Downregulation Of Insulin-like Growth Factor Research Articles

Examining effects of a novel estrogenic perfluoro-alcohol, 1H,1H,8H,8H-Perfluorooctane-1,8-diol (FC8-diol), using the fathead minnow EcoToxChip.

In a previous in vivo study, adult male fathead minnows (Pimephales promelas) were exposed via water for 4 days to 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol). The present study expands on the evaluation of molecular responses to this perfluoro-alcohol by analyzing 26 male fathead minnow liver RNA samples from that study (five from each test concentration: 0, 0.018, 0.051, 0.171, and 0.463 mg FC8-diol/L) using fathead minnow EcoToxChips Ver. 1.0. EcoToxChips are a quantitative polymerase chain reaction array that allows for simultaneous measurement of >375 species-specific genes of toxicological interest. Data were analyzed with the online tool EcoToxXplorer. Among the genes analyzed, 62 and 96 were significantly up- and downregulated, respectively, by one or more FC8-diol treatments. Gene expression results from the previous study were validated, showing an upregulation of vitellogenin mRNA (vtg) and downregulation of insulin-like growth factor 1 mRNA (igf1). Additional genes related to estrogen receptor activation including esr2a (estrogen receptor 2a) and esrrb (estrogen related receptor beta) were also affected, providing further confirmation of the estrogenic nature of FC8-diol. Furthermore, genes involved in biological pathways related to lipid and carbohydrate metabolism, innate immune response, endocrine reproduction, and endocrine thyroid were significantly affected. These results both add confidence in the use of the EcoToxChip tool for inferring chemical mode(s) of action and provide further insights into the possible biological effects of FC8-diol. Environ Toxicol Chem 2024;00:1-9. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Abstract 17196: Genetic and Acquired DHCR7 Deficiency Upregulates 7β-hydroxycholesterol to Drive Endothelial Apoptosis and Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is a fatal disease without a cure, in which endothelial dysfunction drives pathologic remodeling of the pulmonary vasculature. Individuals with Smith-Lemli-Opitz syndrome (SLOS) develop PH, but the underlying mechanisms remain undefined. SLOS is an autosomal recessive disorder of cholesterol synthesis, resulting from loss-of-function mutations in 7-dehydrocholesterol reductase (DHCR7)-the terminal enzyme in the de novo synthesis of cholesterol- that leads to accumulation of 7-dehydrocholesterol (7-DHC) and cytotoxic oxysterol species. The role of DHCR7 in PH, however, is unknown. Hypothesis: Elevated cytotoxic oxysterols resulting from genetic or acquired DHCR7 deficiency promotes endothelial dysfunction and the development of PH. Methods & Results: DHCR7 mRNA and protein expression was decreased in pulmonary artery endothelial cells (ECs) in cellular (P=0.0089), rodent, and human PH models, mirroring DHCR7 reduction in SLOS. Both genetic loss of DHCR7 and hypoxia promoted a global shutdown of de novo cholesterol synthesis, resulting in an accumulation of cholesterol intermediates and increased derivative oxysterols. Specifically, 7β-hydroxycholesterol (7β-HC) was elevated in DHCR7-deficient ECs (P<0.0001), and 7β-HC drove endothelial apoptosis (P<0.0001) via downregulation of insulin-like growth factor 1 (IGF1) in ECs (fold change=0.101 ± 0.034, FDR=6.65E-11). Conversely, overexpression of DHCR7 upregulated IGF1 expression (P<0.0007), and IGF1 delivery reversed apoptosis (P<0.0001). Correspondingly, ECs derived from SLOS patient stem cells with DHCR7 mutations (p.T93M/c.964-1G>C) exhibited IGF1-dependent apoptosis. Finally, EC-specific DHCR7 deletion in chronically hypoxic mice induced more severe PH in vivo (right ventricular systolic pressure 36.4 mmHg vs. 30.8 mmHg, P<0.0130). Conclusions: Resulting either from hypoxic or genetic causes, DHCR7 deficiency promoted EC apoptosis and PH via 7β-HC accumulation and subsequent IGF1 downregulation. Our work is the first to define an oxysterol-dependent mechanism underlying the link between genetic and acquired DHCR7 deficiency and PH.

