IGF2R Research Articles

A novel miRNA located in the GATA4 gene regulates the expression of IGF-1R and AKT1/2 genes and controls cell proliferation.

GATA4 gene is a zinc-finger transcription factor known to be involved in cardiogenesis and the progression of different cancer types. Its diverse functions might be attributed to noncoding RNAs that could be embedded within its sequence. Here, we predicted a stable RNA stem-loop structure that is located in the second intron of the GATA4 gene. Available microRNA (miRNA) sequencing data and molecular genetics tools confirmed the identity of a mature miRNA (named GATA4-miR1) originating from the predicted stem-loop. In silico analysis predicted IGF-1R and AKT1/2 genes as potential targets for GATA4-miR1. Indeed, direct interactions between GATA4-miR1 and 3' untranslated regions sequences of IGF-1R and AKT1/2 genes were documented by dual luciferase assay. In addition, overexpression of GATA4-miR1 in SW480 cells resulted in the reduction of IGF-1R and AKT1/2 genes' expression, detected by reverse transcription quantitative (RT-q) polymerase chain reaction and Western blot analysis. This observation was consistent with a deduced negative correlation between the expression patterns of GATA4-miR1 and IGF-1R genes during cardiomyocyte differentiation. Moreover, overexpressing GATA4-miR1 in SW480 and PC3 cells resulted in a significant increase of the sub-G1 population in both cell lines, as detected by propidium iodide flow cytometry. Further analysis by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay indicated a reduction in the survival and proliferation rates of SW480 cells overexpressing GATA4-miR1, but no impact was observed on apoptosis progression, as indicated by Annexin-V flow cytometry. Overall, GATA4-miR1 represents a promising candidate for further research in the fields of cancer and cardiovascular therapeutics.

Oocyte IVM or vitrification significantly impairs DNA methylation patterns in blastocysts as analysed by single-cell whole-genome methylation sequencing.

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from invivo, fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the invivo group (P<0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and invivo groups, 1578 DMRs were identified between the V_IVF and invivo groups and 151 DMRs were identified between the V_IVF and IVF groups. For imprinted genes, methylation levels of insulin-like growth factor 2 receptor (IGF2R) and protein phosphatase 1 regulatory subunit 9A (PPP1R9A) were lower in the IVF and V_IVF groups than in the invivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and invivo groups. Genes with DMRs between the IVF and invivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and invivo groups were enriched in fertilisation and vitrification-vulnerable pathways. The results of this study indicate that differences in the methylation of critical DMRs may contribute to the differences in quality between invitro- and invivo-derived embryos.

Genome-wide identification of imprinted genes in pigs and their different imprinting status compared with other mammals

Genomic imprinting often results in parent-of-origin specific differential expression of maternally and paternally inherited alleles and plays an essential role in mammalian development and growth. Mammalian genomic imprinting has primarily been studied in mice and humans, with only limited information available for pigs. To systematically characterize this phenomenon and evaluate imprinting status between different species, we investigated imprinted genes on a genome-wide scale in pig brain tissues. Specifically, we performed bioinformatics analysis of high-throughput sequencing results from parental genomes and offspring transcriptomes of hybrid crosses between Duroc and Diannan small-ear pigs. We identified 11 paternally and five maternally expressed imprinted genes in pigs with highly stringent selection criteria. Additionally, we found that the KCNQ1 and IGF2R genes, which are related to development, displayed a different imprinting status in pigs compared with that in mice and humans. This comprehensive research should help improve our knowledge on genomic imprinting in pigs and highlight the potential use of imprinted genes in the pig breeding field.

Development of an IGF1R longevity variant mouse line using CRISPR/Cas9 genome editing.

