Regulation Of Insulin-like Growth Factor 2 Research Articles

Protective Effect and Mechanism of miR-328-3p on Coronary Artery Endothelial Cell Injury Induced by Oxidized Low-density Lipoprotein

To investigate the protective effect of miR-328-3p on oxidized low-density lipoprotein (ox-LDL)-induced coronary artery endothelial cell injury and the potentially relevant mechanisms. Human coronary artery endothelial cells (HCAECs) were induced with ox-LDL, and the cells were divided into a control group consisting of normal cells, an ox-LDL group receiving ox-LDL treatment, an ox-LDL+miR-NC group transfected with miR-NC and treated with ox-LDL, an ox-LDL+miR-328-3p group transfected with miR-328-3p and treated with ox-LDL, and ox-LDL+miR-328-3p+pcDNA group co-transfected miR-328-3p and pcDNA and treated with ox-LDL, and an ox-LDL+miR-328-3p+insulin-like growth factor 2 (IGF2) group co-transfected miR-328-3p and IGF2 and treated with ox-LDL. The expression level of miR-328-3p was determined with RT-qPCR. Cell proliferation was determined by MTT. Cell apoptosis was measured by flow cytometry. Western blot was conducted to examine the protein expression levels of cleaved cas-3 and IGF2. ELISA was performed to determine the levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β. Dual luciferase reporter experiment was performed to verify the targeting relationship between miR-328-3p and IGF2. Compared with those of the control group, miR-328-3p expression level and cell activity were significantly reduced in the ox-LDL group (P<0.05), while the apoptotic rate, the protein expression levels of cleaved cas-3, IGF2, Bax, and Bcl-2, and the levels of TNF-α, IL-6, and IL-1β were significantly increased (P<0.05). Compared with those of the ox-LDL+miR-NC group, miR-328-3p expression level and cell activity significantly increased in the ox-LDL+miR-328-3p group (P<0.05), while the apoptosis rate, the protein expression levels of cleaved cas-3 and IGF2, and the levels of TNF-α, IL-6, and IL-1β were significantly reduced. IGF2 was a functional target of miR-328-3p. Compared with those of the ox-LDL+miR-328-3p+pcDNA co-transfection group, the IGF2 protein level was significantly increased (P<0.05) and cell activity was significantly decreased (P<0.05) in the ox-LDL+miR-328-3p+IGF2 co-transfection group, while the apoptosis rate, cleaved cas-3 protein level, and the levels of TNF-α, IL-6, and IL-1β were significantly elevated (P<0.05). miR-328-3p inhibits ox-LDL-induced apoptosis and inflammatory in coronary artery endothelial cell injury through targeted negative regulation of IGF2.

Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown. To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs. Primary human cumulus GCs in culture. University infertility center. GCs of women undergoing in vitro fertilization. Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic homolog (SMAD) inhibitors. Quantification of mRNA, protein, promoter activity, and DNA methylation. FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P < 0.0001) in a concentration-dependent manner showing a maximal effect at 5 ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P < 0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P < 0.009). For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.

