N-myc Downstream-regulated Gene 2 mRNA Research Articles

Biological and prognostic significance of NDRG2 downregulation in oral squamous cell carcinoma.

Downregulation of N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor gene, has been associated with poor clinical outcomes in various cancers. However, the prognostic significance of NDRG2 in oral squamous cell carcinoma (OSCC) remains unknown. This study aimed to evaluate the prognostic value of NDRG2 downregulation in OSCC and to elucidate the mechanism by which NDRG2 is downregulated and the biological role of NDRG2 in tumor progression. Immunohistochemical and in silico analyses of NDRG2 expression were performed, and the correlation between NDRG2 expression and clinicopathological data was analyzed. The effect of NDRG2 knockdown on the biological behavior of OSCC cells was investigated and the effect of 5-aza-2'-deoxycytidine (5-aza-dC) on NDRG2 expression was determined. NDRG2 expression was significantly downregulated and DNA hypermethylation of NDRG2 was frequently found in head and neck SCC, including OSCC. Low NDRG2 expression was significantly correlated with adverse clinicopathological features and worse survival in OSCC. NDRG2 knockdown could enhance the oncogenic properties of OSCC cells. NDRG2 mRNA levels in OSCC cells could be restored by 5-aza-dC. Downregulation of NDRG2 promotes tumor progression and predicts poor prognosis in OSCC. Therefore, restoration of NDRG2 expression may be a potential therapeutic strategy in OSCC.

TGF-β1 can be regulated by NDRG2 via the NF-κB pathway in hypoxia-induced liver fibrosis

BackgroundThe identification of the important elements that control hepatic stellate cell (HSC) activation will expand our understanding of the mechanism of liver fibrosis induced by hypoxia and affect the outcome of clinical treatment. A previous research demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of fibrosis and a downstream target gene of hypoxia-inducible factor 1 (HIF-1). In this research, we studied the expression and function of NDRG2 in liver fibrosis induced by hypoxia.MethodsLX-2 cells/NF-κB-silenced LX-2 cells were exposed to hypoxic conditions (1% O2) to activate HSCs in vitro. The protein and mRNA expression levels of NDRG2, α-SMA and transforming growth factor beta 1 (TGF-β1) were evaluated by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. Functional studies were performed using adenovirus-mediated gene upregulation.ResultsThe NDRG2 mRNA and protein levels were reduced under hypoxic conditions in LX-2 cells and overexpression of NDRG2 resulted in a decrease in the expression of TGF-β1 and α-SMA. Interestingly, no relationship was observed between NDRG2 and TGF-β1 when the NF-κB pathway was blocked, which indicates that NDRG2 can regulate the expression of TGF-β1 in LX-2 cells via the NF-κB pathway under hypoxic conditions.ConclusionsNDRG2 may regulate the expression of TGF-β1 via the NF-κB pathway and may be a novel therapeutic target for liver fibrosis induced by hypoxia.

NDRG2 gene expression pattern in ovarian cancer and its specific roles in inhibiting cancer cell proliferation and suppressing cancer cell apoptosis

BackgroundThe cancer cell metastasis and the acquisition of chemotherapy resistance remain huge challenge for ovarian cancer treatment. Previously, N-myc downstream-regulated gene 2 (NDRG2) serves as a tumor suppressor for many cancers. Here, we attempted to investigate the specific roles of NDRG2 in ovarian cancer.MethodsThe expression levels of NDRG2 were detected by qRT-PCR or Immunoblotting. CCK-8 assay was employed to examine the cell viability of ovarian cancer cells. The colony formation ability was determined by colony formation assay. Flow cytometry analyses were performed to detect the cell apoptosis and cell cycle. Xenograft tumor assay was performed to detect the in vivo function of NDRG2.ResultsWe revealed that NDRG2 mRNA expression and protein levels were downregulated within both ovarian cancer tissues and cell lines. The overexpression of NDRG2 dramatically inhibited the cell viability and colony formation and tumor growth, whereas promoted the cell apoptosis, cell cycle arrest in G1 phase within ovarian cancer cells. More importantly, NDRG2 overexpression significantly enhanced the suppressive roles of cisplatin (DDP) in ovarian cancer cell viability. On the contrary, NDRG2 silence exerted opposing effects on ovarian cancer cells.ConclusionsIn summary, we provide a solid experimental basis demonstrating the tumor-suppressive effects of NDRG2 in inhibiting the cell proliferation, enhancing the cell apoptosis, eliciting the cell cycle arrest in G1 phase, and promoting the suppressive effects of DDP on the viability of ovarian cancer cells. NDRG2 administration presents a potent adjuvant treatment for ovarian cancer therapy.

