IGFBP-3 mRNA Research Articles

The low doses of SWCNTs affect the expression of proliferation and apoptosis related genes in normal human astrocytes

The unique properties of single-walled carbon nanotubes (SWCNTs) make them viable candidates for versatile implementation in the biomedical devices. They are able to cross the blood–brain barrier, enter cells and accumulate in cell nuclei. We studied the effect of these carbon nanoparticles on the expression of genes associated with endoplasmic reticulum stress and proliferation, cell viability and cancerogenesis as well as microRNAs in normal human astrocytes. We have shown that treatment of normal human astrocytes by small doses of SWCNTs (2 and 8 ng/ml of medium for 24 hrs) affect the expression of DNAJB9, IGFBP3, IGFBP6, CLU, ZNF395, KRT18, GJA1, HILPDA, and MEST mRNAs as well as several miRNAs, which have binding sites at 3′-UTR of these mRNAs. These changes in the expression profile of individual mRNAs introduced by SWCNTs are dissimilar in magnitude and direction and are the result of both transcriptional and posttranscriptional mechanisms of regulation. It is possible that these changes in gene expressions are mediated by the endoplasmic reticulum stress introduced by carbon nanotubes and reflect the disturbance of the genome stability. In conclusion, the low doses of SWCNTs disrupt the functional integrity of the genome and possibly exhibit a genotoxic effect.

The Prognostic Values of the Insulin-Like Growth Factor Binding Protein Family in Ovarian Cancer.

Purpose To assess the expression of insulin-like growth factor binding protein (IGFBP) family and its prognostic impact in ovarian cancer (OC) patients. Materials and Methods The mRNA expression and protein expression of individual IGFBPs in healthy ovarian samples and OC tissues were explored through Oncomine, Gene Expression Profiling Interactive Analysis, and Human Protein Atlas database. Additionally, the prognostic values of the six IGFBP members in patients with OC were evaluated by Kaplan-Meier plotter. Results IGFBP2 and IGFBP4 mRNA expression were remarkably upregulated in patients with OC. To be specific, the mRNA expression of IGFBP2 was upregulated in patients with serous ovarian cancer (SOC), while IGFBP1/3/4/5/6 mRNA levels were downregulated. In addition, the IGFBP4 protein expression was upregulated in SOC, and the IGFBP6 protein expression was upregulated in both of SOC and endometrioid ovarian cancer (EOC) tissues. High IGFBP1 mRNA levels showed favorable overall survival (OS) and progression-free survival (PFS) in all OC. Meanwhile, increased IGFBP5/6 mRNA levels revealed worsen OS and PFS in all OC patients. IGFBP4/6 mRNA levels predicted unfavorable OS and PFS only in SOC patients. Moreover, the aberrant mRNA expression of IGFBP1/2/4/5/6 was correlated with significantly prognosis in patients receiving different chemotherapeutic regimens. Conclusion This study indicates that the IGFBP family reveals distinct prognosis in patients with OC. IGFBP1/2/4/5/6 are useful prognostic predictors for chemotherapeutic effect in OC patients, and IGFBP2/4 are potential tumor markers for the diagnosis of OC.

Impact of Long-Term HFD Intake on the Peripheral and Central IGF System in Male and Female Mice.

The insulin-like growth factor (IGF) system is responsible for growth, but also affects metabolism and brain function throughout life. New IGF family members (i.e., pappalysins and stanniocalcins) control the availability/activity of IGFs and are implicated in growth. However, how diet and obesity modify this system has been poorly studied. We explored how intake of a high-fat diet (HFD) or commercial control diet (CCD) affects the IGF system in the circulation, visceral adipose tissue (VAT) and hypothalamus. Male and female C57/BL6J mice received HFD (60% fat, 5.1 kcal/g), CCD (10% fat, 3.7 kcal/g) or chow (3.1 % fat, 3.4 kcal/g) for 8 weeks. After 7 weeks of HFD intake, males had decreased glucose tolerance (p < 0.01) and at sacrifice increased plasma insulin (p < 0.05) and leptin (p < 0.01). Circulating free IGF1 (p < 0.001), total IGF1 (p < 0.001), IGF2 (p < 0.05) and IGFBP3 (p < 0.01) were higher after HFD in both sexes, with CCD increasing IGFBP2 in males (p < 0.001). In VAT, HFD reduced mRNA levels of IGF2 (p < 0.05), PAPP-A (p < 0.001) and stanniocalcin (STC)-1 (p < 0.001) in males. HFD increased hypothalamic IGF1 (p < 0.01), IGF2 (p < 0.05) and IGFBP5 (p < 0.01) mRNA levels, with these changes more apparent in females. Our results show that diet-induced changes in the IGF system are tissue-, sex- and diet-dependent.