The effects of weight bearing on muscle atrophy early after ACL reconstruction in rats

We examined the effects of weight bearing after anterior cruciate ligament (ACL) reconstruction on muscle atrophy in rats. Rats were divided into the following groups: untreated control, ACL reconstruction (amount of weight bearing was small), ACL reconstruction plus hindlimb unloading (nonweight bearing), and ACL reconstruction plus morphine administration (amount of weight bearing was large) groups. At 7 or 14 days after surgery, atrophy of the rectus femoris and gastrocnemius was assessed. ACL reconstruction induced muscle atrophy in the rectus femoris and gastrocnemius. Unloading facilitated atrophy in the gastrocnemius but not in the rectus femoris. Morphine administration partially prevented atrophy in the gastrocnemius but not in the rectus femoris. After ACL reconstruction, the gene expression of insulin-like growth factor-1 (IGF-1), which is involved in protein synthesis, was downregulated in the gastrocnemius. Unloading decreased the gene expression of IGF-1 and increased the gene expression of atrogin-1, which is involved in protein breakdown, in the gastrocnemius. Morphine administration attenuated the downregulation of IGF-1. Atrophy of the gastrocnemius was more severe with a decrease in weight bearing, although the effect of weight bearing on rectus femoris atrophy was limited in rats. Early weight bearing is effective for reducing gastrocnemius muscle atrophy after ACL reconstruction.

Decreased insulin-like growth factor-1 expression in response to mechanical loading is associated with skeletal muscle anabolic resistance in cancer cachexia

ObjectiveCachexia is a systemic metabolic syndrome characterized by loss of body weight and skeletal muscle mass during chronic wasting diseases, such as cancer. Skeletal muscle in cancer cachexia is less responsive to anabolic factors including mechanical loading; however, the precise molecular mechanism is largely unknown. In this study, we examined the underlying mechanism of anabolic resistance in skeletal muscle in a cancer cachexia model. MethodsCD2F1 mice (male, 8 weeks old) were subcutaneously transplanted (1 × 106 cells per mouse) with a mouse colon cancer-derived cell line (C26) as a model of cancer cachexia. Mechanical overload of the plantaris muscle by synergist tenotomy was performed during the 2nd week and the plantaris muscle was sampled at the 4th week following C26 transplantation. ResultsThe hypertrophic response of skeletal muscle (increased skeletal muscle weight/protein synthesis efficiency and activation of mechanistic target of rapamycin complex 1 signaling) associated with mechanical overload was significantly suppressed during cancer cachexia. Screening of gene expression profile and pathway analysis using microarray revealed that blunted muscle protein synthesis was associated with cancer cachexia and was likely induced by downregulation of insulin-like growth factor-1 (IGF-1) and impaired activation of IGF-1-dependent signaling. ConclusionsThese observations indicate that cancer cachexia induces resistance to muscle protein synthesis, which may be a factor for inhibiting the anabolic adaptation of skeletal muscle to physical exercise in cancer patients.

IGF-1 Microinjection in the Prefrontal Cortex Attenuates Fentanyl-Seeking Behavior in Mice.