An insulin-like growth factor-1 receptor (IGF1R) variant in exon 6 (Arg-407-His) in Ashkenazi Jewish centenarians was previously found to be associated with reduced IGF1R activity. To further study this longevity associated IGF1R variant, we generated a novel mouse line carrying the R407H variant in exon 6 of the Igf1r gene by employing CRISPR/Cas9 genome editing technology. Here, we show that the Igf1r gene can be edited in mouse embryos by zygotic electroporation of Cas9 protein and a single-guide RNAs together with a single stranded oligonucleotide donor containing the desired key nucleotide changes at the Igf1r locus. Sequence analysis of F0 and F1 mice following targeted editing demonstrated the robustness of this approach in mice using CRISPR/Cas9 directed homologous recombination (HDR). Western blot analysis indicates that mice heterozygous for the variant have a significant decrease in IGF1R phosphorylation in various tissues, including skeletal muscle, compared to wildtype. In addition, depletion of IGF1R signaling specifically in skeletal muscle of progeroid Ercc1 -/Δ mice resulted in extended health span and median lifespan providing the rationale for long term lifespan studies in Igf1r hR407H variant mice. This mouse line will be a valuable genetic tool to help determine the impact of IGF1R signaling on aging and longevity. The CRISPR editing approach represents a prototype for generating additional longevity associated gene variant mouse lines to study relevance to human exceptional longevity.

The effects of vitrification after equilibration in different concentrations of cryoprotectants on the survival and quality of bovine blastocysts.

This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG+2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG+16.5% DMSO+0.5M sucrose. After warming, embryos were cultured for 48hr. Re-expansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane-damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p<.05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN-t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression.

Possible key microRNAs and corresponding molecular mechanisms for atrial fibrillation.

Objective:We aimed to find crucial microRNAs (miRNAs) associated with the development of atrial fibrillation (AF), and then try to elucidate the possible molecular mechanisms of miRNAs in AF.Methods:The miRNA microarray, GSE68475, which included 10 right atrial appendage samples from patients with persistent AF and 11 samples from patients with normal sinus rhythm, was used for the analysis. After data preprocessing, differentially expressed miRNAs were screened using limma. Target genes of miRNAs were predicted using miRWalk2.0. We then conducted functional enrichment analyses for miRNA and target genes. Protein-protein interaction (PPI) network and module analyses for target genes were performed. Finally, transcription factors (TFs)-target genes regulatory network was predicted and constructed.Results:Seven genes, including CAMK2D, IGF2R, PPP2R2A, PAX6, POU3F2, YWHAE, and AP2A2, were targeted by TFs. Among these seven genes, CAMK2D (targeted by miR-31-5p), IGF2R (targeted by miR-204-5p), PAX6 (targeted by miR-223-3p), POU3F2 (targeted by miR-204-5p), YWHAE (targeted by miR-31-5p), and AP2A2 (targeted by miR-204-5p) belonged to the top 10 degree genes in the PPI network. Notably, MiR-204-5p, miR-31-5p, and miR-223-3p had more target genes. Besides, CAMK2D was enriched in some pathways, such as adrenergic signaling in cardiomyocytes pathway and cAMP signaling pathway. YWHAE was enriched in the Hippo signaling pathway.Conclusion:miR-31-5p played a crucial role in cardiomyocytes by targeting CAMK2D and YWHAE via cAMP and Hippo signaling pathways. miR-204 was involved in the progression of AF by regulating its target genes IGF2R, POU3F2, and AP2A2. On the other hand, miR-223-3p functioned in AF by targeting PAX6, which was associated with the regulation of apoptosis in AF. This study would provide a theoretical basis and potential therapeutic targets for the treatment of AF.