Relationship of IGF-1 and IGF-Binding Proteins to Disease Severity and Glycemia in Nonalcoholic Fatty Liver Disease.

Growth hormone (GH) and IGF-1 help regulate hepatic glucose and lipid metabolism, and reductions in these hormones may contribute to development of nonalcoholic fatty liver disease (NAFLD). To assess relationships between hepatic expression of IGF1 and IGF-binding proteins (IGFBPs) and measures of glycemia and liver disease in adults with NAFLD. Secondarily to assess effects of GH-releasing hormone (GHRH) on circulating IGFBPs. Analysis of data from a randomized clinical trial of GHRH. Two US academic medical centers. Participants were 61 men and women 18 to 70 years of age with HIV-infection, ≥5% hepatic fat fraction, including 39 with RNA-Seq data from liver biopsy. Hepatic steatosis, inflammation, and fibrosis by histopathology and measures of glucose homeostasis. Hepatic IGF1 mRNA was significantly lower in individuals with higher steatosis and NAFLD Activity Score (NAS) and was inversely related to glucose parameters, independent of circulating IGF-1. Among the IGFBPs, IGFBP2 and IGFBP4 were lower and IGFBP6 and IGFBP7 (also known as IGFBP-related protein 1) were higher with increasing steatosis. Hepatic IGFBP6 and IGFBP7 mRNA levels were positively associated with NAS. IGFBP7 mRNA increased with increasing fibrosis. Hepatic IGFBP1 mRNA was inversely associated with glycemia and insulin resistance, with opposite relationships present for IGFBP3 and IGFBP7. GHRH increased circulating IGFBP-1 and IGFBP-3, but decreased IGFBP-2 and IGFBP-6. These data demonstrate novel relationships of IGF-1 and IGFBPs with NAFLD severity and glucose control, with divergent roles seen for different IGFBPs. Moreover, the data provide new information on the complex effects of GHRH on IGFBPs.

Characterization of Insulin-Like Growth Factor Binding Protein 7 (Igfbp7) and Its Potential Involvement in Shell Formation and Metamorphosis of Pacific Abalone, Haliotis discus hannai.

Insulin-like growth factor binding proteins (IGFBPs) are secreted proteins that play an important role in IGF regulation of growth and development of vertebrate and invertebrates. In this study, the IGFBP7 gene was cloned and characterized from mantle tissues of H. discus hannai, and designated as Hdh IGFBP7. The full-length cDNA sequence transcribed from the Hdh IGFBP7 gene was 1519-bp long with an open reading frame of 720-bp corresponding to a putative polypeptide of 239 amino acids. The molecular mass of its mature protein was approximately 23.44 KDa with an estimated isoelectric point (pI) of 5.35, and it shared significant homology with IGFBP7 gene of H. madaka. Hdh IGFBP7 has a characteristic IGFBP N-terminal domain (22–89 aa), a kazal-type serine proteinase inhibitor domain (77–128), and an immunoglobulin-like C2 domain (144–223). Furthermore, twelve cysteine residues and a signature motif of IGFBPs (XCGCCXXC) were found in its N-terminal domain. Phylogenetic analysis revealed that Hdh IGFBP7 was aligned with IGFBP7 of H. madaka. Tissue distribution analysis showed that the mRNA of Hdh IGFBP7 was expressed in all examined tissues, with the highest expression level observed in the mantle and gill tissues. The expression level of Hdh IGFBP7 mRNA was relatively higher at the juvenile stage during its metamorphosis period. In situ hybridization showed that Hdh IGFBP7 transcript was expressed in epithelial cells of the dorsal mantle pallial and mucus cells of the branchial epithelium in gill. These results provide basic information for future studies on the role of IGFBP7 in IGF regulation of shell growth, development and metamorphosis of abalone.

Short-Term Diet Induced Changes in the Central and Circulating IGF Systems Are Sex Specific.