Abstract 5459: Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis

Abstract Development of hepatocellular carcinoma (HCC), which remains lethal, is associated with alterations in multiple factors including the transforming growth factor beta (TGF-β) signaling pathway. Previously we have uncovered a unique role for TGF-β signaling molecules, Smad3 and its adaptor β2SP, in suppressing stem cell transformation into cancer. Yet, while TGF-β plays a pleiotropic role including regulating stem cell differentiation, proliferation, and inflammation, mechanistic insight into the dichotomy of TGF-β, and its role in stem cell transformation remains poorly understood for these cancers. Here, we took an integrated approach to identify and validate effects of changes in this pathway in HCC and identify potential therapeutic targets such as IGF2. We extended our mechanistic studies associated with the regulation of insulin-like growth factor 2 (IGF2) in the context of the TGF-β-pathway, by utilizing both human liver cancer cell lines and TGF-β signaling-deficient mice (β2SP+/- and β2SP+/-/Smad3+/-) that develop liver cancers. We found the following: 1) IGF2 promotes stemness, overexpression leading to increased expression of stem cell genes that include SOX2 and ALDH1A1, and sphere formation in liver cancer cells; 2) A marked increase in IGF2 mRNA and protein in HepG2 cells and Huh7 cells with β2SP stable knockdown; 3) TGF-β signaling through β2SP/Smad3 significantly inhibits IGF2 transcription; ChIP assays validated binding of Smad3 at the human IGF2 promoter region between -859bp to -688bp upstream of the IGF2 transcriptional start site; 4) TCGA based integrated analyses identify specific HCC patient groups with altered TGF-β members and high expression of IGF2 that could be potentially targetable for IGF2; 5) TCGA analyses of 33 cancers also revealed that potentially “switching off” TGF-β-pathway (through decreased expression levels) leads to increased stemness validating findings of our TGF-β deficient mouse models that phenocopy a human cancer stem cell syndrome. In summary, TGF-β negatively regulates IGF2 transcription: loss of TGF-β signaling through β2SP/Smad3 leads to IGF2 activation, contributing to tumor progression. TGF-β-β2SP/Smad3 are important for IGF2 repression and maintaining stem cell homeostasis, which may result from TGF-β/β2SP/Smad3 and/or CTCF-dependent regulation of IGF2. Our studies provide a framework for new animal models of liver and gastrointestinal cancers and future new therapeutics. Citation Format: Shuyun Rao, Sobia Zaidi, Kazufumi Ohshiro, Jian Chen, Shoujun Gu, Wilma Jogunoori, Jon White, Bibhuti Mishra, Shulin Li, Rehan Akbani, Lopa Mishra. Regulation of IGF2 by TGF-β signaling in liver cancers and stem cell homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5459.

Epigenetic alterations contribute to promoter activity of imprinting gene IGF2

The expression of insulin-like growth factor 2 (IGF2), a classical imprinting gene, didn't completely correlate with its imprinting profiles in hepatocellular carcinoma (HCC). The mechanistic importance of promoter activity in regulation of IGF2 has not been fully clarified. Here we show that histone 3 lysine 4 trimethylation (H3K4me3) modified by menin-MLL complex of IGF2 promoter contributes to promoter activity of IGF2. The strong binding of menin and abundant H3K4me3 at the DNA demethylated P3/4 promoters were observed in Hep3B cells with the robust expression of IGF2. In IGF2-low-expressing HepG2 cells, menin didn't bind to DNA hypermethylated P3/4 regions; however, menin overexpression inhibited DNA methylation and promoted H3K4me3 at the P3/4 as well as IGF2 expression in HepG2. In addition, the H3K4me3 at P3/4 locus was activated in primary HCC specimens with high IGF2 expression. Furthermore, inhibition of the menin/MLL interaction via MI-2/3 reduced IGF2 expression, inhibited the IGF1R-AKT pathway, and significantly repressed HCC with robust expression of IGF2. Taken together, we conclude that H3K4me3 of P3/4 locus mediated by the menin-MLL complex is a novel epigenetic mechanism for releasing IGF2.

Insulin-Like Growth Factor 2 - The Oncogene and its Accomplices.

The era of personalized medicine for cancer treatment has just begun opening up novel molecular targets improving cancer therapy. Members of the insulin-like growth factor 2 (IGF2) axis have been described to be altered and to have prognostic relevance in several different tumor identities. IGF2 belongs to the most complexly regulated growth factors known. As an imprinted gene it is controlled by epigenetic alterations. IGF2 mRNA binding proteins (IMPs/IGF2BPs) further regulate its translation. IGF2 activity is contained through IGF binding proteins (IGFBPs) and differential expression of the target receptors. The necessity of such a complex regulation implies pathophysiological effects of a deregulated expression of IGF2. This review attempts to summarize the different levels of IGF2 regulation, especially in the context of cancer. Members of the IGF2 axis are enlightened from the perspective of novel molecular targets for cancer therapy. Preclinical as well as experimental therapeutic interventions targeting IGF2 in cancer will be surveyed.

Role Of Insulin Like Growth Factor mRNA Binding Protein-3 (IGF2BP3) In Mixed Lineage Leukemia (MLL) Positive B-Cell Lymphomas