English

The multifunctional transmembrane mannose-6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2-R) has been proposed as a putative prognostic marker in breast cancer. Loss of heterozygosity (LOH) with mutations in the remaining allele have been described at the gene level. We previously reported significant decrease of M6P/IGF2-R at the protein level in cancer cells than normal peritumoral ducts of breast tumors which may indicate an increased release of the receptor from the cell membrane to the blood. To assess that hypothesis we measured in this study M6P/IGF2-R levels in serum of women with breast cancer by competitive enzyme-linked immunosorbent assay (ELISA) that allows a detectable sensitivity of 300 ng/mL. The results were obtained referring to a standard curve consisting in varying concentrations of human purified M6P/IGF2-R. Women sera consisted of cancer-free sera (n=39) or serum from women with breast cancer (n=39) were preincubated with anti-M6P/IGF2-R immunoglobulin Y (IgY) antibodies. High levels of M6P/IGF2-R (p<0.01) were found in serum from women with breast cancer in comparison with cancer-free serum. Overall the results support the hypothesis of increased release of the receptor from cancer cells to the blood and add one more argument for M6P/IGF2-R studying as a potential breast cancer marker. Key words: Mannose-6-Phosphate/Insulin-like Growth Factor-2 Receptor (M6P/IGF2-R), loss of heterozygosity (LOH), suppressor gene, breast cancer, competitive enzyme-linked immunosorbent assay (ELISA), IgY, biological marker.

IGFBP-2 in cervical cancer development

ObjectiveIncreased expression of insulin-like growth factor binding protein 2, IGFBP-2, is associated with many cancers, though its role in cervical cancer is unclear. The aim of this study was to investigate the expression of IGFBP-2 protein and the transcriptomics profile of genes involved in the IGF signaling pathway during cervical cancer development. DesignImmunohistochemical expression of IGFBP-2 protein was semi-quantitatively assessed in tissue microarrays containing 9 normal cervix, 10 low grade cervical intraepithelial neoplasia (LGCIN), 10 high grade cervical intraepithelial neoplasia (HGCIN) and 42 squamous cell carcinoma (SCC) cases. The gene expression profiles of IGFBP-2, IGF-1, IGF-1R, PTEN, MDM2, AKT1 and TP53 were determined in three cervical tissue samples each from normal cervix, human papillomavirus (HPV)-infected LGCIN, HGCIN and SCC, using Human Transcriptome Array 2.0. ResultsIGFBP-2 protein was highly expressed in the cytoplasm of SCC cells compared to normal cervix (p = .013). The expression was not significantly associated with CIN grade or SCC stage. Transcriptomics profiling demonstrated upregulation of IGFBP-2 and TP53 in HGCIN and SCC compared to normal cervix. IGF-1, IGF-1R and PTEN genes were downregulated in all histological groups. IGF-1 gene was significantly downregulated in SCC (p = .031), while PTEN gene was significantly downregulated in HGCIN (p = .012), compared to normal cervix. MDM2 and AKT1 genes were downregulated in LGCIN and HGCIN, while upregulated in SCC. ConclusionIn cervical carcinogenesis, IGFBP-2 appears to play an oncogenic role, probably through an IGF-independent mechanism.

Targeted Therapy of Osteosarcoma with Radio-labelled Monoclonal Antibody to an Insulin-like Growth Factor-2 Receptor (IGF2R)

Targeted Therapy of Osteosarcoma with Radio-labelled Monoclonal Antibody to an Insulin-like Growth Factor-2 Receptor (IGF2R)

Differential Effects of Insulin and IGF1 Receptors on ERK and AKT Subcellular Distribution in Breast Cancer Cells.