NELFE promoted pancreatic cancer metastasis and the epithelial‑to‑mesenchymal transition by decreasing the stabilization of NDRG2 mRNA.

Negative elongation factor E (NELFE) has been demonstrated to promote cancer progression as an RNA-binding protein (RBP). However, the expression patterns, biological role and molecular mechanism of NELFE in pancreatic cancer (PC) remain largely unknown. The expression levels of NELFE in 120 pairs of PC tissues and adjacent non-tumor clinical samples collected from patients with PC were examined via reverse transcription-quantitative (RT-q) PCR and immunohistochemistry. The mRNA expression levels of NELFE, N-Myc downstream-regulated gene 2 (NDRG2), c-Myc, survivin and cyclin D1 were detected via RT-qPCR. The protein expression levels of NELFE, NDRG2, total β-catenin, nuclear β-catenin, cytosolic β-catenin, E-cadherin, N-cadherin and Vimentin were measured by western blotting. NELFE and NDRG2 were then knocked-down by short hairpin (sh)RNA. PC cell proliferation was detected by MTT and colony formation assays. Invasion and migration were detected by transwell assays. The interaction between NELFE and NDRG2 was detected by luciferase reporter assays, mRNA decay assays and RNA immunoprecipitation. NELFE expression was increased in PC tissues compared with the paired non-cancerous tissues. NELFE expression was upregulated in PC cells when compared with normal pancreatic cells (HPDE6-C7). The present study revealed that knockdown of NELFE inhibited the proliferation, invasion and migration of PC cells. In addition, transfection of the sh-NELFE vector inhibited the epithelial-to-mesenchymal transition in PC cells by suppressing the expression and nuclear accumulation of β-catenin. Further mechanistic studies revealed that NELFE activates the Wnt/β-catenin signaling pathway by decreasing the stabilization of NDRG2 mRNA in PC. To the best of our knowledge, these results revealed the promotional function of NELFE on PC tumorigenesis and metastasis for the first time, helping to provide a promising strategy for the treatment of patients with PC.

Spatiotemporal expression of NDRG2 in the human fetal brain

N-myc downstream-regulated gene 2 (NDRG2) has been implicated in the development of central nervous system and brain diseases such as brain tumors, ischemic stroke and neurodegenerative disorders. However, it remains unclear that the spatiotemporal distribution of NDRG2 in the human fetal brain. In this study, we examined the expression pattern of NDRG2 in different regions of human fetal brain at 16–28 gestational weeks (GWs) by using RT-PCR, western blot and immunohistochemistry. Firstly, RT-PCR revealed that mRNA of NDRG2 was detected in the human brain regions of fetuses at 16–28 GWs such as medulla oblongata (MdO), mesencephalon (MeE), cerebellum (Cbl), frontal lobe (Fr), ventricular (VZ)/subventricular zone (SVZ) and hippocampus (hip), and the expressions of NDRG2 mRNA in these human fetal brain regions were increased with gestational maturation. Furthermore, western blot and immunohistochemistry results revealed that at 28 GWs, the expression of NDRG2 protein was restricted to the MdO’s olivary nucleus, MeE’s aqueduct, cerebellar internal granular layers, cerebral cortex of the Fr, VZ/SVZ of lateral ventricle, and hippocampal dentate gyrus, and highest expression in the VZ/SVZ, and lowest in the MeE. Finally, double immunohistochemistry results showed that NDRG2 in the MdO, Cbl and VZ/SV at 28 GWS was mainly expressed in neurons (NeuN positive cells), and in some astrocytes (GFAP positive cells). Taken together, these results suggest that NDRG2 is mainly expressed in human fetal neurons of various brain regions during development, which may be involved in neuronal growth and maturation.