Insulin-like growth factor (IGF) 1 exerts a wide range of functions in mammalians participating not only in the control of growth and metabolism, but also in other actions such as neuroprotection. Nutritional status modifies the IGF system, although little is known regarding how diet affects the newest members of this system including pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, proteases that liberate IGF from the IGF-binding proteins (IGFBPs), and stanniocalcins (STCs) that inhibit PAPP-A and PAPP-A2 activity. Here we explored if a 1-week dietary change to either a high-fat diet (HFD) or a low-fat diet (LFD) modifies the central and peripheral IGF systems in both male and female Wistar rats. The circulating IGF system showed sex differences in most of its members at baseline. Males had higher levels of both free (p < 0.001) and total IGF1 (p < 0.001), as well as IGFBP3 (p < 0.001), IGFBP5 (p < 0.001), and insulin (p < 0.01). In contrast, females had higher serum levels of PAPP-A2 (p < 0.05) and IGFBP2 (p < 0.001). The responses to a short-term dietary change were both diet and sex specific. Circulating levels of IGF2 increased in response to LFD intake in females (p < 0.001) and decreased in response to HFD intake in males (p < 0.001). In females, LFD intake also decreased circulating IGFBP2 levels (p < 0.001). In the hypothalamus LFD intake increased IGF2 (p < 0.01) and IGFBP2 mRNA (p < 0.001) levels, as well as the expression of NPY (p < 0.001) and AgRP (p < 0.01), but only in males. In conclusion, short-term LFD intake induced more changes in the peripheral and central IGF system than did short-term HFD intake. Moreover, these changes were sex-specific, with IGF2 and IGFBP2 being more highly affected than the other members of the IGF system. One of the main differences between the commercial LFD employed and the HFD or normal rodent chow is that the LFD has a significantly higher sucrose content, suggesting that this nutrient could be involved in the observed responses.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Modulates Aggressiveness of Ewing Sarcoma by Regulating the CD164-CXCR4 Axis.

Ewing sarcoma (EWS) is the second most common bone and soft tissue-associated malignancy in children and young adults. It is driven by the fusion oncogene EWS/FLI1 and characterized by rapid growth and early metastasis. We have previously discovered that the mRNA binding protein IGF2BP3 constitutes an important biomarker for EWS as high expression of IGF2BP3 in primary tumors predicts poor prognosis of EWS patients. We additionally demonstrated that IGF2BP3 enhances anchorage-independent growth and migration of EWS cells suggesting that IGF2BP3 might work as molecular driver and predictor of EWS progression. The aim of this study was to further define the role of IGF2BP3 in EWS progression. We demonstrated that high IGF2BP3 mRNA expression levels correlated with EWS metastasis and disease progression in well-characterized EWS tumor specimens. EWS tumors with high IGF2BP3 levels were characterized by a specific gene signature enriched in chemokine-mediated signaling pathways. We also discovered that IGF2BP3 regulated the expression of CXCR4 through CD164. Significantly, CD164 and CXCR4 colocalized at the plasma membrane of EWS cells upon CXCL12 stimulation. We further demonstrated that IGF2BP3, CD164, and CXCR4 expression levels correlated in clinical samples and the IGF2BP3/CD164/CXCR4 signaling pathway promoted motility of EWS cells in response to CXCL12 and under hypoxia conditions. The data presented identified CD164 and CXCR4 as novel IGF2BP3 downstream functional effectors indicating that the IGF2BP3/CD164/CXCR4 oncogenic axis may work as critical modulator of EWS aggressiveness. In addition, IGF2BP3, CD164, and CXCR4 expression levels may constitute a novel biomarker panel predictive of EWS progression.

Quercetin induces apoptosis in meningioma cells through the miR-197/IGFBP5 cascade

To investigate the effects of quercetin on cell viability and apoptosis in meningioma cells and to determine the underlying molecular mechanism. HBL-52 meningioma cells were treated with quercetin at doses of 1, 5, 10, 20, and 40 ng/mL for 24, 36 and 48 h, and cell viability was assessed using the Cell Counting kit-8 (CCK-8) test. Apoptosis was determined by flow cytometry. Bax, Bcl-2, and IGFBP5 protein expression was assessed by western blot, and IGFBP5 and miR-197 mRNA levels were measured using quantitative reverse transcription PCR (qRT-PCR). The interaction between miR-197 and IGFBP5 was verified by dual luciferase assay. Quercetin reduces viability and proliferation and increases apoptosis in HBL-52 cells in a dose- and time-dependent manner. Quercetin treatment also decreases Bcl-2 and increases Bax protein expression, and increases miR-197 mRNA while reducing IGFBP5 mRNA expression. A dual luciferase assay showed that miR-197 interacts directly with binding sites in the 3′untranslated region of IGFBP5, and that miR-197 overexpression reduced IGFBP5 expression. Quercetin may reduce meningioma cell proliferation and increase apoptosis by activating the miR-197/IGFBP5 cascade and regulating Bcl-2/Bax.