Insulin and insulin-like growth factor-1 (IGF1) have important roles in breast cancer development. The recent identification of nuclear insulin (INSR) and IGF1 (IGF1R) receptors provides a novel paradigm in the area of signal transduction. The fact that INSR and IGF1R can function as transcription factors, capable of binding DNA and controlling transcription, adds a new layer of biological complexity by conferring upon cell-surface receptors the ability to regulate genomic events. The present study was designed to assess the hypothesis that insulin and IGF1 pathways elicit differential effects on subcellular distribution and activation of ERK1/2 and AKT. To this end, MCF7 breast cancer-derived cell lines with specific INSR or IGF1R disruption were employed. In addition, small interfering RNA technology was used to specifically down-regulate INSR or IGF1R expression in T47D breast cancer cells. DNA affinity chromatography assays were conducted to address the specific binding of ERK1/2 and AKT to the IGF1R promoter region. We demonstrate that both INSR and IGF1R exhibit a nuclear localization in breast cancer-derived cells. In addition, the insulin and IGF1 pathways have different effects on the subcellular distribution (and, particularly, the nuclear presence) of ERK1/2 and AKT molecules. Both cytoplasmic mediators are capable of binding and transactivating the IGF1R promoter. In conclusion, our data are consistent with the notion that, in addition to their classical roles as targets for insulin-like molecules, both ERK1/2 and AKT are involved in transcriptional control of the IGF1R gene. This previously unrecognized regulatory loop may provide mechanistic advantages to breast cancer cells. Given the potential role of INSR and IGF1R as therapeutic targets in oncology, it will be of clinical relevance to address the future use of nuclear receptors and their downstream cytoplasmic mediators as biomarkers for INSR/IGF1R targeted therapy.

Upregulation of IGF2R evades lysosomal dysfunction-induced apoptosis of cervical cancer cells via transport of cathepsins

Cervical cancer is the most common gynecological malignancy in the world; however, the survival rates of advanced-stage and recurrent cervical cancer patients remain poor. The multifaced protein insulin-like growth factor 2 receptor (IGF2R) has various ligands, represented as IGF-2 and mannose-6-phosphate (M6P)-tagged proteins. Regarding its antagonistic activity as an IGF1R signal, IGF2R is currently considered a tumor suppressor gene, whereas its significance as an M6P receptor is still unclear. Here, on the basis of transcriptome analysis of TCGA and GEO open datasets, we show that IGF2R is upregulated and correlated with poor prognosis in cervical cancer. Several experiments using cervical cancer cell lines revealed that IGF2R depletion induced apoptosis, decreased cell viability, and increased vulnerability to certain anticancer drug cisplatin. In contrast to its negligible impact in IGF1R signaling, loss of IGF2R disrupted the Golgi-to-lysosome transport of M6P-tagged cathepsins, resulting in decreased lysosomal activity, with their abnormal accumulation and dysfunction of both autophagy and mitophagy, which cause the accumulation of misfolded proteins and production of reactive oxygen species. Taken together, IGF2R has an oncogenic role through transportation of M6P-tagged cargo in cervical cancer and can be used as a predictive biomarker for prognostic classification.

INTERACTIONS BETWEEN GENES FROM AGING-RELATED PATHWAYS: IMPACT ON HUMAN LONGEVITY

Role of genetic interactions (GxG) in human longevity remains poorly understood. We hypothesized that GxG between genes from biologically connected pathways involved in aging may impact longevity. To test this hypothesis, we selected 53 candidate genes from the aging-related pathways (IGF-1/AKT/FOXO3A, TP53/P21/P16, and mTOR/S6K mediated) that are known to jointly influence outcomes of cell responses to stress and damage, such as apoptosis, senescence, growth/proliferation, and autophagy. We evaluated the effects of interactions between SNPs in these genes on longevity in LLFS and CARe data. RESULTS: The IGF1R, PPARGC1A and BCL2 genes were consistently involved in top GxG effects (p<10-6) on survival in the oldest old (85+ and 95+). One SNP, rs2970870 in PPARGC1A gene, was broadly involved in significant interaction effects on survival 96+ (p<10-7) when paired with SNPs in IGF1R and NFKB1 genes. This SNP individually was associated with survival with nominal significance only; therefore, it would have not been selected in a GWAS. We conclude that interactions between genes from aging-related pathways that regulate cell responses and resilience to damage may have major impact on human longevity and contribute to its genetic heterogeneity. The research was supported by the NIA/NIH grants R01AG062623, U19AG063893, P01AG043352.

