Insulin-like Growth Factor 2 mRNA-binding Protein 1 Expression Research Articles

METTL3 promotes the progression of osteosarcoma through the N6-methyladenosine modification of MCAM via IGF2BP1

BackgroundThe molecular mechanisms of osteosarcoma (OS) are complex. In this study, we focused on the functions of melanoma cell adhesion molecule (MCAM), methyltransferase 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in OS development.MethodsqRT-PCR assay and western blot assay were performed to determine mRNA and protein expression of MCAM, METTL3, IGF2BP1 and YY1. MTT assay and colony formation assay were conducted to assess cell proliferation. Cell apoptosis, invasion and migration were evaluated by flow cytometry analysis, transwell assay and wound-healing assay, respectively. Methylated RNA Immunoprecipitation (MeRIP), dual-luciferase reporter, Co-IP, RIP and ChIP assays were performed to analyze the relationships of MCAM, METTL3, IGF2BP1 and YY1. The functions of METTL3 and MCAM in tumor growth were explored through in vivo experiments.ResultsMCAM was upregulated in OS, and MCAM overexpression promoted OS cell growth, invasion and migration and inhibited apoptosis. METTL3 and IGF2BP1 were demonstrated to mediate the m6A methylation of MCAM. Functionally, METTL3 or IGF2BP1 silencing inhibited OS cell progression, while MCAM overexpression ameliorated the effects. Transcription factor YY1 promoted the transcription level of METTL3 and regulated METTL3 expression in OS cells. Additionally, METTL3 deficiency suppressed tumor growth in vivo, while MCAM overexpression abated the effect.ConclusionYY1/METTL3/IGF2BP1/MCAM axis aggravated OS development, which might provide novel therapy targets for OS.

Inhibition of IGF2BP1 attenuates the progression of endometriosis through PTBP1.

Endometriosis (EMs), manifested by pain and infertility, is a chronic inflammatory disease. The precise pathophysiology of this disease remains uncertain. Insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) and polypyrimidine tract-binding protein 1 (PTBP1) have both been found to regulate proliferation, apoptosis, and invasion. This study aimed to investigate the effects of IGF2BP1/PTBP1 in treating EMs. qRT-PCR and western blotting were employed to quantify IGF2BP1 and PTBP1 expression in six patients with EMs (mean age 33.83 years). The correlation analysis, STRING database prediction, and RNA immunoprecipitation were utilized to identify the relationship between IGF2BP1 and PTBP1. Ectopic endometrial volume, weight, HE staining, and IGF2BP1 silencing were utilized to estimate the effects of IGF2BP1 in EMs model rats. qRT-PCR, CCK-8, 5-ethynyl-2'-deoxyuridine (EDU) labeling, Transwell assay, and flow cytometry were utilized to assess the effects of IGF2BP1/PTBP1 on the proliferation, migration, invasion, and apoptosis of ectopic endometrial stromal cells (eESCs). Furthermore, western blotting was employed to evaluate expressions of PCNA, VEGF, and E-cadherin in EMs rats and eESCs. The mRNA and protein levels of IGF2BP1 and PTBP1 in the ectopic and eutopic endometrium of EMs patients were significantly increased. RNA immunoprecipitation revealed a close interaction of IGF2BP1 with PTBP1. Additionally, the endometrial volume, weight, and histopathologic scores in rats were significantly reduced after IGF2BP1 silencing. IGF2BP1 silencing also decreased the expression of PCNA and VEGF, and increased E-cadherin expression in endometrial tissues of EMs rats. Moreover, IGF2BP1 silencing inhibited proliferation, migration, and invasion and promoted apoptosis through PTBP1 in eESCs. IGF2BP1 exhibits potential beneficial properties in the management of EMs by interacting with PTBP1, thereby highlighting IGF2BP1 as a promising therapeutic target for EMs.

M 6 A reader IGF2BP1 reduces the sensitivity of nasopharyngeal carcinoma cells to Taxol by upregulation of AKT2.