Molecular identification of grass carp igfbp2 and the effect of glucose, insulin, and glucagon on igfbp2 mRNA expression.

The GH (growth hormone)/IGFs (insulin-like growth factors) system has an important function in the regulation of growth. In this system, IGFBPs play a crucial regulatory role in IGF functions. As a member of the IGFBP family, IGFBP2 can bind to IGF and regulate IGF functions to regulate development and growth. In addition, IGFBP2 shows key regulatory functions in cell proliferation and metabolism. In this study, the igfbp2 gene was cloned from grass carp (Ctenopharyngodon idellus) liver. The ORF of grass carp igfbp2 is 834bp long and encodes 277 amino acids. The tissue distribution results showed that igfbp2 is expressed in multiple tissues in grass carp and has a high expression level in the liver. In the OGTT, igfbp2 expression was significantly decreased in the liver and brain after 6h of treatment with glucose. In vitro, igfbp2 expression in grass carp's primary hepatocytes was significantly suppressed by insulin after treatment for 6 and 12h. Moreover, igfbp2 expression was markedly increased in a dose-dependent manner with glucagon incubation in grass carp's primary hepatocytes. To the best of our knowledge, this is the first report about Igfbp2 in grass carp. These results will provide a basis for the in-depth study of grass carp Igfbp2.

Inhibition of miR-19a-3p decreases cerebral ischemia/reperfusion injury by targeting IGFBP3 in vivo and in vitro

BackgroundInflammation and apoptosis are considered to be two main factors affecting ischemic brain injury and the subsequent reperfusion damage. MiR-19a-3p has been reported to be a possible novel biomarker in ischemic stroke. However, the function and molecular mechanisms of miR-19a-3p remain unclear in cerebral ischemia/reperfusion (I/R) injury.MethodsThe I/R injury model was established in vivo by middle cerebral artery occlusion/reperfusion (MCAO/R) in rats and in vitro by oxygen–glucose deprivation and reperfusion (OGD/R) induced SH-SY5Y cells. The expression of miR-19a-3p was determined by reverse transcription quantitative PCR. The infarction volumes, Neurological deficit scores, apoptosis, cell viability, pro-inflammatory cytokines and apoptosis were evaluated using Longa score, Bederson score, TTC, TUNEL staining, CCK-8, ELISA, flow cytometry assays. Luciferase reporter assay was utilized to validate the target gene of miR-19a-3p.ResultsWe first found miR-19a-3p was significantly up-regulated in rat I/R brain tissues and OGD/R induced SH-SY5Y cells. Using the in vivo and in vitro I/R injury model, we further demonstrated that miR-19a-3p inhibitor exerted protective role against injury to cerebral I/R, which was reflected by reduced infarct volume, improved neurological outcomes, increased cell viability, inhibited inflammation and apoptosis. Mechanistically, miR-19a-3p binds to 3′UTR region of IGFBP3 mRNA. Inhibition of miR-19a-3p caused the increased expression of IGFBP3 in OGD/R induced SH-SY5Y cells. Furthermore, we showed that IGFBP3 overexpression imitated, while knockdown reversed the protective effects of miR-19a-3p inhibitor against OGD/R-induced injury.ConclusionsIn summary, our findings showed miR-19a-3p regulated I/R-induced inflammation and apoptosis through targeting IGFBP3, which might provide a potential therapeutic target for cerebral I/R injury.

Overexpression of IGF2BP3 as a Potential Oncogene in Ovarian Clear Cell Carcinoma.