Sensitive glycoprofiling of insulin-like growth factor receptors isolated from colon tissue of patients with colorectal carcinoma using lectin-based protein microarray

Glycosylation of cell receptors influences their function and development of tumour induces changes in glycosylation. Cell growth depends on the activation of receptors which bind growth factors and the insulin-like growth factor (IGF) receptors are among the most important ones. Usually, only small quantities of isolated receptors are available thus there is a need of suitable assay to study receptors glycosylation. Therefore, we developed a lectin-based reverse-phase protein microarray method for screening the glycosylation pattern of receptors in picomolar (pM) concentrations. The method was applied to glycoprofile IGF1 and IGF2 receptors and the solubilised membrane proteins isolated from tumour and non-tumour colon tissue of patients with colorectal cancer. We found that common to both receptors was partial overlapping of the major glycan structures with those present in the entire glycome of membrane proteins. In contrast, receptors possess higher level of α2,3 sialic acid residues and lower level of tri-/tetra-antennary complex type N-glycans and terminal mannose in high-mannose structures. Increased levels of fucosylation and branched mannose structures were observed in both receptors derived from tumour tissue compared to non-tumour tissue. The described method enabling glycan analysis of receptors has a big application potential in e.g. biomarker research, biology and diagnostics.

EP1.03-35 Prevalence, Clinicopathologic Characteristics, and Molecular Associations of IGF1R Mutations in East Asian Patients with NSCLC

IGF1R is a ubiquitous receptor tyrosine kinase that plays critical roles in cell proliferation, growth and survival. Clinical studies have demonstrated upregulation of IGF1R mediated signaling in a number of malignancies including colon, breast, and lung cancers. The aim of this study is to investigate mutations and prognosis of non-small cell lung cancer (NSCLC) harboring IGF1R mutations. A total of 812 patients with non-small-cell lung cancer were recruited between July 2012 and December 2016. The status of IGF1R mutations and other genes were detected by next generation sequencing. IGF1R gene mutation rate was 1.60% (13/812) in non-small cell lung cancer, including N977I (2 patients), S751T (1 patient), E1043D (1 patient), G171W (1 patient), E563K (1 patient), R275C (1 patient), F921[2>1] (1 patient), E712K (1 patient), R222W (1 patient), D1024A (1 patient), A760T (1 patient), and K533N (1 patient), and median overall survival (OS) for these patients was 9.0 months. Among them, all patients were IGF1R gene with co-occurring mutations. Briefly, patients with (n=3) or without (n=10) co-occurring EGFR mutations had a median OS of 6.0 months and 11.0 months respectively (P=0.10); patients with (n=12) or without (n=1) co-occurring TP53 mutations had a median OS of 18.0 months and 8.0 months respectively (P=0.68); patients with (n=4) or without (n=9) co-occurring KRAS mutations had a median OS of 14.5 months and 7.0 months respectively (P=0.76); patients with (n=5) or without (n=8) co-occurring NF1 mutations had a median OS of not up to now and 6.5 months respectively (P=0.24). EGFR, TP53, KRAS, NF1 gene accompanied may have less correlation with IGF1R mutation in NSCLC patients. We report different mutations than those previously reported, which emphasizes the importance of personalized medicine that could be empowered by the use of bioinformatics tools in the diagnostic process and therapeutic approaches.

Regulation of chitosan-mediated differentiation of human olfactory receptor neurons by insulin-like growth factor binding protein-2