Taxol is widely used in the treatment of nasopharyngeal carcinoma (NPC); nevertheless, the acquired resistance of NPC to Taxol remains one of the major obstacles in clinical treatment. In this study, we aimed to investigate the role and mechanism of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in Taxol resistance of NPC. Taxol-resistant NPC cell lines were established by exposing to gradually increased concentration of Taxol. Relative mRNA and protein levels were tested using qRT-PCR and western blot, respectively. NPC cell viability and apoptosis were assessed by cell counting kit-8 and flow cytometry analysis, respectively. Cell migration and invasion capacities were measured using transwell assay. Interaction between IGF2BP1 and AKT2 was examined by RNA immunoprecipitation assay. The N6-methyladenosine level of AKT2 was tested using methylated RNA immunoprecipitation-qPCR. IGF2BP1 expression was enhanced in Taxol-resistant NPC cell lines. Knockdown of IGF2BP1 strikingly enhanced the sensitivity of NPC cells to Taxol and repressed the migration and invasion of NPC cells. Mechanistically, IGF2BP1 elevated the expression of AKT2 by increasing its mRNA stability. Furthermore, overexpression of AKT2 reversed the inhibitory roles of IGF2BP1 silence on Taxol resistance and metastasis. Our results indicated that IGF2BP1 knockdown enhanced the sensitivity of NPC cells to Taxol by decreasing the expression of AKT2, implying that IGF2BP1 might be promising candidate target for NPC treatment.

The m6A reader IGF2BP1 manipulates BUB1B expression to affect malignant behaviors, stem cell properties, and immune resistance of non-small-cell lung cancer stem cells.

N6-methyladenosine (m6A) modification is the most common internal modification in eukaryotic mRNA and an important mechanism for post-transcriptional regulation of genes. This study focuses on the role of the m6A reader insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in the malignant behaviors of non-small-cell lung cancer (NSCLC) cells and especially the cancer stem cells (CSCs). We obtained IGF2BP1 as an aberrantly upregulated gene linking to poor survival of patients with NSCLC by bioinformatics, and then confirmed increased IGF2BP1 expression in NSCLC tissues and cells, especially in the enriched CSCs. Knockdown of IGF2BP1 suppressed proliferation, mobility and epithelial-mesenchymal transition activity of NSCLC cells and CSCs, and it reduced stemness, self-renewal ability, xenograft tumorigenesis and immune resistance of the CSCs. IGF2BP1 was predicted to have a positive correlation with BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B), and it upregulated BUB1B expression through m6A modification. Further overexpression of BUB1B in CSCs counteracted the effects of IGF2BP1 silencing and restored the malignant phenotype, self-renewal, and immune resistance of CSCs in vitro and in vivo. Taken together, this work demonstrates that IGF2BP1 manipulates BUB1B expression to affect malignant behaviors, stem cell properties and immune resistance of NSCLC stem cells.

Ligustrazine inhibits inflammatory response of human endometrial stromal cells through the STAT3/IGF2BP1/RELA axis

Context Endometriosis (EMs) is a gynecological disorder. Ligustrazine has been reported to exert an anti-inflammatory effect on EMs. However, the underlying mechanisms are not completely understood. Objective To investigate the effects of ligustrazine on the progression of EMs and the underlying regulatory mechanisms. Materials and methods Human endometrial stromal cells (HESCs) were isolated from patients with EMs or control subjects. HESCs were treated with 25, 50, 100, or 200 μM ligustrazine for 1, 3, 6, or 12 h. Western blot and enzyme-linked immunosorbent assays were performed to determine the levels of proteins and inflammatory cytokines, respectively. The binding between STAT3 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was assessed by chromatin immunoprecipitation and dual-luciferase reporter assays. The relationship between IGF2BP1 and RELA was assessed by RNA immunoprecipitation and RNA pull-down assay. Results Phosphorylated STAT3, IGF2BP1, RELA, TNF-α, IL-6, and IL-1β were upregulated in EMs tissues compared with control tissues (by 1.79-, 2.55-, 1.58-, 3.01-, 2.55-, and 3.34-fold, respectively). Ligustrazine inhibited the expression of p-STAT3, IGF2BP1, RELA, IL-6, TNF-α, and IL-1β. Overexpression of STAT3 promoted RELA-mediated inflammatory responses, while ligustrazine (100 µM) notably reversed this phenomenon. Ligustrazine also alleviated RELA-induced inflammation via downregulating IGF2BP1. STAT3 bound to the promoter of IGF2BP1, and IGF2BP1 bound to the RELA mRNA. Discussion and conclusion Ligustrazine inhibited inflammation in EMs via regulating the STAT3/IGF2BP1/RELA axis. These findings propose a new agent against EMs and support the development of ligustrazine-based treatment strategies for EMs.

