Erythropoietin Receptor mRNA Levels Research Articles

EPOR mRNA level: A valuable prognostic indicator for patients with ER+ breast cancer

Objective: To explore the expression level and prognostic significance of erythropoietin receptor (EPOR) in patients with breast cancer (BRCA) based on estrogen receptor (ER) status and different molecular subtypes. Methods: The Cancer Genome Atlas (TCGA) and GTEx data were collected in GEPIA initially to identify the dysregulated genes. Further, bc- GenExMiner 4.1 online bioinformatics tool was used to evaluate EPOR mRNA differential expression level according to different classification of clinicopathologic parameters in patients with breast cancer. Additionally, the prognostic value between EPOR mRNA expression and free survival of metastatic relapse (MR) or any event (AE, namely any relapse or death) in patients with breast cancer was done. Results: EPOR mRNA was significantly downregulated in BRCA (1 085 cases) compared to normal tissues (291 cases) (P<0.05). Univariate Cox analysis revealed that high EPOR mRNA expression was remarkably correlated to a decreased risk of MR (HR: 0.79, P<0.000 1) and AE (HR: 0.87, P =0.000 7) especially in breast cancer patients with ER+. Besides, high EPOR mRNA level also associated with a favorable MR- free survival (HR: 0.81, P =0.007 2) and AE-free survival (HR: 0.88, P =0.029 9) in ER+ breast cancer patients. However, no similar above phenomenon was detected in ER- patients. Moreover, the subsequent prognostic adjusted analyses and univariate Cox analysis of AE based on SSP or SCM molecular subtypes validated the above results. Conclusion: EPOR mRNA level is a valuable prognostic indicator for patients with ER+ breast cancer.

Correlation of adrenomedullin with the erythropoietin receptor and microvessel density in hepatocellular carcinoma.

IntroductionUncontrolled angiogenesis plays an essential role in the occurrence, metastasis and malignant progression of hepatocellular carcinoma (HCC). This study aimed to investigate the expression of adrenomedullin (ADM) in human HCC and its correlation with the expression of erythropoietin receptor (EPOR), microvessel density (MVD) and the tumor pathological characteristics.Material and methodsFresh tumor tissues were obtained from 30 HCC patients after hepatectomy. Ten cirrhotic and 10 normal liver tissues were included as controls. Expression of ADM and EPOR was determined by real-time PCR. The MVD was determined by counting the number of microvessels.ResultsThe MVD and the mRNA levels of ADM and EPOR in cancer tissues were significantly higher than those in the non-cancer tissues (p < 0.05). Expression of ADM was significantly correlated with the MVD and EPOR (r = 0.68 and 0.74, p < 0.01). Adrenomedullin and EPOR mRNA levels in HCC tissues were correlated with capsule invasion, pathological differentiation and tumor metastasis (p < 0.05).ConclusionsOur findings suggest that ADM and EPOR may serve as new regulatory factors involved in angiogenesis of HCC and represent novel targets for the treatment of HCC.

Protective effects of erythropoietin on infection induced neonatal rat brain injury using at different times