Oncogenic transformation of early B-cell progenitors leads to the human disease of B-acute lymphoblastic leukemia or B-ALL, which affects both children and adults. Among the different subtypes of B-ALL, defined by particular cytogenetic anomalies, there are two which are difficult to treat and have a dismal prognosis. These are B-ALL with chromosomal translocation t (9; 22)/BCR-ABL and MLL gene rearrangements, which show distinctive gene expression profiles.Gene expression is now known to be significantly regulated by post-transcriptional mechanisms. These involve RNA binding proteins and microRNAs. Deregulation of microRNAs as well as RNA binding protein expression is associated with numerous cancers. Here, we hypothesized that RNA binding proteins may be important in regulating gene expression in MLL rearranged leukemias.To examine this hypothesis, we undertook a microarray study examining the expression of both protein-coding and non-coding genes in B-ALL, including MLL translocations. A total of 44 samples were used for the microarray. Supervised class prediction was carried out using the R library of prediction analysis for microarrays (PAM). One of the most significantly differentially expressed genes was Insulin Like Growth Factor mRNA Binding Protein-3 (IGF2BP3). The expression of IGF2BP3 was highest in the MLL rearranged B-ALL group. IGF2BP3 is an oncofetal protein known to be highly expressed in a number of epithelial malignancies such as glioblastomas. IGF2BP3 has been known to bind to the 5'-UTR and stabilize mRNAs like CD44 the expression of which correlates with epithelial tumors metastasis. IGF2BP3 has been shown to bind to the Insulin like Growth Factor-2 (IGF-2) mRNA and enhance translation in glioblastomas.We confirmed the expression of IGF2BP3 and CD44 in these 44 tumor samples and 90 other B-cell lymphoma samples by RT-qPCR. This corroborated with our previous data showing that the expression of both these genes is significantly higher in the group with MLL translocations. In the MLL rearranged leukemias, there was a significant correlation between the expression of CD44 and IGF2BP3. Interestingly however, there was no significant difference in the expression of IGF2 mRNA between these different subsets, indicating either that IGF2BP3 might be acting on IGF-2 mRNA at the translational level or that IGF-2 regulation may be cell-type specific.To evaluate whether IGF2BP3 affects the growth of B-ALL cells, we used NALM6, a B-ALL cell line which expresses IGF2BP3. We generated microRNA-155 formatted siRNAs against human IGF2BP3 and subcloned them into pHAGE6 based lentiviral vectors. Our preliminary data demonstrates that these vectors are capable of knocking down IGF2BP3 in the NALM6 cell line. In addition, cells with knockdown showed a dramatic decrease in their growth rates, as measured by the MTS assay.The IGF-2 paracrine signaling system is thought to be important in the maintenance of HSCs as well as in lymphocyte development. We separated different precursors of B-cells (Hardy fractions) from murine bone marrow using FACS and measured the mRNA expression of CD44 and IGF2BP3 in these different subsets. The expression of both these genes correlated well with each other and showed a dynamic expression pattern with the highest expression seen in the Hardy Fraction C (late pro-B cells). This indicates that the IGF2BP3/CD44 axis might play a role in regulating normal B-cell development and this may be dysregulated in MLL-translocated B-ALL.To examine whether IGF2BP3 overexpression causes leukemia, we cloned the murine and human IGF2BP3 coding regions in a murine retroviral expression vector, MIG (MSCV-IGF2BP3-IRES-GFP). Retroviral packaging was done using 293T cell line, virus was collected and used to infect 7Oz/3, a murine pre-B ALL cell line. Western blot and qPCR confirmed overexpression of IGF2BP3. We have infected bone marrow cells from CD 45.2 positive wild type donor mice with the virus and transferred them into irradiated CD 45.1 recipient mice. We have confirmed engraftment in these mice using flowcytometry for CD 45.1/2 and are presently following the mice for the development of leukemia.In summary, IGF2BP3 is dysregulated in MLL-rearranged leukemia, and its knockdown can cause decreased growth rates in B-ALL cell lines. The current study explores whether IGF2BP3 is oncogenic and the mechanisms of action of IGF2BP3 in B-cell development and neoplasia. Disclosures:No relevant conflicts of interest to declare.