IGF2BP1-mediated N6-methyladenosine modification promotes intrahepatic cholangiocarcinoma progression

N6-methyladenosine (m6A) RNA methylation and its associated RNA-binding protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) are involved in tumor initiation and progression. Here, we explored the biological function and clinical significance of IGF2BP1 in intrahepatic cholangiocarcinoma (iCCA). We found that IGF2BP1 expression was upregulated by H3K27 acetylation enrichment of its promoter, which positively correlated with poor clinicopathological characteristics and survival. Gain- and loss-of-function experiments showed that IGF2BP1 overexpression (knockdown) enhanced (attenuated) iCCA growth and metastasis in vitro and in vivo. Mechanistically, IGF2BP1 not only regulated the c-Myc/p16 axis to promote iCCA growth and inhibit senescence, but also activated the ZIC2/PAK4/AKT/MMP2 axis to induce tumor metastasis. More importantly, BTYNB, a recently identified IGF2BP1 inhibitor, exerted promising anti-tumor efficacy in a patient-derived xenograft (PDX) model, and IGF2BP1 conditional knockout (cKO) reduced the tumor burden. These results demonstrate the crucial role of IGF2BP1 in iCCA progression via m6A-dependent modification, highlighting IGF2BP1 as a potential therapeutic target in iCCA.

Circular RNA circ_0005667 promotes cisplatin resistance of endometrial carcinoma cells by regulating IGF2BP1 through miR-145-5p.

Circular RNA (circRNA) plays a significant role in cisplatin (DDP) resistance. The purpose of this study was to explore the role of circ_0005667 in DDP resistance of endometrial carcinoma (EC) cells. The expression of circular RNA circ_0005667, microRNA-145-5p (miR-145-5p) and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in DDP-sensitive and DDP-resistant EC tissues and EC cells was determined by quantitative real-time PCR (qRT-PCR). The expression of apoptosis-related proteins, drug resistance-related proteins and IGF2BP1 proteins were detected by western blot. The half-maximal inhibitory concentration (IC 50 ) of DDP was determined using a cell counting kit-8 (CCK-8) assay. For functional assays, cell proliferation, migration, invasion and cell apoptosis were determined using 5-ethynyl-2'-deoxyuridine (EdU) assay, wound healing assay, transwell assay and flow cytometry assay, respectively. The binding relationship between miR-145-5p and circ_0005667 or IGF2BP1 was verified by dual-luciferase reporter assay. A xenograft experiment was applied to clarify the functional role of circ_0005667 in vivo . Levels of circ_0005667 and IGF2BP1 were markedly increased, whereas miR-145-5p was downregulated in DDP-resistant EC tissues and cells. The circ_0005667 deficiency could enhance DDP sensitivity, inhibit cell proliferation, migration and invasion and promote cell apoptosis in DDP-resistant EC cells in vitro . Mechanistically, circ_0005667 modulated IGF2BP1 expression through sponging miR-145-5p. In addition, miR-145-5p depletion attenuated circ_0005667 silencing-induced effects in EC cells. The regulation of miR-145-5p in DDP resistance involved low IGF2BP1 expression. In vivo experiments revealed that circ_0005667 silencing could improve the sensitivity of the tumor to DDP. Circ_0005667 enhanced DDP resistance in EC by elevating IGF2BP1 through sponging miR-145-5p.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 1 (IGF2BP1) Is a Prognostic Biomarker and Associated with Chemotherapy Responsiveness in Colorectal Cancer.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an RNA-binding protein and serves as a post-transcriptional fine-tuner regulating the expression of mRNA targets. However, the clinicopathological roles of IGF2BP1 in colorectal cancer (CRC) remains limited. Thus, we aimed to elucidate the clinical significance and biomarker potentials of IGF2BP1 in CRC. A total of 266 specimens from two sets of CRC patients were collected. IGF2BP1 expression was studied by immunohistochemical (IHC) staining. The Kaplan-Meier survival plot and a log-rank test were used for survival analysis. The Cox proportional hazards model was applied to determine the survival impact of IGF2BP1. Public datasets sets from The Cancer Genome Atlas (TCGA) and Human Cancer Metastasis Database (HCMDB), receiver operating characteristic (ROC) plotter, and two CRC cell lines, HCT-116 and DLD-1, were used for validating our findings. We showed that IGF2BP1 was overexpressed in tumor specimens compared to 13 paired normal parts by examining the immunoreactivity of IGF2BP1 (p = 0.045). The increased expression of IGF2BP1 in primary tumor parts was observed regardless of metastatic status (p < 0.001) in HCMDB analysis. IGF2BP1 expression was significantly associated with young age (59.6% vs. 46.7%, p-value = 0.043) and advanced stage (61.3% vs. 40.0%, p-value = 0.001). After controlling for confounding factors, IGF2BP1 remained an independent prognostic factor (HR = 1.705, p-value = 0.005). TCGA datasets analysis indicated that high IGF2BP1 expression showed a lower 5-year survival rate (58% vs. 65%) in CRC patients. The increased expression of IGF2BP1 in chemotherapy non-responder rectal cancer patients was observed using a ROC plotter. Overexpression of IGF2BP1 promoted the colony-forming capacity and 5-fluorouracil and etoposide resistance in CRC cells. Here, IGF2BP1 was an independent poor prognostic marker in CRC patients and contributed to aggressive phenotypes in CRC cell lines.