The MYB/miR-130a/NDRG2 axis modulates tumor proliferation and metastatic potential in salivary adenoid cystic carcinoma

Increasing evidence has emerged to suggest that N-myc downstream-regulated gene 2 (NDRG2) dysregulation participates in a number of tumor biological processes. However, the role of NDRG2 and miRNA-mediated NDRG2 regulation in salivary adenoid cystic carcinoma (SACC) progression remain unknown. Here, we determined that SACC tissues exhibited decreased level of NDRG2, which was associated with poorer rates of overall survival and distant metastasis-free survival. Silencing NDRG2 promoted SACC cell proliferation and metastasis both in vitro and in vivo. MiRNAs have been reported as vital regulators of NDRG2 expression. Based on micronome sequencing of three paired samples of SACC and normal salivary gland tissue and on an online database analysis, miR-130a was identified as a candidate miRNA that potentially regulates NDRG2. We demonstrated that the expression level of NDRG2 was dramatically reduced by exogenous miR-130a. Moreover, a luciferase assay further validated that miR-130a could degrade NDRG2 mRNA by targeting sites in the NDRG2 3′UTR. A rescue experiment suggested that NDRG2 expression could reverse the miR-130a-mediated promotion of cell proliferation and invasion. The expression of miR-130a has been reported to be regulated by certain transcription factors. In the preset study, we verified that the transcription factor MYB acted as the critical driver in SACC-upregulated miR-130a expression directly and induced NDRG2 downregulation in SACC tissues. Additionally, MYB/miR-130a activated the STAT3 and AKT pathways by downregulating NDRG2. These observations suggest that the MYB/miR-130a/NDRG2 axis, which modulates proliferation and metastasis in SACC, provides promising targets for the treatment of SACC.

NDRG2 knockdown promotes fibrosis in renal tubular epithelial cells through TGF-β1/Smad3 pathway.

Renal fibrosis is a common pathological pathway of various chronic kidney diseases progressing to end-stage renal disease and is characterized by tubular atrophy, fibroblast/myofibroblast activation and excessive deposition of extracellular matrix (ECM). N-Myc downstream-regulated gene-2 (NDRG2) is reported to be associated with liver fibrosis in rats. However, the biological function of NDRG2 in renal fibrosis remains unclear. Therefore, we investigate the effect of NDRG2 on renal fibrosis and the underlying mechanism of NDRG2 in TGF-β1-induced renal tubular epithelial cells (HK-2). Our results show that TGF-β1 down-regulates NDRG2 mRNA and protein expression in HK-2 cells. Moreover, NDRG2 knockdown dramatically reduces the TGF-β1-induced protein and mRNA of E-cadherin and increases the TGF-β1-induced protein and mRNA expression level of α-SMA, Vimentin, Snail, Col-I, Col-III and FN; this is reversed by NDRG2 overexpression. Furthermore, NDRG2 silencing significantly increases the phosphorylation level of Smad3 (p-Smad3), which is decreased by NDRG2 overexpression, although these have no effect on the protein expression of p-Smad2 and Smad7. In addition, SIS3, a specific inhibitor of Smad3 phosphorylation, partly reverses the effect of NDRG2 knockdown on the protein and mRNA expression of epithelial-mesenchymal transition (EMT) markers and ECM components in TGF-β1-induced HK-2 cells. Taken together, our results indicate that NDRG2 knockdown promotes renal fibrosis through its effect on the protein and mRNA expression of EMT markers and ECM components by regulating the downstream Smad3 signaling pathway in renal tubular epithelial cells. Modulation of NDRG2 expression might provide a new therapy for renal fibrosis.