Objective To explore the influence of erythropoietin(EPO) on infection induced neonatal rat brain injury at different starting time and its related mechanism. Methods Postnatal day 2(P2) newborn SD rats were randomly divided into 4 groups: control group(group A), lipopolysaccharide(LPS) group(group B), the early EPO group(group C)and the later EPO group(group D). Pups in group A, B and C were injected different drugs intraperitoneally(group A for saline, group B for 0.6 mg/kg of LPS, and group C for 0.6 mg/kg of LPS and 5 000 U/kg of EPO)once a day for consecutive 5 days(P2-P6). LPS in group D were injected 0.6 mg/kg of LPS intraperitoneally once a day for consecutive 5 days(P2-P6), and with 5 000 U/kg of EPO once a day for consecutive 5 days(P7-P11). Rats in each group were given different drugs starting at corresponding time by intraperitoneal injection for 5 consecutive days.Every 10 newborn rats in group A and B were selected randomly on P2(6 h after intraperitoneal injection of drugs for the first time), P7 and P12, the brains were divided into the left and the right hemispheres marked by sagittal suture, using enzyme-linked immunosorbent assay method to evaluate the erythropoietin receptor(EPOR) protein level with the right cerebral hemisphere and reverse transcription-polymerase chain reaction(RT-PCR) method was used to investigate EPOR mRNA level of the left cerebral hemisphere.Immunohistochemical method was adopted to evaluate the expression of myelin basic protein(MBP), glial fibrillary acidic protein(GFAP) and EPOR at specified time point, and HE dyeing for the pathological changes of brain damage in different groups. Results HE staining of the group A pre-sented the normal structure in the neonatal rat brain.Reduced numbers of hippocampal pyramidal cells, expansion of the lateral ventricles and periventricular leukomalacia were found in group B. No leukomalacia or lateral ventricles's expansion in EPO administrated groups and it was more obvious in group C. The EPOR protein and mRNA of group B was increased compared with the group A. The EPOR protein and mRNA levels had a tendency to decline with the increase of age.The MBP expression of group B(107.46±3.65)was significantly reduced compared with the group A(146.78±3.13)(P 0.05). Conclusions EPO shows a protective effect on the cerebral white matter injury caused by postpartum infection, it is superior to administer EPO at early time than later time.The mechanism of the protective effect may be connected with the fact that the infection can induce the expression of brain EPOR and the EPOR expression level has a tendency to decline with the increase of age. Key words: Erythropoietin; Erythropoietin receptor; Myelin basic protein; Glial fibrillary acidic protein; Neonatal rat

Erythropoietin attenuates oxidative stress and apoptosis in Schwann cells isolated from streptozotocin-induced diabetic rats

High glucose-evoked oxidative stress and apoptosis within Schwann cells (SCs) are mechanisms facilitating the procession of diabetic peripheral neuropathy (DPN). Although erythropoietin (EPO) was demonstrated to have neuroprotective effects in neurodegenerative diseases, the effects of EPO on glucose-evoked oxidative stress and apoptosis of SCs remain unknown. Primary cultured SCs isolated from streptozotocin (STZ)-induced diabetic peripheral neuropathic rats and normal control rats were exposed to high or normal glucose condition with or without EPO incubation for 72 h. Cell viability, apoptotic rate, cellular reactive oxygen species (ROS) level, total glutathione (GSH) level, EPO mRNA and erythropoietin receptor (EPOR) mRNA levels were assayed. SCs from diabetic rats showed a lower cell viability and a higher apoptotic rate. High glucose culture condition elevated ROS level and diminished total GSH level of SCs. EPO improved cell viability and decreased cell apoptotic rate of SCs. EPO also elevated total GSH level and decreased intracellular ROS level. SCs from diabetic rats exhibited higher EPO mRNA and EPOR mRNA levels than SCs from normal control rats. The data of this study offered fresh viewpoints for interpreting the pathogenesis of DPN and novel pharmacological principles implicit in the therapeutic effect of EPO.

Hypoxia differentially upregulates the expression of embryonic, fetal and adult hemoglobin in human glioblastoma cells

Hemoglobin is produced mainly in erythroid cells. However, it has been reported in non-erythroid cells of human and rodents. We have shown previously that neuroglobin, cytoglobin and hemoglobin are expressed in human glioblastoma multiforme (GBM) cells. We sought to determine whether hemoglobin expression is upregulated by hypoxia, and whether its expression is restricted to the cancer stem cell populations in different GBM cell lines or GBM brain tumor initiating cells (BTICs). Flow cytometry, magnetic cell sorting and qRT-PCR were used to examine the hypoxic upregulation of hemoglobins aswellas erythropoietin (EPO) and erythropoietin receptor (EPOR) in GBM cell lines (M006x, M059J, M059K, U87R and U87T) and GBM-BTICs. The data showed significantly increased expression in globins (α, β, γ, δ, ζ and ε), EPO and EPOR mRNA levels under hypoxia. Globin expression is not limited to the stem cell populations or GBM-BTICs but is a property of the entire GBM population. We assumed that the total expression of mRNA of different normalized globins (α, β, γ, δ, ζ and ε) at different time‑points for the same cell line is 100%. Under aerobic conditions, εglobin was predominantly expressed, and then decreased gradually with increasing time in hypoxia. This was coupled to a concomitant increase in α and γglobins. Our findings suggest that hypoxic upregulation of hemoglobin expression in GBM cells may be a part of a repertoire of active defence and adaptation mechanisms enabling these cells to acquire resistance to aggressive multimodality treatments of chemotherapy and radiotherapy. New therapeutic strategies to interfere with hemoglobin expression or function in GBM cells are required.