Silence of Insulin-Like Growth Factor 2 mRNA-Binding Protein 1 Prevents Vascular Smooth Muscle Cells Proliferation via Nuclear Factor of Activated T Cells Isoform-3/Ca2+/Calmodulin Pathway

Increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of atherosclerosis (AS), and the insulin like growth factor 2 (IGF2) is involved in AS through effects on VSMCs growth and migration. The IGF2 mRNA-binding protein 1(IGF2BP1) is a secreted protein that can bind to IGF2 and regulate its localization, however, whether IGF2BP1 could regulate VSMCs proliferation remains to be elucidated. This study aimed to investigate the role of IGF2BP1 in VSMCs proliferation and uncover the potential mechanism. Primary human aortic VSMCs that transfected with or without shRNA-IGF2BP1 were stimulated by platelet-derived growth factor-BB (PDGF-BB), and then cell proliferation, intracellular Ca2+ level, cell apoptosis and the expression of IGF2BP1, calmodulin (CaM) and cell cycle-related proteins were detected. RNA pull down assay was used to determine the interaction between IGF2BP1 and nuclear factor of activated T cells isoform-3 (NFATc3). We found that PDGF-BB promoted cell proliferation and enhanced IGF2BP1 protein expression in a concentration-dependent manner. The 10 μg/L PDGF-BB significantly increased intracellular Ca2+ level, NFATc3, CaM and calcineurin A protein expression, TUNEL-positive cells, and the expression of cell cycle-related proteins cyclin D1/E1/B1. However, knockdown of IGF2BP1 significantly blunted all these effects induced by PDGF-BB. In addition, IGF2BP1 could bind to NFATc3 RNA. Collectively, knockdown of IGF2BP1 could inhibit PDGFBB- induced VSMCs proliferation via targeting NFATc3/Ca2+/calmodulin pathway and disturbing the effect of NFATc3/ on cell cycle.

LINC00483 is regulated by IGF2BP1 and participates in the progression of breast cancer.

To explore the role of long intergenic non-coding ribonucleic acid 483 (LINC00483) in the development of breast cancer (BC) and its possible mechanism of action. LINC00483 expression level in BC tissues and cell lines was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). The association between LINC00483 expression and survival rate of BC patients was analyzed using Kaplan-Meier survival analysis. The binding relation between LINC00483 and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was verified via RNA immunoprecipitation (RIP) and RNA pull-down assays. The expression of IGF2BP1 in BC patients was determined using qRT-PCR. Moreover, the role of LINC00483 on the proliferative ability of BC cells was detected via cell counting kit-8 (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Whether LINC00483 exerts its effects under the regulation of IGF2BP1 was verified via reversal assay. The results of qRT-PCR showed that LINC00483 had a significantly high expression in BC tissues and corresponding cell lines, and it rose with the increase in tumor stage, which was higher in patients with metastasis. CCK8/EdU assay revealed that the proliferative ability of MDA-MB-231 and MCF-7 cell lines was enhanced by overexpression of LINC00483. It was confirmed by RIP and pull-down assays that IGF2BP1 could bind to LINC00483, and the expression of LINC00483 was significantly promoted after up-regulation of IGF2BP1. It was found via qRT-PCR that the expression of IGF2BP1 evidently rose in BC patients, which was positively related with the expression level of LINC00483. The results of reversal assay manifested that the function of LINC00483 on cell proliferation was regulated by IGF2BP1. LINC00483 has a significantly higher expression in BC tissues than that in para-carcinoma tissues, and its effect of promoting proliferation of BC cells may be regulated by IGF2BP1.