N-myc downstream-regulated gene 2 (NDRG2) promoter methylation and expression in pituitary adenoma

BackgroundPituitary adenoma (PA) is a benign primary tumor that arises from the pituitary gland and is associated with ophthalmological, neurological and endocrinological abnormalities. However, causes that increase tumor progressing recurrence and invasiveness are still undetermined. Several studies have shown N-myc downstream regulated gene 2 (NDRG2) as a tumor suppressor gene, but the role of NDRG2 gene in pituitary adenoma pathogenesis has not been elucidated. The aim of our research has been to examine NDRG2 mRNA expression in PA and to determine the associations between the NDRG2 gene epigenetic changes and the development of recurrence or invasiveness of PA and patient clinical data.MethodsThe MS-PCR was used for NDRG2 promoter methylation analysis and gene mRNA expression levels were evaluated by qRT-PCR in 68 non-functioning and 73 functioning adenomas. Invasiveness was evaluated using magnetic resonance imaging with Hardy’s modified criteria. Statistical analysis was performed to find correlations between NDRG2 gene mRNA expression, promoter methylation and patient clinical characteristics and PA activity.ResultsThe NDRG2 mRNA expression was significantly lower in the case of acromegaly (GH and IGF-1 hypersecretion) than in other diagnoses of PAs (p < 0.05). Also, the NDRG2 expression was significantly higher in prolactinoma (PRL hypersecretion) than in in other diagnoses of PAs (p < 0.05). The promoter of NDRG2 was methylated in 22.69% (12/58 functioning and 15/61 non-functioning) of patients with PA. However, the NDRG2 gene mRNA expression was not significantly related to its methylation status. Clinical factors, such as: age, gender, relapse and diagnoses of Cushing syndrome were of no significance for NDRG2 promoter methylation and mRNA expression levels, as well as secreting or non-secreting PAs and the invasiveness of PAs.ConclusionThe different NDRG2 promoter methylation and expression levels in PA samples showed tumor heterogeneity and indicates a potential role of this gene in pituitary adenoma pathogenesis, but the corresponding details require intensive research.

NDRG2 gene copy number is not altered in colorectal carcinoma.

AIMTo investigate if the down-regulation of N-myc Downstream Regulated Gene 2 (NDRG2) expression in colorectal carcinoma (CRC) is due to loss of the NDRG2 allele(s).METHODSThe following were investigated in the human colorectal cancer cell lines DLD-1, LoVo and SW-480: NDRG2 mRNA expression levels using quantitative reverse transcription-polymerase chain reaction (qRT-PCR); interaction of the MYC gene-regulatory protein with the NDRG2 promoter using chromatin immunoprecipitation; and NDRG2 promoter methylation using bisulfite sequencing. Furthermore, we performed qPCR to analyse the copy numbers of NDRG2 and MYC genes in the above three cell lines, 8 normal colorectal tissue samples and 40 CRC tissue samples.RESULTSAs expected, NDRG2 mRNA levels were low in the three colorectal cancer cell lines, compared to normal colon. Endogenous MYC protein interacted with the NDRG2 core promoter in all three cell lines. In addition, the NDRG2 promoter was heavily methylated in these cell lines, suggesting an epigenetic regulatory mechanism. Unaltered gene copy numbers of NDRG2 were observed in the three cell lines. In the colorectal tissues, one normal and three CRC samples showed partial or complete loss of one NDRG2 allele. In contrast, the MYC gene was amplified in one cell line and in more than 40% of the CRC cases.CONCLUSIONOur study suggests that the reduction in NDRG2 expression observed in CRC is due to transcriptional repression by MYC and promoter methylation, and is not due to allelic loss.

Epigenetic silencing of NDRG2 promotes colorectal cancer proliferation and invasion.

