Egl-9 Family Hypoxia Inducible Factor Research Articles

Long non-coding RNA LINC00662 promotes proliferation and migration of breast cancer cells via regulating the miR-497-5p/EglN2 axis.

Previous reports indicated that long noncoding RNA 662 (LINC00662) plays a crucial role in several human cancers. Here, we studied the expression pattern of LINC00662 and explored its function in human breast cancer. The expression level of LINC00662 was determined in human breast cancer cell lines and tissues by real-time quantitative polymerase chain reaction (RT-qPCR). Cytoplasmic and nuclear RNA from MDA-MB-157 cells were extracted to analyze the subcellular location of LINC00662. Moreover, the MTT assay, wound-healing assay, colony-forming assay and transwell assay were employed in MDA-MB-157 cells to detect the effect of LINC00662 on cell apoptosis, invasion, migration and proliferation, respectively. LINC00662-specific miRNA and miRNA-gene axis were examined in a dual-luciferase reporter assay and Western blot. We found that LINC00662 was overexpressed in both breast cancer cell lines and tissue compared to normal breast cell lines and healthy breast tissue. Analysis of subcellular localization revealed that LINC00662 was mainly found in the cytoplasm. Furthermore, LINC00662 silencing reduced cell viability and inhibited the proliferation, migration and invasion of MDA-MB-157 cells. Bioinformatics analysis predicted that LNC00662 binds to miR-497-5p. A series of studies confirmed that LINC00662 directly interacted with miR-497-5p and downregulated its expression in MDA-MB-157 cells. MiR-497-5p knockdown significantly reversed the inhibitory effect of shLINC00662. Moreover, egl-9 family hypoxia inducible factor 2 (EglN2) was verified as a target of miR-497-5p. Overall, our results demonstrated that overexpression of LINC00662 accelerated the malignant growth of breast cancer cells via sponging miR-497-5p and upregulating EglN2 expression, and indicate that targeting LINC00662 may represent a novel strategy for breast cancer therapy.

Genetic Analysis of EGLN1 C127S Variant in Taiwanese Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder related to nigrostriatal dopaminergic neuron degeneration and iron accumulation. As a cellular oxygen sensor, prolyl hydroxylase domain containing protein 2 (PHD2, encoded by egl-9 family hypoxia inducible factor 1, EGLN1) modifies hypoxia-inducible factor alpha (HIF-α) protein for proteasomal destruction under normoxic condition. In addition, 2-oxoglutarate- (OG-) dependent dioxygenase activity of PHD2 is involved in the oxygen and iron regulation of iron-responsive element binding protein 2 (IRP2) stability. Previously increased expression of EGLN1 was found in the substantia nigra of the parkinsonian brain. We investigated the possible role of c.380 G > C (p.C127S) of EGLN1 gene in Taiwanese patients with PD. 479 patients and 435 healthy controls were recruited. Polymerase chain reaction and BsmAI restriction enzyme analysis were applied for analysis. An association between CC genotype and reduced PD risk in the recessive model (CC vs. GG + GC) was found. Our study provides a link between EGLN1 c.380 G > C SNP and the development of PD.

Effect of miR-873 on cardiomyocyte apoptosis induced by hypoxia reoxygenation and its mechanism

To explore the role of miR-873 in cardiomyocyte injury induces by hypoxia reoxygenation (H/R) and its related mechanisms. Methods: H/R model was established by culturing mouse cardiac H9c2 cells in vitro, and miR-873 mimic was transfected. The experiments were divided into a control group, a H/R group, a negative control group and a miR-873 mimic group. The expression of miR-873 was measured using real-time PCR. The protein expression levels of egl-9 family hypoxia inducible factor 3 (Egln3), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were evaluated by Western blotting. Cell apoptosis ELISA kit and cysteine-containing, aspartate-specific proteases-3 (caspase-3) activity kit was used to detect cell apoptosis and caspase-3 activity, respectively. The targeting effect of miR-873 on Egln3 were examined by the dual luciferase report gene assay, and the experiments were divided into a negative control group, a Egln3 3'-untranslated regions (3'-UTR) WT group (WT group) and a Egln3 3'-UTR MUT group (MUT group). In order to further detect the effects of Egln3 on miR-873 mimics, the Egln3 overexpressed cells were constructed, and the experiments were divided into a H/R group, a H/R+miR-873 mimic group, a H/R+pcDNA3-Egln3 (pcEgln3) group and a H/R+ miR-873 mimic+pcEgln3 group. Results: Compared with the control group, the expression level of miR-873 was significantly decreased in the H/R group (P<0.05). Compared with the H/R group, H9c2 cell apoptosis, caspase-3 activity and the ratio of Bax/Bcl-2 were significantly reduced in the miR-873 mimic group (all P<0.05). Compared with the negative control group, the luciferase activity was significantly down-regulated in the WT group (P<0.05), while the luciferase activity was not significantly changed in the MUT group (P>0.05). In the over-expression experiment, compared with the H/R group, the cell apoptosis and the ratio of Bax/Bcl-2 were significantly reduced in the miR-873 mimic group (both P<0.05). Compared with miR-873 mimic group, the cell apoptosis and the ratio of Bax/Bcl-2 were significantly up-regulated in the H/R+pcEgln3 group and the H/R+miR-873 mimic+pcEgln3 group (all P<0.05). Conclusion: MiR-873 can inhibit H/R- induced apoptosis of cardiomyocyte via targeting Egln3.