IGF2BP1 overexpression stabilizes PEG10 mRNA in an m6A-dependent manner and promotes endometrial cancer progression.

Rationale: N6-methyladenosine (m6A) mRNA methylation is the most abundant chemical posttranscriptional modification in mRNA and is involved in the regulation of a number of biological processes. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) has recently been reported as having the capacity to recognize m6A sites in mRNA and plays a role in regulating mRNA metabolization. However, it is unclear which genes IGF2BP1 targets to identify m6A sites and what are their respective functions in endometrial cancer (EC).Methods: Quantitative PCR, western blot and immunohistochemistry were used to measure IGF2BP1 expression in EC cell lines and tissues. Xenograft experiments were performed to examine the in vivo role of IGF2BP1 in EC cell growth. RNA-binding protein immunoprecipitation sequencing, methylated RNA-binding protein immunoprecipitation sequencing and RNA-sequencing were also conducted to identify potential IGF2BP1 targets involved in EC regulation. Co-immunoprecipitation and mass spectrometry were used to identify IGF2BP1-interacting proteins.Results: IGF2BP1 expression increased in EC, and high expression of this protein correlated with poor prognosis. IGF2BP1 overexpression/knockdown can promote (and inhibit) cell proliferation and regulate the tumor cell cycle and cancer progression, both in vivo and in vitro. Mechanistically, IGF2BP1 can recognize m6A sites in the 3' untranslated region (3'UTR) of Paternally Expressed Gene 10 (PEG10) mRNA and recruits polyadenylate-binding protein 1 (PABPC1) to enhance PEG10 mRNA stability, which consequently promotes PEG10 protein expression. Additionally, it would appear that a large number of PEG10 proteins bind p16 and p18 gene promoter sequences, thereby repressing expression and accelerating the cell cycle.Conclusion: This investigation found that IGF2BP1 has a crucial role in the m6A-dependent regulatory mechanism for endometrial cancer. This study provides new insights into our understanding of disease progression and provides another potential route for understanding biological functions.

LncRNA PCAT6 Regulated by YY1 Accelerates the Progression of Glioblastoma via miR-513/IGF2BP1.

Glioblastoma is one of the most frequent and aggressive primary tumor of glial brain tumors. Long non-coding RNA Prostate cancer-associated ncRNA transcript 6 (PCAT6) has been identified to influence the progression of many cancers, but its expression and functions in glioblastoma remain unclear. In this study, we intended to investigate the expression, functions and the corresponding mechanisms of PCAT6 in glioblastoma. We observed that PCAT6 expression was upregulated in glioblastoma tissues and cell lines and its high expression was due to the transcriptional activation by Yin Yang 1. miR-513 was a target of PCAT6 and Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) was a target of miR-513. Hence, PCAT6 upregulated IGF2BP1 expression via miR-513 in a competing endogenous RNAs manner. PCAT6 and IGF2BP1 functioned as oncogenes while miR-513 acted as a tumor suppressor gene in glioblastoma. PCAT6 and miR-513 modulated the proliferation and survival of glioblastoma cells via AKT signaling by mediating IGF2BP1. IGF2BP1 raised the expression of PCAT6 by increasing its stability. In conclusion, our results indicate that PCAT6/miR-513/IGF2BP1 positive feedback loop plays a crucial role in facilitating glioblastoma progression.

Mesenchymal stem cell-derived exosomes carrying microRNA-150 suppresses the proliferation and migration of osteosarcoma cells via targeting IGF2BP1.