Opioid use disorder (OUD) is a chronic relapsing psychiatric disorder with an enormous socioeconomic burden. Opioid overdose deaths have reached an epidemic level, especially for fentanyl. One of the biggest challenges to treat OUD is the relapse to drug seeking after prolonged abstinence. Abnormalities in insulin-like growth factor-1 (IGF-1) have been reported in various neurological and psychiatric disorders, including OUD. However, whether IGF-1 and its downstream signaling pathways are associated with relapse to fentanyl seeking remains unclear. Mice were subjected to daily 2-hour fentanyl (10 μg/mL, 27 μL/infusion) oral self-administration training for 14 days, followed by 14-day fentanyl cessation. Expression levels of IGF-1/IGF-1 receptor and downstream signaling pathways in the dorsomedial prefrontal cortex (dmPFC) were detected. Then, IGF-1 was bilaterally microinjected into the dmPFC from fentanyl cessation day 9 to day 13. Fentanyl-seeking behavior and excitatory synaptic transmission of pyramidal neurons in PFC were evaluated. We found that 14-day cessation from fentanyl oral self-administration caused significant downregulation of IGF-1 and IGF-1 receptor phosphorylation in the dmPFC. These changes were accompanied by inhibition of the downstream Akt and S6 signaling pathway. In addition, local administration of IGF-1 in the dmPFC attenuated context-induced fentanyl-seeking behavior. Furthermore, electrophysiology and immunohistochemistry analyses showed that IGF-1 blocked fentanyl-induced reduction of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors-mediated excitatory synaptic transmission as well as synaptic expression of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and N-methyl-D-aspartate receptor subunits. These results suggest that IGF-1 in the PFC plays a pivotal role in regulating fentanyl seeking after prolonged cessation from fentanyl oral self-administration.

CTCF variant begets to short stature by down-regulation of IGF1.

Pathogenic variants in the transcription factor CCCTC-binding factor (CTCF) are associated with mental retardation, autosomal dominant 21 (MRD21, MIM#615502). Current studies supported the strong relationship between CTCF variants and growth, yet the mechanism of CTCF mutation leading to short stature is not known. Clinical information, treatment regimens, and follow-up outcomes of a patient with MRD21 were collected. The possible pathogenic mechanisms of CTCF variants leading to short stature were investigated using immortalized lymphocyte cell lines (LCLs), HEK-293T, and immortalized normal human liver cell lines (LO2). This patient received long-term treatment with recombinant human growth hormone (rhGH) which resulted in an increased height of 1.0 SDS. She had low serum insulin-like growth factor 1 (IGF1) before the treatment and the IGF1 level was not significantly increased during the treatment (-1.38 ± 0.61 SDS). The finding suggested that the CTCF R567W variant could have impaired IGF1 production pathway. We further demonstrated that the mutant CTCF had a reduced ability to bind to the promoter region of IGF1, consequently significantly reducing the transcriptional activation and expression of IGF1. Our novel results demonstrated a direct positive regulation of CTCF on the transcription of the IGF1 promoter. The impaired IGF1 expression due to CTCF mutation may explain the substandard effect of rhGH treatment on MRD21 patients. This study provided novel insights into the molecular basis of CTCF-associated disorder.

Expression of signaling molecules in ameloblastoma using cDNA microarray: A systematic review

Aim: Ameloblastoma is a benign but locally aggressive epithelial odontogenic tumor. The present review aimed to identify the most commonly expressed signaling molecule in ameloblastoma detected using cDNA microarray. Materials and Methods: It is a systematic review. In this review, articles were searched using databases—PubMed and Google Scholar and were also searched using Hand Search. All the studies which used cDNA microarray to detect the expression of signaling molecules were included. All the variants of ameloblastoma were included for the review. A total of 22 ameloblastoma cases were included in this systematic review. Out of three studies, 67% of the cases demonstrated upregulation of smoothened (SMO) and sonic hedgehog (SHH). Results: Out of three studies, 33% of the cases showed upregulation of toll-like receptor 2 (TLR2), fos proto-oncogene (FOS), Wnt family member 10a (Wnt10a), and patched and down-regulation of insulin-like growth factor 2 (IGF2). Of the 67% of the studies which described SMO and SHH, significant expression of SMO (73 times fold change) was noted. SHH expression was noted to have 0.88 times fold change. Of the 33% of the studies included, the highest average fold change was noted to be associated with FOS (14-fold change time) followed by Wnt10a (5.1-fold change times) and TLR2 (3.29-fold change times). However, downregulation of IGF2 by 0.09-fold change times was observed in 33% of the studies. Conclusion: The SMO gene from the SHH pathway was the highest expressed signaling molecule in ameloblastoma.