Olfaction is normally taken for granted in our lives, not only assisting us to escape from dangers, but also increasing our quality of life. Although olfactory neuroepithelium (ON) can reconstitute its olfactory receptor neurons (ORNs) after injury, no adequate treatment for olfactory loss has yet emerged. The present study investigates the role of glycosaminoglycans (GAGs) in modulating olfactory neuronal homeostasis and elucidates the regulatory mechanism. This work isolates and cultures human olfactory neuroepithelial cells (HONCs) with various GAGs for 7 days, and find that chitosan promotes ORN maturation, expressing olfactory marker protein (OMP) and its functional components. Growth factor protein array, ELISA and western blot analysis reveal that insulin-like growth factor binding protein 2 (IGFBP2) shows a higher level in chitosan-treated HONCs than in controls. Biological activity of insulin-like growth factor-1 (IGF-1), IGF-2 and IGF-1 receptor (IGF1R) is further investigated. Experimental results indicate that IGF-1 and IGF-2 enhance the growth of immature ORNs, expressing βIII tubulin, but decrease mature ORNs. Instead, down-regulation of phosphorylated IGF1R lifts the OMP expression, and lowers the βIII tubulin expression, by incubation with the phosphorylated inhibitor of IGF1R, OSI-906. Finally, the effect of chitosan on ORN maturity is antagonized by concurrently adding IGFBP2 protease, matrix metallopeptidase-1. Overall, our data demonstrate that chitosan promotes ORN differentiation by raising the level of IGFBP2 to sequestrate the IGFs-IGF1R signaling. Statement of SignificanceOlfactory dysfunction serves as a crucial alarm in neurodegenerative diseases, and one of its causes is lacking of sufficient mature olfactory receptor neurons to detect odorants in the air. However, the clinical treatment for olfactory dysfunction is still controversial. Chitosan is the natural linear polysaccharide and exists in rat olfactory neuroepithelium. Previously, chitosan has been demonstrated to mediate the differentiation of olfactory receptor neurons in an in vitro rat model, but the mechanism is unknown. The study aims to evaluate the role and mechanism of chitosan in an in vitro human olfactory neurons model. Overall, these results reveal that chitosan is a potential agent for treating olfactory disorder by the maintenance of olfactory neural homeostasis. This is the first report to demonstrate that chitosan promotes differentiation of olfactory receptor neurons through increasing IGFBP2 to sequestrate the IGFs-IGF1R.

Detection and targeting insulin growth factor receptor type 2 (IGF2R) in osteosarcoma PDX in mouse models and in canine osteosarcoma tumors

Osteosarcoma (OS) represents 3.4% of all childhood cancers with overall survival of 70% not improving in 30 years. The consistent surface overexpression of insulin-like growth factor-2 receptor (IGF2R) has been reported in commercial and patient-derived xenograft (PDX) OS cell lines. We aimed to assess efficacy and safety of treating PDX and commercial OS tumors in mice with radiolabeled antibody to IGF2R and to investigate IGF2R expression on canine OS tumors. IGF2R expression on human commercial lines 143B and SaOS2 and PDX lines OS-17, OS-33 and OS-31 was evaluated by FACS. The biodistribution and microSPECT/CT imaging with 111Indium-2G11 mAb was performed in 143B and OS-17 tumor-bearing SCID mice and followed by radioimmunotherapy (RIT) with 177Lutetium-2G11 and safety evaluation. IGF2R expression in randomly selected canine OS tumors was measured by immunohistochemistry. All OS cell lines expressed IGF2R. Biodistribution and microSPECT/CT revealed selective uptake of 2G11 mAb in 143B and OS-17 xenografts. RIT significantly slowed down the growth of OS-17 and 143B tumors without local and systemic toxicity. Canine OS tumors expressed IGF2R. This study demonstrates the feasibility of targeting IGF2R on OS in PDX and spontaneous canine tumors and sets the stage for further development of RIT of OS using comparative oncology.

Effects of 2,3',4,4'5-pentachlorobiphenyl exposure during pregnancy on epigenetic imprinting and maturation of offspring's oocytes in mice.