Cell type and context-specific function of PLAG1 for IGF2 P3 promoter activity

The fetal transcription factor PLAG1 is found to be overexpressed in cancers, and has been suggested to bind the insulin like growth factor 2 (IGF2) P3 promoter, and to activate the IGF2 gene. The expression of IGF2 has partly been linked to loss of CTCF-dependent chromatin insulator function at the H19 imprinting control region (ICR). We investigated the role of PLAG1 for IGF2 regulation in Hep3B and JEG-3 cell lines. Chromatin immunoprecipitation revealed cell type-specific binding of PLAG1 to the IGF2 P3 promoter, which was substantially insensitive to recombinant PLAG1 overexpression in the endogenous context. We hypothesized that the H19 chromatin insulator may be involved in the cell type-specific PLAG1 response. By using a GFP reporter gene/insulator assay plasmid construct with and without the H19 ICR and/or an SV40 enhancer, we confirm that the effect of the insulator is specifically associated with the activity of the IGF2 P3 promoter in the GFP reporter system, and furthermore, that the reporter insulator is functional in JEG-3 but not in Hep3B cells. FACS analysis was used to assess the function of PLAG1 in low endogenously expressing, but Zn-inducible stable PLAG1 expressing JEG-3 cell clones. Considerable increase in IGF2 expression upon PLAG1 induction with a partial insulator overriding activity was found using the reporter constructs. This is in contrast to the effect of the endogenous IGF2 gene which was insensitive to PLAG1 expression in JEG-3, while modestly induced the already highly expressed IGF2 gene in Hep3B cells. We suggest that the PLAG1 binding to the IGF2 P3 promoter and IGF2 expression is cell type-specific, and that the PLAG1 transcription factor acts as a transcriptional facilitator that partially overrides the insulation by the H19 ICR.

Insulin-like growth factor-2 activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases.

Insulin-like growth factor-2 (IGF-2) plays a pivotal role in regulating intestinal epithelial metabolism, growth, and proliferation, but its regulatory effects on mucosal cell amino acid transport have not been well studied. The purpose of this in vitro study was to investigate the regulatory mechanisms and intracellular signaling pathways involved in the regulation of IGF-2 on glutamine transport in cultured intestinal cells. Continuous incubation with IGF-2 stimulated glutamine transport activity in cultured IEC-6 cells in a dose- and time-dependent fashion. Prolonged incubation (up to 48 hours) resulted in a 50% increase in transport activity (0.81+/-0.21 nmole/mg protein/min in IGF-2 cells vs. 0.57+/-0.15 nmole/mg protein/min in control cells) and a threefold increase in glutamine transporter ATB(0) mRNA levels. IGF-2 stimulated transport activity by increasing transport maximal capacity (V(max) 4.31+/-0.36 nmole/mg protein/min in IGF-2 cells vs. 2.51+/-0.23 nmole/mg protein/min in control cells) without affecting the transport affinity (K(m) 0.31+/-0.03 mmol/L glutamine in IGF-2 cells vs. 0.28+/-0.03 mmol/L glutamine in control cells). This IGF-2-induced glutamine transport activity was attenuated by actinomycin-D or cycloheximide. The levels of mitogen-activated protein kinases p42/44, MEK1/2, and p38 as well as protein kinase C levels were elevated in IGF-2-treated cells and inhibitors of mitogen-activated protein kinase MEK1 (PD 98059), mitogen-activated protein kinase p38, and protein kinase C (chelerythrine chloride) individually attenuated the IGF-2-induced glutamine transport. These data suggest that IGF-2 stimulates intestinal glutamine uptake in cultured rat intestinal epithelial cells via a mechanism that involves transcription and translation of the transporter. Activation of mitogen-activated protein kinases and protein kinase C cascades are involved in the regulation. This increase in glutamine uptake may occur to support intestinal cell growth and proliferation.

Adrenal Medullary Nodules in Beckwith-Wiedemann Syndrome Resemble Extra-Adrenal Paraganglia.

Clues to mechanisms regulating development and tumorigenesis may be provided by studies of unusual diseases. Beckwit-Wiedemann syndrome (BWS) is a rare congenital disorder apparently related to abnormal regulation of insulin-like growth factor-2 (IGF-2) production. IGF2 mRNA has been previously localized to the chief cells of extra-adrenal paraganglia and to adult, but not fetal, adrenal medulla. Expression of IGF-2 by neuroblastomas has been hypothesized to reflect extra-adrenal paraganglionic differentiation. In the adrenals of a fetus with 8W5, we have observed both increased numbers of chromaffin cells and organoid nodules resembling extra-adrenal paraganglia. Immunoreactive IGF-2 was observed in both cell types, but was also observed in chromaffin cells in the normal fetal adrenal. The findings suggest autocrine or paracrine influences of IGF-2 in regulating the number and phenotype of cells derived from sympathoadrenal precursors in the developing adrenal medulla as well as in extra-adrenal paraganglia. These results have implications for the interpretation of data from neuroblastoma studies.