Exosome-transported lncRNA 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 discovered 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 lncRNA 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 lncRNA H19 were positively correlated with plasma and cerebral lncRNA H19. In a cell co-culture model, we confirmed that lncRNA H19 was transported from neurons to astrocytes by exosomes to induce downregulation of insulin-like growth factor-1 through the H19/let-7a/insulin-like growth factor-1 receptor axis. This study provides the first evidence for the transportation of lncRNA H19 by exosomes and the relationship between lncRNA H19 and insulin-like growth factor-1.

Down-Regulation of Insulin Like Growth Factor 1 Involved in Alzheimer's Disease via MAPK, Ras, and FoxO Signaling Pathways.

The inability to halt or even delay the course of Alzheimer's disease (AD) forces the development of new molecular signatures and therapeutic strategies. Insulin like growth factor 1 (IGF1) is a promising target for AD treatment, yet exact mechanisms of AD ascribed to IGF1 remain elusive. Herein, gene expression profiles of 195 samples were analyzed and 19,245 background genes were generated, among which 4,424 differentially expressed genes (DEGs) were overlapped between AD/control and IGF1-low/high groups. Based on such DEGs, seven co-expression modules were established by weight gene correlation network analysis (WGCNA). The turquoise module had the strongest correlation with AD and IGF1-low expression, the DEGs of which were enriched in GABAergic synapse, long-term potentiation, mitogen-activated protein kinase (MAPK), Ras, and forkhead box O (FoxO) signaling pathways. Furthermore, cross-talking pathways of IGF1, including MAPK, Ras, and FoxO signaling pathways were identified in the protein-protein interaction network. According to the area under the curve (AUC) analysis, down-regulation of IGF1 exhibited good diagnostic performance in AD prediction. Collectively, our findings highlight the involvement of low IGF1 in AD pathogenesis via MAPK, Ras, and FoxO signaling pathways, which might advance strategies for the prevention and therapy of AD based on IGF1 target.

SURG-05. Α5Β1 INTEGRIN TARGETING LIPOSOME DELIVERY OF MIR-603 SUPPRESSED GLIOBLASTOMA STEM CELL STATE AND ENHANCED RADIATION SENSITIVITY

Abstract INTRODUCTION Despite the potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a surgery mediated polyethylenimine (PEI)/liposome-based strategy for the delivery of miR-603, a master regulatory miRNA that suppresses glioblastoma stem cell state by simultaneous down-regulation of insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). METHODS miR-603 was complexed with PEI, a cationic polymer designed to facilitate miRNA from the endolysosomal compartment. The miR-603/PEI complex was encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5β1 integrin that is overexpressed in glioblastoma. RESULTS Patient-derived glioblastoma cells internalized PR_b coated liposomes but not the non-coated liposomes. The internalization of PR_b liposomes encapsulating miR-603/PEI was associated with orders of magnitude increase in intra-cellular miR-603 levels and decreased IGF1 and IGF1R mRNA/protein levels. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI showed altered morphology and decreased expression of stem cell marker, suggesting the treated cells have exited the cancer stem cell state. Finally, treatment of the PR_b liposomes encapsulating miR-603/PEI sensitized glioblastoma cells to ionizing radiation (IR) in patient-derived glioblastoma cells. These results were not observed in liposomes missing the PR_b peptide, PEI, or miR-603. CONCLUSION These results suggest that intra-tumoral injection of PR_b functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a strategy for glioblastomas therapy. A first-in-human trial is currently underway to test this strategy.