Genome-wide methylation arrays have revealed aberrant methylation of N-Myc downstream-regulated gene 2 (NDRG2) promoter in colorectal cancer (CRC). This study investigated the role of NDRG2 in colorectal carcinogenesis. The aberrant promoter methylation, mRNA, and protein expression of NDRG2 were evaluated in 27 pairs of human CRC and adjacent normal tissues and seven human CRC-derived cell-lines. After stable NDRG2 over-expressed RKO and DLD-1 human CRC cell-lines were constructed, in vitro functional assays, including colony formation, cell viability, proliferation, invasion and migration assays, and in vivo xenograft models were performed. The promoter of NDRG2 was methylated in 89% human CRC tissue compared to adjacent normal colonic mucosa (7.4%; P < 0.001). High-level methylation of NDRG2 promoter was more prevalent in proximal CRC (P = 0.022) and advanced T stage (P = 0.039). NDRG2 mRNA and protein expression was down-regulated in 89% and 100% human CRC tissue, respectively. In human CRC cell-lines, the promoter of NDRG2 was methylated aberrantly and mRNA, and protein expression of NDRG2 was down-regulated. NDRG2 mRNA expression was reactivated by 5-aza-2'-deoxycytidine. Colony formation of NDRG2 over-expressing RKO cells was inhibited (P = 0.012), as was the viability, proliferation, and invasion of NDRG2 over-expressing DLD-1 cells (P < 0.001, P = 0.003, and P = 0.044, respectively). Tumor volume in xenograft mice transplanted with NDRG2 over-expressing RKO and DLD-1 cells was smaller than that in controls (P = 0.002 and P = 0.001, respectively). Epigenetic silencing of NDRG2 induces proliferation and invasion of CRC and may be associated with proximal CRC and advanced T stage. NDRG2 methylation might serve as novel biomarker of CRC.

Tumor suppressor candidate gene, NDRG2 is frequently inactivated in human glioblastoma multiforme.

N‑myc downstream regulated gene2 (NDRG2) is highly expressed in numerous normal tissues, while it is marginally expressed or undetectable in various tumors, including lung and colon cancer. In order to investigate the expression of NDRG2 in human glioma and its downstream regulatory mechanisms, quantitative polymerase chain reaction (qPCR), immunohistochemistry and western blot analyses were used to assess NDRG2 mRNA and protein expression in different grades of human glioma and adjacent normal tissues. The methylation status of the NDRG2 promoter region was also determined using bisulfite sequencing. NDRG2 mRNA expression was observed to be significantly lower in glioma tissues than in adjacent normal tissues (P<0.05). Furthermore, a significant negative correlation was found between the glioma tumor grade and NDRG2 expression (P<0.05), at the mRNA and protein levels. Moreover, the methylation rate of the NDRG2 promoter region was 46.3% in the glioma tissues compared with 18.2% in the adjacent normal tissues (P<0.05). These findings show that NDRG2 expression is downregulated in human glioma and that the level of NDRG2 expression negatively correlates with the glioma grade. Furthermore, these findings indicate that NDRG2 downregulation may be due to aberrant methylation of the NDRG2 promoter region and subsequent transcriptional inactivation.

Glioma Malignancy-Dependent NDRG2 Gene Methylation and Downregulation Correlates with Poor Patient Outcome.