Abstract 764: MicroRNA-1205 contributes to the risk of castration-resistant prostate cancer

Abstract Although death rates for prostate cancer are down about 50% as a result of improvements in early detection and treatment, it is still the second leading cause of death (26,120 cases) for American men. However, the genetic mechanism underlying the development and progression of prostate cancer remains poorly understood. It is therefore important to identify the genetic risk factors for prostate cancer. Genome-wide association studies (GWAS) showed evidence for an association of prostate cancer risk with independent genetic variants on chromosome 8q24, conferring prostate cancer susceptibility at this locus. Nevertheless, at the locus with these genetic variants, no specific gene has been identified as responsible for the risk of prostate cancer. At 8q24.21, there is a long-range interaction of these genetic variants with c-MYC or long non-coding RNAs (lncRNAs) in a tissue-specific manner, including prostate, suggesting master genetic factors at 8q24.21 that contribute to this genetic risk. The chromosome 8q24.21 locus, which contains the proto-oncogene c-MYC, long non-coding RNA PVT1, and microRNAs (miRs), is the most commonly amplified region in human prostate cancer. Here, the copy numbers and expressions of miRs-1204~1208 were investigated using quantitative PCR for prostate cancer cell lines and primary tumors. The data revealed that copy numbers and expression of miR-1205 were increased in both castration-resistant prostate cancer cell lines and primary tumors. In castration-resistant prostate cancer specimens, the copy number at the miR-1205 locus correlated with expression of miR-1205. Furthermore, functional analysis with a miR-1205 mimic, a miR-1205 inhibitor, and CRISPR/Cas9 knockout revealed that, in human prostate cancer cells, miR-1205 promoted cell proliferation and cell cycle progression but not apoptosis. In these cells, miR-1205 downregulated expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) gene and targeted a site in its 3’-untranslated region to downregulate its transcriptional activity. Thus, by targeting EGLN3, miR-1205 has an oncogenic role and may contribute to the genetic risk of castration-resistant prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Wen Wen, Yicun Wang, Yue Zhang, Xuelian Cui, Xiaoguang M. Liu, Runhua Liu. MicroRNA-1205 contributes to the risk of castration-resistant prostate cancer [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 764.

MicroRNA-1205, encoded on chromosome 8q24, targets EGLN3 to induce cell growth and contributes to risk of castration-resistant prostate cancer.

The chromosome 8q24.21 locus, which contains the proto-oncogene c-MYC, long non-coding RNA PVT1, and microRNAs (miRs), is the most commonly amplified region in human prostate cancer. A long-range interaction of genetic variants with c-MYC or long non-coding PVT1 at this locus contributes to the genetic risk of prostate cancer. At this locus is a cluster of genes for six miRs (miR-1204, −1205, −1206, −1207–3p, −1207–5p, and −1208), but their functional role remains elusive. Here, the copy numbers and expressions of miRs-1204~1208 were investigated using quantitative PCR for prostate cancer cell lines and primary tumors. The data revealed that copy numbers and expression of miR-1205 were increased in both castration-resistant prostate cancer cell lines and in primary tumors. In castration-resistant prostate cancer specimens, the copy number at the miR-1205 locus correlated with expression of miR-1205. Furthermore, functional analysis with an miR-1205 mimic, an miR-1205 inhibitor, and CRISPR/Cas9 knockout revealed that, in human prostate cancer cells, miR-1205 promoted cell proliferation and cell cycle progression and inhibited hydrogen peroxide-induced apoptosis. In these cells, miR-1205 downregulated expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) gene and targeted a site in its 3’-untranslated region to downregulate its transcriptional activity. Thus, by targeting EGLN3, miR-1205 has an oncogenic role and may contribute to the genetic risk of castration-resistant prostate cancer.