Targeted Liposomes Encapsulating miR-603 Complexes Enhance Radiation Sensitivity of Patient-Derived Glioblastoma Stem-Like Cells.

Despite potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a strategy for miRNA delivery in the treatment of glioblastoma, the most common form of adult brain cancer, that involves complexation of miRNA with polyethylenimine (PEI) and encapsulation in targeted liposomes. miRNA 603 (miR-603) is a master regulatory miRNA that suppresses glioblastoma radiation resistance through down-regulation of insulin-like growth factor 1 (IGF1) signaling. miR-603 was complexed with PEI, a cationic polymer, and encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5β1 integrin that is overexpressed in glioblastomas. Cultured patient-derived glioblastoma cells internalized PR_b-functionalized liposomes but not the non-targeted liposomes. The integrin targeting and complexation of the miRNA with PEI were associated with a 22-fold increase in intracellular miR-603 levels, and corresponding decreases in IGF1 and IGF1 receptor (IGF1R) mRNA expression. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI sensitized the cells to ionizing radiation (IR), a standard of care treatment for glioblastomas. These results suggest that PR_b-functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a therapeutic platform for glioblastomas.

Exogenous insulin-like growth factor 1 attenuates cisplatin-induced muscle atrophy in mice.

BackgroundA reduction in the skeletal muscle mass worsens the prognosis of patients with various cancers. Our previous studies indicated that cisplatin administration to mice caused muscle atrophy. This is a concern for human patients receiving cisplatin. The insulin‐like growth factor 1 (IGF‐1)/phosphoinositide 3‐kinase (PI3K)/Akt pathway stimulates the rate of protein synthesis in skeletal muscle. Thus, IGF‐I can be a central therapeutic target for preventing the loss of skeletal muscle mass in muscle atrophy, although it remains unclear whether pharmacological activation of the IGF‐1/PI3K/Akt pathway attenuates muscle atrophy induced by cisplatin. In this study, we examined whether exogenous recombinant human IGF‐1 attenuated cisplatin‐induced muscle atrophy.MethodsMale C57BL/6J mice (8–9 weeks old) were injected with cisplatin or saline for four consecutive days. On Day 5, quadriceps muscles were isolated. Mecasermin (recombinant human IGF‐1) or the vehicle control was subcutaneously administered 30 min prior to cisplatin administration. A dietary restriction group achieving weight loss equivalent to that caused by cisplatin administration was used as a second control. C2C12 myotubes were treated with cisplatin with/without recombinant mouse IGF‐1. The skeletal muscle protein synthesis/degradation pathway was analysed by histological and biochemical methods.ResultsCisplatin reduced protein level of IGF‐1 by about 85% compared with the vehicle group and also reduced IGF‐1/PI3K/Akt signalling in skeletal muscle. Under this condition, the protein levels of muscle ring finger protein 1 (MuRF1) and atrophy gene 1 (atrogin‐1) were increased in quadriceps muscles (MuRF1; 3.0 ± 0.1 folds, atrogin‐1; 3.0 ± 0.3 folds, P < 0.001, respectively). The administration of a combination of cisplatin and IGF‐1 significantly suppressed the cisplatin‐induced downregulation of IGF‐1/PI3K/Akt signalling and upregulation of MuRF1 and atrogin‐1 (up to 1.6 ± 0.3 and 1.5 ± 0.4 folds, P < 0.001, respectively), resulting in diminished muscular atrophy. IGF‐1 showed similar effects in cisplatin‐treated C2C12 myotubes, as well as the quadriceps muscle in mice.ConclusionsThe downregulation of IGF‐1 expression in skeletal muscle might be one of the factors playing an important role in the development of cisplatin‐induced muscular atrophy. Compensating for this downregulation with exogenous IGF‐1 suggests that it could be a therapeutic target for limiting the loss of skeletal muscle mass in cisplatin‐induced muscle atrophy.