Polychlorinated biphenyls (PCBs) are a class of organic pollutants that have been widely found in the environment. The chemical 2,3',4,4'5-pentachlorobiphenyl (PCB118) is an important dioxin-like PCB compound with strong toxicity. PCB118 can accumulate in adipose tissue, serum and milk in mammals, and it is highly enriched in the follicular fluid. In this study, pregnant mice were exposed to 0, 20 and 100μg/kg/day of PCB118 during pregnancy at the fetal primordial germ cell migration stage. The methylation patterns of the imprinted genes H19, Snrpn, Peg3 and Igf2r as well as the expression levels of Dnmt1, 3a, 3b and 3l, Uhrf1, Tet2 and Tet3 in fully grown germinal vesicle oocytes were measured in offspring. The rates of in vitro maturation, in vitro fertilization, oocyte spindle and chromosomal abnormalities were also calculated. The results showed that prenatal exposure to PCB118 altered the DNA methylation status of differentially methylated regions in some imprinted genes, and the expression levels of Dnmt1, 3a, and 3l, Uhrf1 and Tet3 were also changed. In addition, PCB118 disturbed the maturation process of progeny mouse oocytes in a dose-dependent manner. Therefore, attention should be paid to the potential impacts of PCB118-contaminated dietary intake during pregnancy on the offspring's reproductive health.

Chlorella vulgaris Modulates Genes and Muscle-Specific microRNAs Expression to Promote Myoblast Differentiation in Culture.

Background Loss of skeletal muscle mass, strength, and function due to gradual decline in the regeneration of skeletal muscle fibers was observed with advancing age. This condition is known as sarcopenia. Myogenic regulatory factors (MRFs) are essential in muscle regeneration as its activation leads to the differentiation of myoblasts to myofibers. Chlorella vulgaris is a coccoid green eukaryotic microalga that contains highly nutritious substances and has been reported for its pharmaceutical effects. The aim of this study was to determine the effect of C. vulgaris on the regulation of MRFs and myomiRs expression in young and senescent myoblasts during differentiation in vitro. Methods Human skeletal muscle myoblast (HSMM) cells were cultured and serial passaging was carried out to obtain young and senescent cells. The cells were then treated with C. vulgaris followed by differentiation induction. The expression of Pax7, MyoD1, Myf5, MEF2C, IGF1R, MYOG, TNNT1, PTEN, and MYH2 genes and miR-133b, miR-206, and miR-486 was determined in untreated and C. vulgaris-treated myoblasts on Days 0, 1, 3, 5, and 7 of differentiation. Results The expression of Pax7, MyoD1, Myf5, MEF2C, IGF1R, MYOG, TNNT1, and PTEN in control senescent myoblasts was significantly decreased on Day 0 of differentiation (p<0.05). Treatment with C. vulgaris upregulated Pax7, Myf5, MEF2C, IGF1R, MYOG, and PTEN in senescent myoblasts (p<0.05) and upregulated Pax7 and MYOG in young myoblasts (p<0.05). The expression of MyoD1 and Myf5 in young myoblasts however was significantly decreased on Day 0 of differentiation (p<0.05). During differentiation, the expression of these genes was increased with C. vulgaris treatment. Further analysis on myomiRs expression showed that miR-133b, miR-206, and miR-486 were significantly downregulated in senescent myoblasts on Day 0 of differentiation which was upregulated by C. vulgaris treatment (p<0.05). During differentiation, the expression of miR-133b and miR-206 was significantly increased with C. vulgaris treatment in both young and senescent myoblasts (p<0.05). However, no significant change was observed on the expression of miR-486 with C. vulgaris treatment. Conclusions C. vulgaris demonstrated the modulatory effects on the expression of MRFs and myomiRs during proliferation and differentiation of myoblasts in culture. These findings may indicate the beneficial effect of C. vulgaris in muscle regeneration during ageing thus may prevent sarcopenia in the elderly.