Genodynamics: A New Biophysical Approach to Modeling Adaptation in Human Populations

Using <i>genodynamics</i>, the Howard University biophysics research and interdisciplinary development group transforms genomic sequence data into genomic energy measures to explore the science of genome variation in population diversity and human biology. Genodynamics utilizes the statistical distribution of single nucleotide polymorphism (SNP) data from the Haplotype Map project to mathematically model whole genome-environment interactions in human adaptation to environmental stressors/stimuli by functionally parameterizing the interplay between the biophysical and environmental factors in a quantifiable manner. Our double-blind computer program flagged smooth mathematical function relationships between allelic energies of two SNPs in intron one of the egl-9 family hypoxia inducible factor 1 (EGLN1) and the environmental parameter averaged ancestral annual ultraviolet radiation exposure. EGLN1 is a gene on chromosome 1 known to play an essential role in the regulation of the hypoxia inducible factor pathway. We have demonstrated that our genodynamics approach can quantify, through adaptive forces, the effects that environmental stressors/stimuli have had on patterns of common variation in the human genome and by doing so offer an alternative means of investigating the implications of SNP information dynamics on natural selection in human populations.

TCF7L2 positively regulates aerobic glycolysis via the EGLN2/HIF-1\u03b1 axis and indicates prognosis in pancreatic cancer

Patients with pancreatic ductal adenocarcinoma have much worse prognoses, and much effort has been directed toward understanding the molecular biological aspects of this disease. Accumulated evidence suggests that constitutive activation of the Wnt/β-catenin signalling contributes to the oncogenesis and progression of pancreatic cancer. Transcription factor 7-like2/transcription factor 4 (TCF7L2/TCF4), a β-catenin transcriptional partner, plays a vital role in the Wnt/β-catenin signalling pathway. In the present study, we investigated the clinicopathological significance of TCF7L2 in pancreatic cancer. Our results demonstrated that patients with higher TCF7L2 expression had worse prognosis. Our in vitro studies demonstrated that TCF7L2 positively regulated aerobic glycolysis by suppressing Egl-9 family hypoxia inducible factor 2 (EGLN2), leading to upregulation of hypoxia inducible factor 1 alpha subunit (HIF-1α). The impact of TCF7L2 on aerobic glycolysis was further confirmed in vivo by assessing 18FDG uptake in pancreatic cancer patients and in a subcutaneous xenograft mouse model. In summary, we identified novel predictive markers for prognosis and suggest a previously unrecognized role for TCF7L2 in control of aerobic glycolysis in pancreatic cancer.

BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

Association of PHD3 and HIF2α gene expression with clinicopathological characteristics in human hepatocellular carcinoma.

Egl-9 family hypoxia-inducible factor (HIF)3/prolyl hydroxylase 3 (EGLN3/PHD3) serves a role in the progression and prognosis of cancer. PHD3 is able to induce apoptosis in HepG2 cells. In the present study, the protein levels of PHD3 and HIF2α were analyzed by western blot analysis and immunohistochemistry in 84 paired hepatocellular carcinoma (HCC) tissues and adjacent non-tumor liver tissues. The mRNA levels of PHD3 and HIF2α were analyzed by reverse transcription-quantitative polymerase chain reaction. PHD3 was overexpressed in HCC tissues compared with adjacent liver tissues (mRNA expression: P<0.001; protein expression: P=0.003; immunohistochemistry positive rate: P=0.001). The high level of expression of PHD3 in HCC tissues was associated with good differentiation (mRNA expression: P=0.002; protein expression: P<0.001) and small tumor size (mRNA expression: P<0.001; protein expression: P=0.002). In addition, HIF2α expression was lower in HCC tissues compared with adjacent liver tissues (mRNA expression: P<0.001; protein expression: P=0.002; immunohistochemistry positive rate: P=0.002). No statistically significant associations were identified between HIF2α expression and clinicopathological characteristics. Pearson's and Spearman's correlation coefficients revealed no correlation between HIF2α and PHD3 expression in HCC. In conclusion, PHD3 expression acts as a favorable prognostic marker for patients with HCC. There is no correlation between PHD3 and HIF2α expression in HCC.

Potential regulation of glioma through the induction of apoptosis signaling via Egl-9 family hypoxia-inducible factor 3.

Glioma is an aggressive form of brain cancer that occurs following the abnormal proliferation of glial cells. Although glioma cannot spread to other organs, the morbidity and mortality rates of the disease are high, even following surgery, radiotherapy and chemotherapy. The function of Egl-9 family hypoxia-inducible factor 3 (Egln3) in cancer is controversial, and it is debated as to whether Egln3 positively or negatively regulates tumors. In the present study, a mouse model of low-grade glioma was successfully established. Through the use of immunohistochemical and western blot analyses, it was demonstrated that Egln3 expression in glioma tissue performed an important role in regulation by amplifying the signals for apoptosis, as determined by an increase in DNA fragments. Furthermore, Egln3 expression was inhibited by the administration of dimethyloxalylglycine, and the downregulated expression of Egln3 had marked effects on the regulation of glioma through apoptosis. The present study therefore provides evidence of an association between Egln3 expression and apoptosis in low-grade glioma.