Aims: NDRG2 (N-myc downstream regulated gene 2) gene is involved in important biological processes: cell differentiation, growth and apoptosis. Several molecular studies have shown NDRG2 as a promising diagnostic marker involved in brain tumor pathology. The aim of the study was to investigate how changes in epigenetic modification and activity of NDRG2 reflect on glioma malignancy and patient outcome. Methods: 137 different malignancy grade gliomas were used as the study material: 14 pilocytic astrocytomas grade I, 45 diffuse astrocytomas grade II, 29 anaplastic astrocytomas grade III, and 49 grade IV astrocytomas (glioblastomas). Promoter methylation analysis has been carried out by using methylation-specific PCR, whereas RT-PCR and Western-blot analyses were used to measure NDRG2 expression levels. Results: We demonstrated that NDRG2 gene methylation frequency increased whereas expression at both mRNA and protein levels markedly decreased in glioblastoma specimens compared to the lower grade astrocytomas. NDRG2 transcript and protein levels did not correlate with the promoter methylation state, suggesting the presence of alternative regulatory gene expression mechanisms that may operate in a tissue-specific manner in gliomas. Kaplan-Meier analyses revealed significant differences in survival time in gliomas stratified by NDRG2 methylation status and mRNA and protein expression levels. Conclusions: Our findings highlight the usefulness of combining epigenetic data to gene expression patterns at mRNA and protein level in tumor biomarker studies, and suggest that NDRG2 downregulation might bear influence on glioma tumor progression while being associated with higher malignancy grade.

FXR Controls the Tumor Suppressor NDRG2 and FXR Agonists Reduce Liver Tumor Growth and Metastasis in an Orthotopic Mouse Xenograft Model

The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR−/− mice develop hepatocellular carcinoma (HCC) and show an increased prevalence for intestinal malignancies, suggesting a role of FXR as a tumor suppressor in enterohepatic tissues. The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.We show reduced NDRG2 mRNA in livers of FXR−/− mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options.

DNA Methylation of NDRG2 in Gastric Cancer and Its Clinical Significance

Gastric cancer is one of the most common digestive malignancies worldwide. N-myc downstream-regulated gene 2 (NDRG2) is a differentiation-related gene that is considered to be a metastasis suppressor gene. In this study, we examined the expression and DNA methylation of NDRG2 in gastric cancer cell lines and tissues, as well as its clinical significance. Six gastric cancer cell lines and 42 paired normal and gastric cancer tissue samples were used to assess NDRG2 mRNA expression using RT-PCR. NDRG2 DNA methylation status was evaluated by methylation-specific PCR (MSP) in gastric cancer cell lines and tissues. The suppression of NDRG2 in BGC823 cells by siRNA transfection was utilized to detect the role of NDRG2 in gastric cancer progression. NDRG2 mRNA was down-regulated in gastric cancer cell lines and tissues, and its expression was just related to lymph node metastasis (p=0.032). MSP showed methylation of NDRG2 in 54.0% (47/87) of primary gastric cancer specimens and in 20.0% (16/80) of corresponding nonmalignant gastric tissues. NDRG2 methylation was related to depth of tumor invasion, Borrmann classification and TNM stage (p<0.05). Upon treatment with 5-aza-2'-deoxycytidine and trichostatin A, NDRG2 expression was upregulated in HGC27 cells, and demethylation of the highly metastatic cell line, MKN45, inhibited cell invasion. Furthermore, the suppression of NDRG2 by siRNA transfection enhanced BGC823 cells invasion. Our results suggest that the aberrant methylation of NDRG2 may be mainly responsible for its downregulation in gastric cancer, and may play an important role in the metastasis of gastric cancer.

NDRG2 Ameliorates Hepatic Fibrosis by Inhibiting the TGF-β1/Smad Pathway and Altering the MMP2/TIMP2 Ratio in Rats

Liver fibrosis is a worldwide clinical issue. It has been well established that activated hepatic stellate cells (HSCs) are responsible for excessive extracellular matrix (ECM) deposition in chronically damaged livers. The identification of key elements that control HSCs activation will help to further our understanding of liver fibrosis and improve the outcome of clinical treatment. This study demonstrates that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of liver fibrosis as NDRG2 mRNA and protein levels were reduced during HSCs activation. In addition, enhanced NDRG2 expression reduced Smad3 transcription and phosphorylation, which inhibited HSCs activation by blocking the TGF-β1 signal. Moreover, NDRG2 contributed to an increase in the ratio of matrix metalloproteinase 2 (MMP2) to tissue inhibitor of matrix metalloproteinase 2 (TIMP2), which may facilitate the degradation of the ECM. In dimethylnitrosamine (DMN)-induced fibrotic rat livers, adenovirus-mediated NDRG2 overexpression resulted in decreased ECM deposition and improved liver function compared with controls. In conclusion, the present findings indicate that the modulation of NDRG2 is a promising strategy for the treatment of liver fibrosis.