Ovarian Clear Cell Carcinoma (OCCC) displays distinctive clinical and molecular characteristics and confers the worst prognosis among all ovarian carcinoma histotypes when diagnosed at advanced stage, because of the lack of effective therapy. IGF2BP3 is an RNA binding protein that modulates gene expression by post-transcriptional action. In this study, we investigated the roles of IGF2BP3 in the progression of OCCC. We used 328 OCCCs from the AOVT (the Alberta Ovarian Tumor Type study) and the COEUR (the Canadian Ovarian Experimental Unified Resource) cohorts to elucidate the associations between IGF2BP3 expression and clinicopathological parameters, with positive IGF2BP3 expression defined as diffuse block staining, being more frequently observed at stage III (P = 0.0056) and significantly associated with unfavorable overall survival (HR = 1.59, 95% CI 1.09–2.33) in multivariate analysis. IGF2BP3 mRNA gene expression was markedly increased in OCCC cell lines compared to normal tissues such as ovarian surface epithelium. We chose two IGF2BP3-overexpressing cell lines ES2 and OVMANA for in vitro and in vivo knockdown experiments. The proliferation and viability of both cell lines were significantly inhibited by two IGF2BP3 siRNAs and similar suppression was observed in cell migration and invasion by Wound Healing and Transwell assays. The percentage of apoptotic cancer cells was enhanced by both IGF2BP3 siRNAs. In vivo experiments showed significantly reduced sizes of tumors when treated with IGF2BP3 siRNA compared to controls. Furthermore, cancer metastasis-indicators MMP2 and MMP9 proteins were down-regulated. In conclusion, our study shows that IGF2BP3 expression is a promising biomarker for prognostication of women diagnosed with OCCC with multiple effects on key cell functions, supporting its role as an important cellular regulator with potential oncogenic activity, and as a potential target for future intervention strategies.

Decidual NK cell-derived conditioned medium from miscarriages affects endometrial stromal cell decidualisation: endocannabinoid anandamide and tumour necrosis factor-α crosstalk.

What are the effects of endocannabinoid anandamide (AEA) in uterine natural killer (unK) cells from miscarriage decidua, regarding their cytokine profile and endometrial stromal cell (ESC) crosstalk? uNK-conditioned media from miscarriage samples present high TNF-α levels which inhibit ESC decidualisation. AEA plasma levels are higher in women who have suffered a miscarriage. Moreover, AEA inhibits ESC proliferation and differentiation, although the levels and impact on the uNK cell cytokine profile at the feto-maternal interface remain elusive. This laboratory-based study used human primary uNK cells which were isolated from first-trimester decidua (gestational age, 5-12weeks) derived from 8 women with elective pregnancy termination and 18 women who suffered a miscarriage. The first-trimester placental tissues were assayed for AEA levels by UPLC-MS/MS and respective enzymatic profile by western blot. The uNK cells were isolated and maintained in culture. The expression of angiogenic markers in uNK cells was examined by quantitative PCR (qPCR). The uNK-conditioned medium was analysed for IFN-γ, TNF-α and IL-10 production by enzyme-linked immunosorbent assay, and the impact on ESC differentiation was assessed by measuring decidual markers Prl, Igfbp-1 and Fox01 mRNA expression using qPCR. AEA levels were higher in miscarriage decidua compared with decidua from elective terminations. The uNK cell-conditioned medium from the miscarriage samples exhibited high TNF-α levels and interfered with the decidualisation of ESCs. Exacerbated inflammation and elevated TNF-α levels at the feto-maternal interface may trigger AEA signalling pathways that, in turn, may impact decidualisation and the angiogenic ability of uNK cells. N/A. Primary uNK cell responses are based on a simple in vitro model. Thus, in complex microenvironments, such as the feto-maternal interface, the mechanisms may not be exactly the same. Also, the inflammatory events of miscarriage that, in this study, have happened prior to processing of the samples may cause different responses to that observed. In addition, the magnitude of the inflammatory response, required to trigger the AEA pathways that impact decidualisation and the uNK angiogenic ability in vivo, is still unclear. The endocannabinoid AEA is a modulator of reproductive competence. AEA not only may contribute to neuroendocrine homeostasis but also can take part in uterine changes occurring during early pregnancy. The work was supported by UID/MULTI/04378/2019 with funding from Fundação para a Ciência e a Tecnologia (FCT)/MCTES through national funds and PORTUGAL 2020 Partnership Agreement, NORTE-01-0145-FEDER-000024. S.C. Cunha acknowledges FCT for the IF/01616/2015 contract. There are no conflicts of interest.