BackgroundMicroRNA-150 (miR-150) plays a critical role in varied types of human cancers. In this study, we explored the effect and mechanism of mesenchymal stem cell (MSC)-derived exosomes (exo) carrying miR-150 (MSC-Exo-150) on the proliferation, migration, invasion, and apoptosis of osteosarcoma (OS) cells.MethodsMiR-150 expression in OS cell lines was assessed by quantitative reverse-transcription PCR (qRT-PCR). MSCs were transfected with cell-miR-67 or has-miR-150, and grouped as MSC-67 or MSC-150. Exosomes were isolated from each group, and separately named MSC-Exo-67, MSC-Exo-150 and MSC-Exo. MTT or flow cytometry assay was used to analyze the proliferation or apoptosis of U2SO and HOS cells, respectively. Wound healing or transwell assay was utilized to examine the migration or invasion of U2SO and HOS cells, respectively. The target relationship of miR-150 and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) was established using StarBase2.0 and verified by dual-luciferase reporter gene analysis. Xenografted tumor model was established in rats to confirm the inhibitory effect of MSC-Exo-150 on the growth of xenografted tumor in vivo.ResultsThe expression of miR-150 was downregulated in OS cell lines, and significantly higher in MSC-150 cells than that in MSCs. MiR-150 was overexpressed in MSC-Exo-150 group compared with MSC-Exo group. After transfection of MSC-Exo-150 into U2SO and HOS cells, cell viability, mobility and invasion rate were decreased, and the cell apoptosis was increased. MiR-150 targeted IGF2BP1 and IGF2BP1 expression was negatively modulated by miR-150. Overexpression of IGF2BP1 reversed the anti-tumor effect of MSC-Exo-150 on HOS cells.ConclusionsMSC-Exo-150 inhibited proliferation, migration, invasion, and induced apoptosis of OS cells by targeting IGF2BP1.

The mRNA-Binding Protein IGF2BP1 Restores Fetal Hemoglobin in Cultured Erythroid Cells from Patients with β-Hemoglobin Disorders

Sickle cell disease (SCD) and β-thalassemia are caused by structural abnormality or inadequate production of adult hemoglobin (HbA, α2β2), respectively. Individuals with either disorder are asymptomatic before birth because fetal hemoglobin (HbF, α2γ2) is unaffected. Thus, reversal of the switch from HbF to HbA could reduce or even prevent symptoms these disorders. In this study, we show that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is one factor that could accomplish this goal. IGF2BP1 is a fetal factor that undergoes a transcriptional switch consistent with the transition from HbF to HbA. Lentivirus delivery of IGF2BP1 to CD34+ cells of healthy adult donors reversed hemoglobin production toward the fetal type in culture-differentiated erythroid cells. Analogous studies using patient-derived CD34+ cells revealed that IGF2BP1-dependent HbF induction could ameliorate the chain imbalance in β-thalassemia or potently suppress expression of sickle β-globin in SCD. In all cases, fetal γ-globin mRNA increased and adult β-globin decreased due, in part, to formation of contacts between the locus control region (LCR) and γ-globin genes. We conclude that expression of IGF2BP1 in adult erythroid cells has the potential to maximize HbF expression in patients with severe β-hemoglobin disorders by reversing the developmental γ- to β-globin switch.

Insulin-like growth factor 2 mRNA-binding protein 1 promotes cell proliferation via activation of AKT and is directly targeted by microRNA-494 in pancreatic cancer.

BACKGROUNDStudies have shown that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) plays critical roles in the genesis and development of human cancers.AIMTo investigate the clinical significance and role of IGF2BP1 in pancreatic cancer.METHODSExpression levels of IGF2BP1 and microRNA-494 (miR-494) were mined based on Gene Expression Omnibus datasets and validated in both clinical samples and cell lines by quantitative real-time polymerase chain reaction and Western blot. The relationship between IGF2BP1 expression and clinicopathological factors of pancreatic cancer patients was analyzed. The effect and mechanism of IGF2BP1 on pancreatic cancer cell proliferation were investigated in vitro and in vivo. Analyses were performed to explore underlying mechanisms of IGF2BP1 upregulation in pancreatic cancer and assays were carried out to verify the post-transcriptional regulation of IGF2BP1 by miR-494.RESULTSWe found that IGF2BP1 was upregulated and associated with a poor prognosis in pancreatic cancer patients. We showed that downregulation of IGF2BP1 inhibited pancreatic cancer cell growth in vitro and in vivo via the AKT signaling pathway. Mechanistically, we showed that the frequent upregulation of IGF2BP1 was attributed to the downregulation of miR-494 expression in pancreatic cancer. Furthermore, we discovered that reexpression of miR-494 could partially abrogate the oncogenic role of IGF2BP1.CONCLUSIONOur results revealed that upregulated IGF2BP1 promotes the proliferation of pancreatic cancer cells via the AKT signaling pathway and confirmed that the activation of IGF2BP1 is partly due to the silencing of miR-494.

IGF2BP1 Significantly Enhances Translation Efficiency of Duck Hepatitis A Virus Type 1 without Affecting Viral Replication.