Erythropoietin Receptor Expression Is a Potential Prognostic Factor in Human Lung Adenocarcinoma

Recombinant human erythropoietins (rHuEPOs) are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR) signaling in human non-small cell lung cancer (NSCLC) also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III–IV adenocarcinoma (ADC) and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC) proliferation was determined by 5-bromo-2’-deoxy-uridine (BrdU) incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine) did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.

Limitations of a Transgenic Breast Cancer Model for Studies Erythropoietin-Induced Tumor Progression

Adverse effects of erythropoietin (EPO) on tumor progression and survival were observed in recent phase 3 oncology trials. However, mechanisms remain poorly understood. We tested the effects of exogenous EPO on murine B16F10 melanoma growth in a subcutaneous tumor transplant model, and for the first time, in a model of spontaneous tumor formation within autochthonous epithelial tissues using murine mammary tumor virus promoter polyoma virus middle T antigen (MMTV-PyMT) transgenic mice. EPO receptor (EPOR) messenger RNA (mRNA) was detectable in both B16F10 tumors and mammary tumors from MMTV-PyMT mice but was 0.12 ± 0.02% and 1.3 ± 0.91% of the EPOR mRNA level in murine erythroid HCD-57 cells, respectively. B16F10 tumor growth rates in mice treated for 3 weeks with 30 μg/kg per week of darbepoetin α, 0.41 inverse days (range, 0.05–0.69 inverse days; n = 16), were similar to tumor growth rates observed in mice treated with PBS, 0.42 inverse days (range, 0.10–0.69 inverse days; n = 17). In contrast, darbepoetin a raised hematocrit levels to 0.593 (maximum, 0.729) compared with 0.448 (maximum, 0.532) in PBS-treated mice (P = .0004). In MMTV-PyMT mice, the weights of tumor-bearing mammary glands in mice treated for 6 weeks with 30N μg/kg per week of darbepoetin α, 3.37 g (range, 1.94-5.81 g; n = 27), did not significantly differ from the weights in PBS-treated mice, 3.76 g (range, 2.30–6.33 g; n = 26). In contrast, darbepoetin α raised hematocrit levels to 0.441 (maximum, 0.606) compared with 0.405 (maximum, 0.492) in PBS-treated mice (P = .05). Thus, effects of exogenous EPO on tumor growth were not recapitulated in these murine tumor models.

Involvement of Erythropoietin in Retinal Ischemic Preconditioning

The purpose of this study was to examine the role of erythropoietin in retinal ischemic preconditioning (IPC). Rats were subjected to retinal ischemia after IPC. Electroretinography assessed functional recovery after ischemia; retinal sections were examined to determine loss of retinal ganglion cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to assess apoptosis. Levels of downstream mediators were measured in retinal homogenates by Western blotting. To assess the involvement of erythropoietin in IPC, Western blotting was used to measure levels of erythropoietin and its receptor (EPO-R) in retinal homogenates after IPC. To examine erythropoietin's role in IPC, the impact of blocking erythropoietin via intravitreal injection of soluble EPO-R (sEPO-R) before IPC was studied. Erythropoietin levels did not change after IPC, but EPO-R increased. Intravitreal injection of sEPO-R significantly attenuated both the functional and histologic neuroprotection produced by IPC in comparison to control injection of denatured sEPO-R. Apoptotic damage after ischemia was enhanced in the sEPO-R-treated retinas as indicated by fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Phosphorylated extracellular-signal-regulated kinase and heat shock protein 27, but not protein kinase B, upregulated in denatured sEPO-R-treated retinae, were attenuated in eyes injected with sEPO-R. These results indicate that EPO-R upregulation is a critical component of the functional, histologic, and antiapoptotic protective effect of IPC on ischemia in the retina and that several downstream effectors may be involved in the neuroprotective actions of erythropoietin.