Exosomal miR-486-5p derived from human placental microvascular endothelial cells regulates proliferation and invasion of trophoblasts via targeting IGF1

Preeclampsia (PE) is a serious complication of pregnancy. Exosomes are known to be upregulated in PE. In this study, we sought to investigate the effect of miR-486-5p from human placental microvascular endothelial cells, on the function of trophoblast cells. To investigate the function of human placental microvascular endothelial cell (HPVEC)-derived exosomes on trophoblast cells, HPVECs were treated with hypoxia/reoxygenation (H/R). The separation efficiency of exosomes was determined by transmission electron microscopy, nanosight and Western blot. Cell Counting Kit-8, EdU staining, wound-healing, and transwell assay were performed to detect the effect of exosomally transferred miR-486-5p inhibitor on proliferation, migration and invasion of trophoblast cells. MiRDB and dual-luciferase report assay were used to find the target of miR-486-5p. Our data revealed that miR-486-5p was significantly upregulated in H/R-treated HPVEC-Exo, and miR-486-5p was enriched in HPVEC-Exo. miR-486-5p inhibitor carried by HPVEC-Exo significantly inhibited the proliferation, migration and invasion of trophoblast cells. Insulin-like growth factor 1 (IGF1) was found to be the target of miR-486-5p, and IGF1 overexpression notably reversed the effect of miR-486-5p inhibitor from HPVEC-Exo on trophoblast cell function. In summary, H/R-treated HPVEC-derived exosomally expressing miR-486-5p inhibitor significantly inhibited the proliferation, migration and invasion of trophoblast cells via downregulation of IGF1. The findings from the present study may be useful in the development of treatments for PE.

Downregulation of IGF2 expression in third trimester placental tissues from Zika virus infected women in Brazil

ObjectivesScreening for genes differentially expressed in placental tissues, aiming to identify transcriptional signatures that may be involved in ZIKV congenital pathogenesis. MethodsTranscriptome data from placental tissues of pregnant women naturally infected with Zika virus during the third trimester were compared to those from women who tested negative for Zika infection. The findings were validated using both a cell culture model and an immunohistochemistry/morphological analysis of naturally infected placental tissues. ResultsTranscriptome analysis revealed that Zika virus infection induces downregulation of insulin-like growth factor II (IGF2) gene, an essential factor for fetal development. The Caco-2 cell culture model that constitutively expresses IGF2 was used for the transcriptome validation. Asiatic and African Zika virus strains infection caused downregulated IGF2 gene expression in Caco-2 cells, whereas other flaviviruses, such as dengue serotype 1, West Nile and wild-type yellow fever viruses, had no effect on this gene expression. Immunohistochemical assays on decidual tissues corroborated our transcriptome analysis, showing that IGF2 is reduced in the decidua of Zika virus-infected women. ConclusionsOur results draw attention to IGF2 modulation in uterine tissues, and this finding is expected to support future studies on strategies to ameliorate the harmful effects of Zika virus infection during pregnancy.

MiRNA-98-5p Targeting IGF2BP1 Induces Mesenchymal Stem Cell Apoptosis by Modulating PI3K/Akt and p53 in Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common hematological autoimmune disease, in which defective mesenchymal stem cells (MSCs) are potentially involved. Our previous study suggested that MSCs in ITP patients displayed enhanced apoptosis. MicroRNAs (miRNAs) play important roles in ITP by affecting megakaryopoiesis, platelet production and immunoregulation, whereas the roles of miRNAs in ITP-MSCs remain unknown. In a previous study, we performed microarray analysis to obtain mRNA and miRNA profiles of ITP-MSCs. In the present study, we reanalyze the data and identify miR-98-5p as a candidate miRNA contributing to MSC deficiency in ITP. miR-98-5p acts through targeting insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), and the subsequent downregulation of insulin-like growth factor 2 (IGF-2) causes inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in the process of MSC deficiency. Furthermore, miR-98-5p upregulates p53 by inhibiting β-transducin repeat-containing protein (β-TrCP)-dependent p53 ubiquitination. Moreover, miR-98-5p overexpression impairs the therapeutic effect of MSCs in ITP mice. All-trans retinoic acid (ATRA) protects MSCs from apoptosis by downregulating miR-98-5p, thus providing a potential therapeutic approach for ITP. Our findings demonstrate that miR-98-5p is a critical regulator of ITP-MSCs, which will help us thoroughly understand the pathogenesis of ITP.