As a disease characterized by severe liver necrosis and hemorrhage, duck viral hepatitis (DVH) is mainly caused by duck hepatitis A virus (DHAV). The positive-strand RNA genome of DHAV type 1 (DHAV-1) contains an internal ribosome entry site (IRES) element within the 5′ untranslated region (UTR), structured sequence elements within the 3′ UTR, and a poly(A) tail at the 3′ terminus. In this study, we first examined that insulin-like growth factor-2 mRNA-binding protein-1 (IGF2BP1) specifically interacted with the DHAV-1 3′ UTR by RNA pull-down assay. The interaction between IGF2BP1 and DHAV-1 3′ UTR strongly enhanced IRES-mediated translation efficiency but failed to regulate DHAV-1 replication in a duck embryo epithelial (DEE) cell line. The viral propagation of DHAV-1 strongly enhanced IGF2BP1 expression level, and viral protein accumulation was identified as the key point to this increment. Collectively, our data demonstrated the positive role of IGF2BP1 in DHAV-1 viral proteins translation and provided data support for the replication mechanism of DHAV-1.

MicroRNA-4500 inhibits human glioma cell progression by targeting IGF2BP1

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) overexpression promotes glioma cell progression. The aim of the current study is to silence IGF2BP1 in glioma cells by the microRNA (miRNA) strategy. The bio-informatic analyses identified that microRNA-4500 (miR-4500) putatively targets 3′-UTR (3′-untranslated region) of IGF2BP1. In A172 cells and primary human glioma cells ectopic overexpression of the wild-type miR-4500 (but not the mutant form) downregulated IGF2BP1 and its target genes (Gli1, IGF2 and c-Myc). Functional studies show that ectopic miR-4500 overexpression inhibited glioma cell growth, survival, proliferation, migration and invasion. Conversely, in A172 cells miR-4500 inhibition, by a lentiviral construct, increased expression of IGF2BP1 and its targets, promoting cell survival, proliferation and migration. Furthermore, IGF2BP1 knockout by the CRISPR/Cas9 method inhibited A172 cell progression. Significantly, miR-4500 overexpression or miR-4500 inhibition was ineffective in IGF2BP1 knockout A172 cells. At last, we show that miR-4500 levels are downregulated in human glioma tissues, correlating with IGF2BP1 upregulation. Together, we conclude that miR-4500 inhibits human glioma cell progression by targeting IGF2BP1.

IGF2BP1 promotes LPS-induced NFκB activation and pro-inflammatory cytokines production in human macrophages and monocytes

BackgroundLipopolysaccharide (LPS)-induced macrophage/monocyte activation and pro-inflammatory cytokines production are important mediators for periodontitis progression. The current study tested the potential role of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in the process. MethodsTHP-1 human macrophages and primary human peripheral blood mononuclear cells (PBMCs) were treated with LPS. mRNA and protein expression of IGF2BP1 were tested by qPCR and Western blotting assay. IGF2BP1 expression was altered by shRNAs or CRISPR/Cas-9 gene editing methods. LPS-induced cytokine production was tested by ELISA assay. Cytokine mRNA expression was tested by the quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay. ResultsIn THP-1 human macrophages and PBMCs, treatment with LPS induced mRNA and protein expression of IGF2BP1. IGF2BP1 silencing (by targeted shRNAs) or CRISPR/Cas-9 knockout largely inhibited LPS-induced production of multiple pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Conversely, forced over-expression of IGF2BP1 facilitated LPS-induced pro-inflammatory cytokines production in THP-1 cells. For the mechanism study, we show that IGF2BP1 co-immunoprecipitated with p65-p52 nuclear factor kappa B (NFκB) complex in nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced p65-p52 nuclear translocation and NFκB activation were inhibited by IGF2BP1 silencing or CRISPR/Cas-9 knockout. ConclusionIGF2BP1 promotes LPS-induced NFκB signalling and transcriptional activation in human macrophages and monocytes.