Spatial–temporal expression of NDRG2 in rat brain after focal cerebral ischemia and reperfusion

N-myc downstream regulated gene 2 (NDRG2) was reported to be widely expressed in the nervous system. However, the expression and potential role of NDRG2 in focal cerebral ischemia brain remain unclear. Herein, we investigated spatial–temporal expression of NDRG2 in the rat brain following transient focal cerebral ischemia. Male Sprague–Dawley rats underwent a 120-min transient occlusion of middle cerebral artery. Rats were killed and brain samples were harvested at 4, 12, 24, and 72h after reperfusion. Expression of NDRG2 in the brain was determined by reverse transcriptase–polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemical staining. Cellular apoptosis was assessed by TUNEL staining. The results showed that NDRG2 was expressed on cells with an astrocytes-like morphology in ischemic penumbra. NDRG2 mRNA and protein expression began to increase at 4h after reperfusion and peaked at 24h in the ischemic penumbra. By using immunofluorescence, NDRG2 signals were co-localized with GFAP-positive astrocytes, and NDRG2 expression in astrocytes translocated from a cytoplasm to a nuclear localization at 24h after reperfusion. Double immunofluorescent staining for TUNEL and NDRG2 showed that some NDRG2 signals co-localized with TUNEL-positive cells, and that the apoptotic cells increased with enhancement of NDRG2-positive signals. In conclusion, NDRG2 expression is up-regulated in ischemic penumbra following transient focal cerebral ischemia. NDRG2 expression in astrocytes may play important pathological roles in cell apoptosis after stroke.

Expression profile of the N-myc Downstream Regulated Gene 2 (NDRG2) in human cancers with focus on breast cancer

BackgroundSeveral studies have shown that NDRG2 mRNA is down-regulated or undetectable in various human cancers and cancer cell-lines. Although the function of NDRG2 is currently unknown, high NDRG2 expression correlates with improved prognosis in high-grade gliomas, gastric cancer and hepatocellular carcinomas. Furthermore, in vitro studies have revealed that over-expression of NDRG2 in cell-lines causes a significant reduction in their growth. The aim of this study was to examine levels of NDRG2 mRNA in several human cancers, with focus on breast cancer, by examining affected and normal tissue.MethodsBy labelling a human Cancer Profiling Array with a radioactive probe against NDRG2, we evaluated the level of NDRG2 mRNA in 154 paired normal and tumor samples encompassing 19 different human cancers. Furthermore, we used quantitative real-time RT-PCR to quantify the levels of NDRG2 and MYC mRNA in thyroid gland cancer and breast cancer, using a distinct set of normal and tumor samples.ResultsFrom the Cancer Profiling Array, we saw that the level of NDRG2 mRNA was reduced by at least 2-fold in almost a third of the tumor samples, compared to the normal counterpart, and we observed a marked decreased level in colon, cervix, thyroid gland and testis. However, a Benjamini-Hochberg correction showed that none of the tissues showed a significant reduction in NDRG2 mRNA expression in tumor tissue compared to normal tissue. Using quantitative RT-PCR, we observed a significant reduction in the level of NDRG2 mRNA in a distinct set of tumor samples from both thyroid gland cancer (p = 0.02) and breast cancer (p = 0.004), compared with normal tissue. MYC mRNA was not significantly altered in breast cancer or in thyroid gland cancer, compared with normal tissue. In thyroid gland, no correlation was found between MYC and NDRG2 mRNA levels, but in breast tissue we found a weakly significant correlation with a positive r-value in both normal and tumor tissues, suggesting that MYC and NDRG2 mRNA are regulated together.ConclusionExpression of NDRG2 mRNA is reduced in many different human cancers. Using quantitative RT-PCR, we have verified a reduction in thyroid cancer and shown, for the first time, that NDRG2 mRNA is statistically significantly down-regulated in breast cancer. Furthermore, our observations indicate that other tissues such as cervix and testis can have lower levels of NDRG2 mRNA in tumor tissue compared to normal tissue.