Expression Pattern of Erythropoietin and Erythropoietin Receptor in Experimental Model of Retinal Detachment

Purpose: Erythropoietin (EPO), known for its role in erythroid cell differentiation, has been suggested to have a neuroprotective effect on retinal neurons. The aim of the current study was to investigate the expression pattern of EPO and erythropoietin receptor (EPOR) in the detached retina of a rat model of retinal detachment (RD). Methods: Forty-eight albino Sprague-Dawley rats were randomized into normal control group (n = 6) and RD model group (n = 42). The rat RD model was established by slow injection of 1.4% sodium hyaluronate into the upper half of the subretinal space, in which the upper half of the neurosensory retina was detached from the underlying retinal pigment epithelium (RPE). The expression of EPO and EPOR mRNA/protein was determined at 1, 3, 6, 12, 24, 48, and 72 hr after sodium hyaluronate injection by semiquantitative RT-PCR and Western blot analysis, respectively. Meanwhile, the distributions of EPO and EPOR were examined by immunohistochemistry. Results: EPO and EPOR mRNA levels increased gradually after sodium hyaluronate injection and peaked 48 hr later. EPO mRNA level became significantly higher than that in the normal control group at 12 hr after injection, and EPOR mRNA level became significantly higher at 6 hr (p < 0.05). Moreover, the protein levels of EPO and EPOR presented a similar tendency to the mRNA level and became significantly higher than those in normal control group at 3 hr after injection (p < 0.05). Immunohistochemical results showed that both EPO and EPOR were strongly expressed all over the neurosensory retina 48 hr after injection, but EPOR was not found in the rod outer segment. Only weak expression of EPO and EPOR was noticed in the retina of normal control group. Conclusions: The levels of EPO and EPOR in detached retina increase with the duration of retinal detachment and reach their peaks at 48 hr; most layers of detached neurosensory retina can express EPO and EPOR. It is indicated that the EPO/EPOR system might play an important role in protection of retinal neurons during RD; supplementation with exogenous EPO might promote the survival of retinal neurons.

A Distinct Function of STAT Proteins in Erythropoietin Signal Transduction

The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is an important signaling pathway of interferons and cytokines. We examined the activation of STAT proteins induced by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) using the human leukemia cell line, UT-7, which requires these cytokines for growth. IL-3, GM-CSF, and EPO induced DNA-binding activity to the oligonucleotides corresponding to the sis-inducible elements (SIE) of c-fos, in addition to the beta-casein promoter (beta-CAP), SIE- and beta-CAP-binding proteins were identical to Stat1alpha and Stat3 complex and to Stat5 protein, respectively. This indicates that IL-3, GM-CSF, and EPO commonly activated Stat1alpha, Stat3, and Stat5 proteins in UT-7. However, EPO hardly activated Stat1alpha and Stat3 in UT-7/GM, which is a subline of UT-7 that grows slightly in response to EPO. Transfection studies revealed that UT-7/GM cells constitutively expressing Stat1alpha, but not Stat3, can grow as well in response to EPO as GM-CSF, suggesting that Stat1alpha is involved in the EPO-induced proliferation of UT-7. Thus, although Stat1alpha, Stat3, and Stat5 proteins are activated by GM-CSF, IL-3, and EPO, our data suggest that each STAT protein has a distinctive role in the actions of cytokines.

Cell-Cycle–Dependent Regulation of Erythropoietin Receptor Gene

AbstractTo understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF ), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

Localization of specific erythropoietin binding sites in defined areas of the mouse brain.