Endometrial genome-wide DNA methylation patterns of Guanzhong dairy goats at days 5 and 15 of the gestation period

Uterine receptivity for the embryo is established and maintained through a series of precise cellular and molecular events, such as DNA methylation. There have been no studies to elucidate entire genome DNA methylation changes associated with embryo receptivity development of the endometrium (RE). In the present study, there was development of a complete genome-wide DNA methylome maps of the RE using whole-genome bisulphite sequencing and bioinformatics analysis. As many as 163.06 Gb of sequencing data averaging 81.53 Gb per sample were obtained for genome bisulphite sequencing of endometrium samples. There were distinct genome-wide DNA methylation patterns in pre-receptive endometrium (PE; Day 5 of gestation) and RE (Day 15 of gestation). There were as many as 16,467 differentially methylated regions (DMRs); 21,391 DMRs were less methylated in RE samples compared with PE samples (P-values ≤ 0.05 and |log2 (fold change)| ≥ 2). Compared with PE samples, methylation ratios of IGF2BP2, ACOX2, PTGDS, VEGFB and PTGDR2 genes were markedly less in RE samples (P-value ≤ 0.05 and |log2 (fold change)| ≥ 2). Conversely, in RE samples there was a markedly greater methylation ratio of IGFBP3 and IGF1R genes. The results of KEGG analysis indicated that these genes were involved in the signalling pathways for insulin, mitogen-activated protein kinase, gonadotropin-releasing hormone, vascular endothelial growth factor and progesterone-mediated oocyte maturation, which participated in differential regulation of goat endometrial development during receptive and prereceptive phases. The results of previous and the present study indicate resulting proteins of IGF2BP2, PTGDS, VEGFB, PGR, IGFBP3 and IGF1R gene expression may have important functions in regulating endometrial receptivity for the embryo.

Abstract 2479: A survey of molecular factors of DNA methylation and demethylation in a broad range of cancer cell lines

Abstract DNA methylation has a powerful influence on cellular processes. It plays important roles in cancer initiation and progression. Cancer cells exhibit frequent epigenomic alterations that affect expression of genes involved in apoptosis, cell proliferation, and DNA repair. We used expression information of known methylating and demethylating genes in cancer cell lines to analyze their association with methylation levels of individual probes and median epigenome-wide methylation levels. We utilized RNA-seq gene expression measures and Illumina Infinium HumanMethylation450 BeadChip DNA methylation data of 645 cancer cell lines from 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer resources. We analyzed 73 genes, products of which are involved in methylation and demethylation processes, and examined correlations between expression levels of these genes and methylation status of individual probes and epigenome-wide. In a combined analysis across all cancer types, we observed statistically significant correlations between expression of global DNA methyltransferase genes, DNMT1, DNMT3A, and DNMT3B, and probe methylation levels in 566, 909, and 230 genes, respectively. In particular, DNMT3A expression was associated with methylation status of growth factor and receptor genes FGF1, FGFR4, and EGFR. Expression of hydroxymethylating genes TET1, TET2, and TET3 was correlated with methylation status of probes in 363, 1, and 1055 genes, respectively, including associations of TET3 expression with methylation of probes in the FGF1, KIT, FHIT, IGF1R, IGF2BP2, and PARP3 genes, which may affect growth, proliferation, and DNA repair of tumor cells. APOBEC1, CBX2, UHRF1 and ZBTB38, products of which are involved in various molecular steps of DNA methylation or demethylation, had the largest numbers of associations in different cancer types. For example, in chronic lymphocytic leukemia cell lines we observed a strong negative correlation between ZBTB38 expression, the product of which binds methylated DNA, and methylation levels of probes in 204 genes. We also found a strong correlation between UHRF1 gene expression and methylation levels of probes in 150 genes in chronic lymphocytic leukemia cell lines, in agreement with reported roles of the product of this gene in binding DNA methyltransferases and affecting DNMT1 methylation activity. These results provide a better understanding of molecular mechanisms of epigenetic dysregulation in cancer cells and may provide strategies for future analysis of molecular targets of drug response that may be affected via epigenetic processes. Citation Format: Suleyman Vural, Julia Krushkal. A survey of molecular factors of DNA methylation and demethylation in a broad range of cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2479.