DNA Methylation-based Diagnostic and Prognostic Biomarkers for Glioblastoma

Glioblastomas are the most common primary central nervous system malignancy tumor in adults. Glioblastoma patients have poor prognosis, with an average survival period of approximately 14 mo after diagnosis. To date, there are a limited number of effective treatment methods for glioblastoma, and its molecular mechanisms remain elusive. In this article, we analyzed the key biomarkers and pathways in glioblastoma patients based on gene expression and DNA methylation datasets. The 60 hypomethylated/upregulated genes and 110 hypermethylated/downregulated genes were identified in GSE50923, GSE50161, and GSE116520 microarrays. Functional enrichment analyses indicated that these methylated-differentially expressed genes were primarily involved in collagen fibril organization, chemical synaptic transmission, extracellular matrix-receptor interaction, and GABAergic synapse. The hub genes were screened from a protein–protein interaction network; in selected genes, increased NMB mRNA level was associated with favorable overall survival, while elevated CHI3L1, POSTN, S100A4, LOX, S100A11, IGFBP2, SLC12A5, VSNL1, and RGS4 mRNA levels were associated with poor overall survival in glioblastoma patients. Additionally, CHI3L1, S100A4, LOX, and S100A11 expressions were negatively correlated with their corresponding methylation status. Furthermore, the receiver-operator characteristic curve analysis indicated that CHI3L1, S100A4, LOX, and S100A11 can also serve as highly specific and sensitive diagnostic biomarkers for glioblastoma patients. Collectively, our study revealed the possible methylated-differentially expressed genes and associated pathways in glioblastoma and identified four DNA methylation-based biomarkers of glioblastoma. These results may provide insight on diagnostic and prognostic biomarkers, and therapeutic targets in glioblastoma.

Igfbp3 in grass carp (Ctenopharyngodon idellus): Molecular identification and mRNA expression under glucose, insulin and glucagon.

IGFBPs play a pivotal role in regulating the physiological function of IGFs (insulin-like growth factors). As an important member of IGFBPs, IGFBP3 is involved in the regulation of physiological functions of IGFs. To investigate the functional role of Igfbp3 in a herbivorous fish species, grass carp igfbp3 (GenBank accession no. MN251843) was isolated from the liver by molecular cloning. The ORF of grass carp igfbp3 was 882bp, which encoded 293 amino acids, and the first 22 amino acids comprised the signal peptide. The predicted molecular weight of grass carp Igfbp3 is 31.95kDa, and the theoretical isoelectric point is 8.32. Grass carp Igfbp3 displayed a high identity with its counterparts of common carp and zebrafish. And phylogenetic tree analysis showed that the grass carp Igfbp3 was clustered into the Igfbp3 subgroups of common carp and zebrafish. Tissue distribution study showed that igfbp3 was ubiquitously expressed in all tissues examined in the present study. High expression level of igfbp3 was detected in the heart, brain and fat of grass carp. The OGTT demonstrated that igfbp3 mRNA expression was significantly elevated in the liver of grass carp in response to glucose treatment. In vitro study showed that insulin could markedly stimulated igfbp3 mRNA expression in primary grass carp hepatocytes. Moreover, igfbp3 mRNA levels were also regulated by glucagon in grass carp primary hepatocytes. These results may provide the theoretical foundation for investigating the role of Igfbp3 in fish metabolism.

PSI-19 Poor maternal nutrition during gestation alters placental IGF-I, IGF-II, and IGFBP-3 mRNA expression in sheep

Abstract Insulin-like growth factors (IGF) modulate placental and fetal growth and development through nutrient sensing and endocrine signaling. We hypothesized that poor maternal nutrition during gestation would alter IGF-I, IGF binding protein (IGFBP)-2, and IGFBP-3 mRNA expression in the ovine placenta, but would not affect IGF-II mRNA expression. Pregnant ewes (n = 57) were individually fed: 60% (RES), 100% (CON), or 140% (OVER) of National Research Council requirements for TDN starting at day 30±0.2 of gestation. Ewes were euthanized and cotyledon and caruncle samples were collected at days 45, 90, and 135 of gestation. Relative mRNA expression of IGF-I, IGF-II, IGFBP-2, and IGFBP-3 was quantified using real-time PCR. Data were analyzed using the MIXED procedure in SAS. Relative IGF-I mRNA expression increased during gestation in the caruncle (d45: 0.96±0.06; d90: 1.28±0.06; d135: 1.38±0.05; P &amp;lt; 0.001). In the caruncle, IGFBP-2 expression was greater at d90 and d135 than d45 (d45: 0.67±0.20; d90: 1.90±0.20; d135: 1.65±0.18; P &amp;lt; 0.001). There was no observed effect of diet or day of gestation on IGF-II or IGFBP-3 expression in the caruncle. In the cotyledon, IGF-I expression tended to be greater in RES than OVER, which was similar to CON (CON: 0.96±0.07; RES: 1.10±0.06; OVER: 0.89±0.07; P = 0.08). Relative IGF-II mRNA expression was greater in RES cotyledons than OVER (CON: 1.30±0.35; RES: 1.96±0.31; OVER: 0.54±0.32; P = 0.01). During gestation, IGFBP-2 expression decreased in the cotyledon (d45: 1.26±0.12; d90: 0.93±0.12; d135: 0.59±0.11; P &amp;lt; 0.001). Relative IGFBP-3 mRNA expression was less in RES cotyledons than in OVER or CON (CON: 1.84±0.64; RES: 0.03±0.57; OVER: 3.62±0.66;P &amp;lt; 0.001). The changes in IGF expression in the cotyledon to a greater extent than in the caruncle in response to poor maternal diet suggest a potential mechanism by which maternal-fetal exchange may be modified to restrict placental and fetal growth.