Effects of down-regulation of IGF2 gene on the biological characteristics of HCT116 colon cancer stem cells

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 G(2)/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 G(0)/G(1) 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.

Effects of TGF-β1 on the expression and secretion of cytokines induced by Aβ1-42 in hippocampal neuron and microglia co-cultures

To investigate the neuroprotective effects of transforming growth factor beta 1(TGF-β1) on the expression and secretion of cytokines induced by Aβ1-42 in hippocampal neurons and microglial co-cultures. Hippocampal neurons and microglia obtained from SD rat were co-cultured. TGF-β1 was applied on day 5 after the neurons and microglia co-cultures were incubated at the concentrations of 5 or 20 ng/ml, Aβ1-42 was added 1 h following TGF-β1 application at a concentration of 5 μmol/L. They were incubated for 72 h and then assessed for further studies. Western blot analyses were employed to examine the expression of inducible nitric oxide synthase (iNOS); Real-time PCR and ELISA were used to detect the mRNA expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and insulin-like growth factor-1 (IGF-1). In the hippocampal neuron-microglia co-cultures, Aβ1-42 induced upregulation of iNOS, TNF-α and IL-1β, downregulation of IGF-1. TGF-β1 pretreatment ameliorated the pro-inflammatory effects caused by Aβ1-42. TGF-β1 significantly inhibits the increase in inflammatory cytokines and the decrease in neurotrophic factor which are caused by Aβ1-42-induced microglia activation.

Esculetin exerts antitumor effect on human gastric cancer cells through IGF-1/PI3K/Akt signaling pathway

In this study, we aimed to investigate the antitumor effect of esculetin, a coumarin derivative extracted from natural plants, on human gastric cancer cells, and to illustrate the potential mechanisms. The results showed that esculetin exhibited anti-proliferative effects against gastric cancer cells and induced their apoptosis in a dose dependent manner with lower toxicity against normal gastric epithelial cells. Mechanism study indicated that esculetin induced gastric cancer MGC-803 cells apoptosis by triggering the activation of mitochondrial apoptotic pathway through reducing the mitochondrial membrane potential (MMP), increasing Bax/Bcl-2 ratio, activating caspase-3 and caspase-9 activity, and increasing cytochrome c release from mitochondria. Further study showed that the pro-apoptotic effects of esculetin were associated with down-regulation of insulin-like growth factor-1/ phosphatidylinositide 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling pathway. Activation of IGF-1/PI3K/Akt pathway by IGF-1 abrogated the pro-apoptotic effects of esculetin, while inhibition of IGF-1/PI3K/Akt pathway by triciribine or LY294002 enhanced the pro-apoptotic effects of esculetin. In addition, esculetin inhibited in vivo tumor growth with no obvious toxicity following subcutaneous inoculation of MGC-803 cells in nude mice, and inhibited activation of IGF-1/PI3K/Akt pathway in tumor tissue. ConclusionThese results indicate that esculetin could inhibit cell proliferation and induce apoptosis of gastric cancer cells through IGF-1/PI3K/Akt mediated mitochondrial apoptosis pathway, and may be a novel effective chemotherapeutic agent against gastric cancer.