The main physiological regulator of erythropoiesis is the hematopoietic growth factor erythropoietin (EPO), which is induced in response to hypoxia. Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily, controls the terminal maturation of red blood cells. So far, EPO has been reported to act mainly on erythroid precursor cells. However, we have detected mRNA encoding both EPO and EPO-R in mouse brain by reverse transcription-PCR. Exposure to 0.1% carbon monoxide, a procedure that causes functional anemia, resulted in a 20-fold increase of EPO mRNA in mouse brain as quantified by competitive reverse transcription-PCR, whereas the EPO-R mRNA level was not influenced by hypoxia. Binding studies on mouse brain sections revealed defined binding sites for radioiodinated EPO in distinct brain areas. The specificity of EPO binding was assessed by homologous competition with an excess of unlabeled EPO and by using two monoclonal antibodies against human EPO, one inhibitory and the other noninhibitory for binding of EPO to EPO-R. Major EPO binding sites were observed in the hippocampus, capsula interna, cortex, and midbrain areas. Functional expression of the EPO-R and hypoxic upregulation of EPO suggest a role of EPO in the brain.

Abundance and Stability of Erythropoietin Receptor mRNA in Mouse Erythroid Progenitor Cells

The abundance and stability of erythropoietin (EPO) receptor mRNA was measured in Friend virus-infected mouse splenic erythroblast (FVA) cells. FVA cells correspond to the colony-forming unit-erythroid (CFU-E) and cluster-forming unit-erythroid stages of differentiation and they mature in vitro into reticulocytes during 45 to 60 hours of culture. After 20 hours, there was a rapid decline in the EPO receptor mRNA level through the 45-hour culture period. Levels of actin and beta-globin mRNAs were monitored as two representative proteins that are actively synthesized in early versus late stages of terminal erythroid differentiation. A general decrease of actin mRNA level was apparent, whereas globin mRNA levels increased throughout the culture period. The greatest decrease in both EPO receptor and actin mRNA was observed when the cell population was at the stage of basophilic and polychromatophilic erythroblasts. The half-life for EPO receptor mRNA was approximately 75 minutes, as determined using the transcriptional inhibitor actinomycin D. The half-life measured at several times during the 48-hour culture period remained constant. These results indicate that EPO receptor mRNA must be transcribed continuously until late in the maturation process of FVA cells in order to maintain the levels seen by Northern analysis. The number of copies of EPO receptor mRNA at 0 hours was determined to be 25 copies per cell. This low number and the decline of EPO receptor mRNA correlate with the low receptor numbers on FVA cells, as well as the decline of binding sites with cell maturation.

Abundance and stability of erythropoietin receptor mRNA in mouse erythroid progenitor cells

The abundance and stability of erythropoietin (EPO) receptor mRNA was measured in Friend virus-infected mouse splenic erythroblast (FVA) cells. FVA cells correspond to the colony-forming unit-erythroid (CFU- E) and cluster-forming unit-erythroid stages of differentiation and they mature in vitro into reticulocytes during 45 to 60 hours of culture. After 20 hours, there was a rapid decline in the EPO receptor mRNA level through the 45-hour culture period. Levels of actin and beta- globin mRNAs were monitored as two representative proteins that are actively synthesized in early versus late stages of terminal erythroid differentiation. A general decrease of actin mRNA level was apparent, whereas globin mRNA levels increased throughout the culture period. The greatest decrease in both EPO receptor and actin mRNA was observed when the cell population was at the stage of basophilic and polychromatophilic erythroblasts. The half-life for EPO receptor mRNA was approximately 75 minutes, as determined using the transcriptional inhibitor actinomycin D. The half-life measured at several times during the 48-hour culture period remained constant. These results indicate that EPO receptor mRNA must be transcribed continuously until late in the maturation process of FVA cells in order to maintain the levels seen by Northern analysis. The number of copies of EPO receptor mRNA at 0 hours was determined to be 25 copies per cell. This low number and the decline of EPO receptor mRNA correlate with the low receptor numbers on FVA cells, as well as the decline of binding sites with cell maturation.