Down-regulation of insulin-like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway.

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down‐regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT‐PCR and Western blot analyses were performed to examine the levels of apoptosis‐related factors, including Bax, caspase‐3 and Bcl2. The silencing of IGFBP5 up‐regulated the levels of Bax and caspase‐3 and down‐regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.

Dietary restriction in sheep: Uterine functionality in ewes with different body reserves during early gestation

The aim of this study was to characterize insulin, leptin and IGF-1 profiles after undernutrition in pregnant ewes with different initial body condition (iBCS) and to investigate embryo quality, uterine gene expression and presence and location of proteins during early gestation (day 5). Thirty-six Rasa Aragonesa ewes were divided into 2 groups with different BCS: BCS> 2.75 (high, H, n = 19) and BCS <2.25 (low, L, n = 17) and they were randomly assigned to two nutritional treatments: 1.5 maintenance (M) (control, C) or 0.5 (M) (undernourishment, U) times the daily maintenance requirements establishing four groups: high-iBCS control (HC, n = 9), high-iBCS undernourished (HU, n = 10), low-iBCS control (LC, n = 9) and low-iBCS undernourished (LU, n = 8). High-iBCS ewes presented higher concentration of IGF-1, reflecting a better metabolic status in these animals. There was a greater proportion of high-iBCS ewes presenting more than one CL (P < 0.05), and associated greater P4 plasma concentration, number of recovered embryo and a tendency for higher embryo viability rate (P = 0.13). In uterus, undernourished ewes tended to present lower P4 (P = 0.09) and higher E2 concentration (P = 0.10). Inmunostaining of uterine progesterone and estrogen receptors (PR and ERα) was not affected by iBCS and nutritional treatment. Ewes with low-iBCS tended to have more INSR mRNA, and undernourished ewes tended to have more IGFBP2 mRNA expression (P < 0.08). An interesting finding was that the uterine response to undernutrition was dependent on iBCS: a higher expression of GHR (P < 0.05) and a tendency in IGFBP5 (P = 0.09) mRNA was found in undernourished than control ewes but only in the high-iBCS group. In summary, the present study demonstrates that the endocrine response and the uterine gene expression to undernutrition depend on the initial body energy reserves (iBCS) and appears to be associated with a differential embryo quality.

Airway smooth muscle cells are insensitive to the anti-proliferative effects of corticosteroids: The novel role of insulin growth factor binding Protein-1 in asthma

Airway remodeling in asthma manifests, in part, as enhanced airway smooth muscle (ASM) mass, due to myocyte proliferation. While the anti-proliferative effects of glucocorticoid (GC) were investigated in normal ASM cells (NASMC), little is known about such effects in ASM cells derived from asthma subjects (AASMC). We posit that GC differentially modulates mitogen-induced proliferation of AASMC and NASMC. Cells were cultured, starved, then treated with Epidermal growth factor (EGF) (10 ng/ml) and Platelet-derived growth factor (PDGF) (10 ng/ml) for 24 h and/or fluticasone propionate (FP) (100 nM) added 2 h before. Cell counts and flow cytometry analyses showed that FP failed to decrease the cell number of and DNA synthesis in AASMC irrespective of mitogens used. We also examine the ability of Insulin Growth Factor Binding Protein-1 (IGFBP-1), a steroid-inducible gene that deters cell growth in other cell types, to inhibit proliferation of AASMC where FP failed. We found that FP increased IGFBP1 mRNA and protein levels. Interestingly, the addition of IGFBP1 (1 μg/ml) to FP completely inhibited the proliferation of AASMC irrespective to the mitogens used. Further investigation of different signaling molecules involved in ASM growth and GC receptor functions (Protein kinase B (PKB/AKT), Mitogen-activated protein kinases (MAPKs), Focal Adhesion Kinase (FAK)) showed that IGFBP-1 selectively decreased mitogen-induced p38 phosphorylation in AASMC. Collectively, our results show the insensitivity of AASMC to the anti-proliferative effects of GC, and demonstrate the ability of IGFBP1 to modulate AASMC growth representing, hence, a promising strategy to control ASM growth in subjects with GC insensitive asthma.