Inhibition of N-methyl-N-nitrosourea-induced gastric tumorigenesis by Liuwei Dihuang Pill in db/db mice

AIMTo investigate the inhibitory effect of Liuwei Dihuang Pill (LDP) on gastric tumorigenesis induced by N-methyl-N-nitrosourea (MNU) in diabetic mice.METHODSFour-week-old mice were divided into four groups: A, 12 db/m mice treated with MNU and saline, as the non-diabetic control; B, 12 db/db mice treated with MNU and saline, as the diabetic control; C, 12 db/db mice treated with MNU and metformin, as the positive control; and D, 12 db/db mice treated with MNU and LDP. MNU was administrated for 20 wk to induce gastric carcinogenesis. LDP was administrated for 10 wk for improvement of insulin resistance. Body weight and food intake were measured every week. Blood samples were collected for assays of fasting blood glucose, insulin, insulin-like growth factor (IGF)-1, adiponectin and leptin. Stomach tissues were collected for histopathological analysis, immunohistochemical staining of Ki67, quantitative reverse transcription-polymerase chain reaction and western blotting.RESULTSThe incidence of MNU-induced gastric dysplasia was significantly elevated in diabetic (db/db) mice relative to the control (db/m) mice. The incidence of gastric dysplasia was significantly reduced by LDP with suppression of cell proliferation, as demonstrated by a decrease in Ki67 staining. Hyperglycemia, hyperinsulinemia and serum IGF-1 were inhibited by LDP. Expression of IGF-1 and insulin receptor mRNAs was decreased, phosphorylation of IGF-1 receptor and AKT protein was reduced in the stomach tissues by LDP. In addition, adiponectin was increased and leptin was decreased in the serum by LDP.CONCLUSIONLDP decreased risk of gastric dysplasia in type 2 diabetic mice by down-regulation of IGF and insulin activity and correction of adipokines disorders.

Downregulation of Insulin-Like Growth Factor-1 via Nitric Oxide Production in a Hypergalactosemic Model of Neonate Skin Fibroblast Cultures

Background: Galactosemia is a severe metabolic disorder known to cause hepatosplenomegaly, jaundice and cataracts in neonates, and many patients develop later complications such as mental retardation, disorders of motor function or speech, and hypergonadotrophic hypogonadism. The pathogenetic mechanisms of classical galactosemia are unclear; however, nitric oxide (NO) has been suggested to play a role. Objectives: Insulin-like growth factor-1 (IGF-1) is important for the growth and development of children, and the aim of this study was to examine the association of NO production with IGF-1 gene expression under galactosemic conditions. Methods: Serum levels of IGF-1 and nitrite were measured in 15 galactosemia patients and 15 age- and gender-matched healthy controls. Fibroblast cultures established from postcircumcision foreskin of 3- to 8-day-old healthy neonates were treated for 72 h with 0-10 m<smlcap>M</smlcap> of galactose or 0-5 m<smlcap>M</smlcap> of galactose-1-phosphate (Gal-1-P) in the presence or absence of NO synthase inhibitor (<smlcap>L</smlcap>-NAME), and inducible NO synthase (iNOS) protein was measured using Western blot analysis. RT-PCR was performed to assess the IGF-1 gene expression. Results: Galactosemia patients were observed to have significantly (p < 0.01) elevated serum nitrites and markedly decreased levels (p < 0.01) of serum IGF-1 as compared to healthy controls. The cotreatment of neonate skin fibroblast cultures with galactose and Gal-1-P significantly (p < 0.01) increased cellular levels of NO and iNOS protein expression, and decreased (p < 0.01) IGF-1 mRNA levels. Treatment with <smlcap>L</smlcap>-NAME, a NOS inhibitor, significantly (p < 0.05) alleviated a galactose/Gal-1-P-induced decrease in IGF-1 mRNA levels. Conclusion: These results suggest that NO mediates the downregulation of IGF-1 by Gal-1-P/galactose, thereby providing a new molecular mechanism and possible therapeutic insight for galactosemia-related complications.