Relaxin deficiency results in increased expression of angiogenesis- and remodelling-related genes in the uterus of early pregnant mice but does not affect endometrial angiogenesis prior to implantation.

BackgroundExtensive uterine adaptations, including angiogenesis, occur prior to implantation in early pregnancy and are potentially regulated by the peptide hormone relaxin. This was investigated in two studies. First, we took a microarray approach using human endometrial stromal (HES) cells treated with relaxin in vitro to screen for target genes. Then we aimed to investigate whether or not relaxin deficiency in mice affected uterine expression of representative genes associated with angiogenesis and uterine remodeling, and also blood vessel proliferation in the pre-implantation mouse endometrium.MethodsNormal HES cells were isolated and treated with recombinant human relaxin (10 ng/ml) for 24 h before microarray analysis. Reverse transcriptase PCR was used to analyze gene expression of relaxin and its receptor (Rxfp1) in ovaries and uteri; quantitative PCR was used to analyze steroid receptor, angiogenesis and extracellular matrix remodeling genes in the uteri of wild type (Rln+/+) and Rln-/- mice on days 1-4 of pregnancy. Immunohistochemistry localized endometrial endothelial cell proliferation and mass spectrometry measured steroid hormones in the plasma.ResultsMicroarray analysis identified 63 well-characterized genes that were differentially regulated in HES cells after relaxin treatment. Expression of some of these genes was increased in the uterus of Rln+/+ mice by day 4 of pregnancy. There was significantly higher vascular endothelial growth factor A (VegfA), estrogen receptor 1 (Esr1), progesterone receptor (Pgr), Rxfp1, egl-9 family hypoxia-inducible factor 1 (Egln1), hypoxia inducible factor 1 alpha (Hif1α), matrix metalloproteinase 14 (Mmp14) and ankryn repeat domain 37 (Ankrd37) in Rln-/- compared to Rln+/+ mice on day 1. Progesterone receptor expression and plasma progesterone levels were higher in Rln-/- mice compared to Rln+/+ mice. However, endometrial angiogenesis was not advanced as pre-implantation endothelial cell proliferation did not differ between genotypes.ConclusionsRelaxin treatment modulates expression of a variety of angiogenesis-related genes in HES cells. However, despite accelerated uterine gene expression of steroid receptor, progesterone and angiogenesis and extracellular matrix remodeling genes in Rln-/- mice, there was no impact on angiogenesis. We conclude that although relaxin deficiency results in phenotypic changes in the pre-implantation uterus, endogenous relaxin does not play a major role in pre-implantation angiogenesis in the mouse uterus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0148-y) contains supplementary material, which is available to authorized users.

Loss of TRIM62 expression is an independent adverse prognostic factor in acute myeloid leukemia.

Tripartite motif (TRIM)-62 is a putative tumor suppressor gene whose role in leukemia is unknown. We evaluated the effect of TRIM62 protein expression in patients with acute myeloid leukemia (AML). We used reverse-phase protein array methodology to determine TRIM62 levels in leukemia-enriched protein samples from 511 patients newly diagnosed with AML. TRIM62 levels in AML cells were significantly lower than in normal CD34-positive cells, suggesting that TRIM62 loss might be involved in leukemogenesis, but was not associated with specific karyotypic abnormalities or Nucleophosmin (NPM1), Fms-like Tyrosine Kinase-3 (FLT3), or rat sarcoma viral oncogene (RAS) mutational status. Low TRIM62 levels were associated with shorter complete remission duration and significantly shorter event-free and overall survival rates, particularly among patients with intermediate-risk cytogenetics. In that AML subgroup, age and TRIM62 levels were the most powerful independent prognostic factors for survival. TRIM62 protein levels further refined the risk associated with NPM1 and FLT3 mutational status. TRIM62 loss was associated with altered expression of proteins involved in leukemia stem cell homeostasis (β-catenin and Notch), cell motility, and adhesion (integrin-β3, ras-related C3 botulinum toxin substrate [RAC], and fibronectin), hypoxia (Hypoxia-inducible factor 1-alpha [HIF1α], egl-9 family hypoxia-inducible factor 1 [Egln1], and glucose-regulated protein, 78 kDa [GRP78]), and apoptosis (B-cell lymphoma-extra large (BclXL) and caspase 9). Low TRIM62 levels, consistent with a tumor suppressor role, represent an independent adverse prognostic factor in AML.