Defining the Significance of Insulin‐like Growth Factor 2 mRNA‐Binding Protein 1 (IGF2BP1) Expression in both Human and Canine Osteosarcoma

Osteosarcoma is a malignant bone tumor that afflicts over 10,000 dogs and 450 adolescents yearly. Most dogs and approximately 25% of children succumb to metastatic disease. We identified elevated insulin‐like growth factor II mRNA binding protein 1 (IGF2BP1) as a biomarker of poor prognosis in canine osteosarcoma. IGF2BP1 is an oncofetal protein that regulates mRNA subcellular localization, nuclear export, stability and translation. This empowers IGF2BP1 to control expression of oncogene targets that contribute to cellular proliferation and resistance to chemotherapy. IGF2BP1 expression correlates with poor outcome in a variety of human cancers. We over‐expressed IGF2BP1 in a canine osteosarcoma cell line and used shRNA knockdown constructs to decrease expression in a human osteosarcoma cell line. IGF2BP1 over‐expressing clones show a 1.67 fold increase in doxorubicin resistance compared to control and IGF2BP1 expression correlates to IC50 values (r2 = 0.89). IGF2BP1 knockdown increased sensitivity to doxorubicin by ≥ 10 fold compared to control and significant reductions in cellular migration, invasion, proliferation, and tumor growth in nude mice were also observed (p &lt; 0.05). These results support the hypothesis that elevated expression of IGF2BP1 may contribute to tumor progression and inadequate therapeutic response. Microarray analysis of control and IGF2BP1 over‐expressing cells was used to detect global changes in gene expression and to identify potential targets for IGF2BP1. Differentially expressed genes were cross‐referenced to the RNA‐Binding Protein Immunoprecipitation database to identify direct mRNA targets bound by IGF2BP1 in osteosarcoma. We identified 162 genes that were differentially expressed (FC ≥2, FDR&lt; 0.05). Pathway analysis identified enrichment for genes involved in regulation of cell adhesion, migration, and extracellular matrix. We identified 13 differentially expressed genes that directly bind IGF2BP1 and are involved in cell adhesion and protein kinase activity. We investigated mechanisms that contribute to increased IGF2BP1 expression in panels of human and canine osteosarcoma cell lines and tumors. Using qPCR analysis, we observed genomic amplification in 35% of canine tumors and cell lines and correlated amplification with IGF2BP1 transcript expression (p &lt; 0.05, r2 = 0.8). Genomic amplification was not observed in human cell lines. Significant loss of 3’UTR regulatory sequences was observed in 20% of canine cell lines (p&lt;0.05). IGF2BP1 promoter analysis indicated that most regulatory elements were located within 580 bp of the start site of translation in both species including transcriptional response elements for Myc and β‐catenin. Overall, our data suggests that increased IGF2BP1 expression contributes to the development and progression of human and canine osteosarcoma. Multiple mechanisms contribute to elevated IGF2BP1 expression including gene amplification and altered mRNA expression and stability. These results can be used to develop new treatment strategies that target elevated IGF2BP1.Support or Funding InformationMorris Animal Foundation, CVMBS Research Council, Cancer League of ColoradoThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-423–5p inhibits the progression of trophoblast cells via targeting IGF2BP1

IntroductionPreeclampsia (PE) is one of the leading causes of maternal and fetal mortality globally. The imbalance of trophoblast homeostasis is closely linked with the pathogenesis of PE. MicroRNA-423–5p (miR-423–5p) has been reported to be abnormally expressed in placenta and blood plasma of pregnant women with PE. In the present study, miR-423–5p expression in blood plasma of pregnant women with PE and healthy pregnant women was detected. Also, the roles and molecular mechanisms of miR-423–5p in the development of trophoblast cells were further investigated. MethodsExpression of miR-423–5p and insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) mRNA was detected by RT-qPCR assay. Protein expression of IGF2BP1, Bcl-2 and Bax was determined using western blot assay. Cell migratory and invasive capacities were assessed by transwell migration and invasion assay. Cell apoptotic rate was determined using flow cytometry via the double-staining of Annexin V-FITC/Propidium Iodide. The interaction between miR-423–5p and IGF2BP1 was demonstrated by bioinformatics analysis and luciferase reporter assay. ResultsMiR-423–5p was highly expressed in blood plasma of pregnant women with PE. MiR-423–5p inhibited migration, invasion and proliferation as well as induced apoptosis in HTR-8/SVneo cells. Further investigation revealed that IGF2BP1 was a target of miR-423–5p. Moreover, IGF2BP1 overexpression promoted migration, invasion and proliferation, suppressed apoptosis, and weakened miR-423–5p function in HTR-8/SVneo cells. DiscussionMiR-423–5p inhibited migration, invasion and proliferation as well as induced apoptosis by targeting IGF2BP1 in HTR-8/SVneo cells, presenting a novel molecular basis implicated in PE pathogenesis.