Overexpression of IGF2BP3 As a Potential Oncogene in Ovarian Clear Cell Carcinoma

Ovarian Clear Cell Carcinoma (OCCC) displays distinctive clinical and molecular characteristics and confers the worst prognosis among all ovarian carcinoma histotypes when diagnosed at advanced stage because of the lack of effective therapy. IGF2BP3 is an RNA binding protein that modulates gene expression by posttranscriptional action. In this study, we investigated the roles of IGF2BP3 in the progression of OCCC. We used 328 OCCCs from the AOVT (the Alberta Ovarian Tumor Type study) and the COEUR (the Canadian Ovarian Experimental Unified Resource) cohorts to elucidate the associations between IGF2BP3 expression and clinicopathological parameters, with positive IGF2BP3 expression defined as diffuse block staining, being more frequently observed at stage III (P=0.0056) and significantly associated with unfavorable overall survival (HR=1.59, 95% CI 1.09 - 2.33) in multivariate analysis. IGF2BP3 mRNA gene expression was markedly increased in OCCC cell lines compared to normal tissue such as ovarian surface epithelium. We chose two IGF2BP3-overexpressing cell lines ES2 and OVMANA for in vitro and in vivo knockdown experiments. The proliferation and viability of both cell lines were significantly inhibited by 2 IGF2BP3 siRNAs and similar suppression was observed in cell migration and invasion by Wound Healing and Transwell assays. The percentage of apoptotic cancer cells was enhanced by both IGF2BP3 siRNAs. In vivo experiments showed a significantly reduced size of tumors when treated with IGF2BP3 siRNA compared to controls. Furthermore, cancer metastasis-indicators MMP2 and MMP9 proteins were down-regulated. In conclusion, our study shows that IGF2BP3 expression is a promising biomarker for prognostication of women diagnosed with OCCC with multiple effects on key cell functions, supporting its role as an important cellular regulator with potential oncogenic activity, and as a potential target for future intervention strategies. Funding Statement: This study used resources provided by the Canadian Ovarian Cancer Research Consortium’s COEUR biobank funded by the Terry Fox Research Institute (grant #2009-15) and supervised by the Centre hospitalier de l’Universite de Montreal (CHUM). The Consortium acknowledges contributions to its COEUR biobank from institutions across Canada. Wenrang Kan participated in basic experimental work as a volunteer. This work was supported by grants of the National Natural Science Foundation of China (NSFC30970119, 81030029, 81271786, NSFC-NIH 81161120416, 81671980, 81871623) and College students’ Innovation & Entrepreneurship project in Heilongjiang Province (201410226047 J.J.K, D.Y.; 201510226020 D.S.L, L.Y., T.L., L.Q., L.L.G.; 201610226095 H.Y.W., Z.H.S. T.T.G., S.J.H., S.G.; 201610226094 Y.Y.Q., M.Y.; 201710226073 S.J.H., S.G.). H.D.L. is supported by a scholarship from China Scholarship Council, CSC No. 201508230143, for an academic visit to the University of Calgary (Univ. of Calgary ID number: 30016355). We also thank the Health and Family Planning Commission of Heilongjiang Province (2016-188), the Fundamental Research Funds for the Provincial Universities (2017JCZX57), the Young Innovative Talents in Universities of Heilongjiang Province (UNPYSCT-2018064), the China Postdoctoral Science Foundation (2018M630380) and the Heilongjiang Postdoctoral Financial Assistance (LBH-Z18198) program for their support. Declaration of Interests: There are no competing interest concerns with any of the authors. Ethics Approval Statement: All animal experiments were executed in accordance with the Institutional Ethics Committee of Harbin Medical University.