total, 33 SNPs were reported in the 3′UTR, of which only five SNPs (rs8191959, rs200237825, rs3832385, rs201568808, rs1050015) had available minor allele frequency (MAF) values ( >0.05).And only the effect of rs1050015 variant on IGF2R mRNA expression level had significant difference (P=0.010). Conclusion The expression of IGF2R gene can be up-regulated by rs10500105 variant in the 3′UTR, which might support its use as markers of cancer risk and individualized treatment.

IGF2 expression in blood is not associated with its imprinting status in healthy pregnant Chinese women

Loss of Imprinting (LOI) of IGF2 and over-expressed IGF2 are associated with tumorigenesis. Our previous epidemiological study found a relatively high frequency of IGF2 LOI in healthy mid-gestation pregnant women. The aim of this study is to determine whether the expression of IGF2 is associated with its imprinting status in healthy Chinese pregnant women. The IGF2 imprinting status of 300 pregnant women was analyzed. 20 cases of IGF2 LOI and 20 cases of IGF2 retention of imprinting (ROI) were selected randomly for IGF2 expression analysis. The expression pattern of IGF2 between the group with IGF2 ROI and group with IGF2 LOI in healthy Chinese pregnant women was evaluated by real time PCR and western blot. The result showed no significant differences between IGF2 ROI and LOI groups in mRNA and protein levels. These results imply that IGF2 imprinting status has no obvious impact on its expression. There may be some unknown important factors other than imprinting status driving IGF2 expression.

The insulin polymorphism -23Hph increases the risk for type 1 diabetes mellitus in the Romanian population

The insulin -23Hph and IGF2 Apa polymorphisms were genotyped in Romanian patients with T1DM (n = 204), T2DM (n = 215) or obesity (n = 200) and normoponderal healthy subjects (n = 750). The genotypes of both polymorphisms were distributed in concordance with Hardy-Weinberg equilibrium in all groups. The -23Hph AA genotype increased the risk for T1DM (OR: 3.22, 95 percentCI: 2.09-4.98, p < 0,0001), especially in patients without macroalbuminuria (OR: 4.32, 95 percentCI: 2.54-7.45, p < 0,0001). No other significant association between the alleles or genotypes of insulin -23Hph and IGF2 Apa and diabetes or obesity was identified.