Evaluation of NDRG2 gene expression in primary papillary thyroid carcinoma and in metastases of this neoplasm to regional lymph nodes

BackgroundAt present, researchers' attention has been concentrating on NDRG2 (N-Myc downstream-regulated gene 2) as a new gene candidate in the development and progression of papillary thyroid carcinoma (PTC). NDRG2, together with NDRG1, NDRG3 and NDRG4 are members of the NDRG family, a new class of genes, inhibited by N-Myc oncogene.AimThe aim of our study was to evaluate NDRG2 mRNA expression in the primary PTC and its metastases to regional lymph nodes.Materials and methodsPostoperative tissue and macroscopically changed lymph nodes of sixteen (16) patients with PTC constituted the studied material. In this group, metastases of the cancer to regional lymph nodes were confirmed histopathologically in 8 cases. Quantitative evaluation of NDRG2 mRNA expression was performed by the real-time polymerase chain reaction (real-time PCR) method.ResultsThe mean values of NDRG2 mRNA expression in the primary tumour tissues were statistically significantly lower vs. the levels of NDRG2 mRNA expression in macroscopically unchanged thyroid tissue (p < 0.0001). A comparison of the mean NDRG2 mRNA expression of primary tumours and that of their metastases to regional lymph nodes did not demonstrate any statistical differences (p > 0.05). A positive correlation was observed between NDRG2 mRNA expression in primary tumour cells and in the cancer metastases to lymph nodes (Rs = 0.7857; p < 0.05). Factors, such as age, sex, tumour stage in TNM system, were of no significance for NDRG2 mRNA expression level (p > 0.1).ConclusionThe results of our study demonstrated decreased NDRG2 mRNA expression levels in PTC, when compared to macroscopically unchanged thyroid tissue, which may point to the potential role of NDRG2 in the development and progression of cancer in question.

NDRG2 is a candidate tumor-suppressor for oral squamous-cell carcinoma

Oral cancer is one of the most common cancers worldwide, and squamous-cell carcinoma (OSCC) is the most common phenotype of oral cancer. Although patients with OSCC have poor survival rates and a high incidence of metastasis, the molecular mechanisms of OSCC development have not yet been elucidated. This study investigated whether N-myc downstream-regulated gene 2 (NDRG2) contributes to the carcinogenesis of OSCC, as NDRG2 is reported to be a candidate tumor-suppressor gene in a wide variety of cancers. The down-regulation of NDRG2 mRNA, which was dependent on promoter methylation, was seen in the majority of OSCC cases and in several cases of precancerous leukoplakia with dysplasia. Induction of NDRG2 expression in an HSC-3/OSCC cell line significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The majority of OSCC cell lines showed an activation of PI3K/Akt signaling, and enforced expression of NDRG2 in HSC-3 cells decreased the level of phosphorylated Akt at Serine 473 (p-Akt). Immunohistochemical p-Akt staining was detected in 56.5% of the OSCC tumors, and 80.4% of the tumors were negative for NDRG2 staining. Moreover, positive p-Akt staining was inversely correlated with decreased NDRG2 expression in OSCC tumors with moderate to poor differentiation ( p < 0.005). Therefore, NDRG2 is a candidate tumor-suppressor gene for OSCC development and probably contributes to the tumorigenesis of OSCC partly via the modulation of Akt signaling.

Frequent promoter hypermethylation and transcriptional downregulation of the NDRG2 gene at 14q11.2 in primary glioblastoma

The N-myc downstream-regulated gene 2 (NDRG2) at 14q11.2 has been reported to be downregulated in glioblastoma, and NDRG2 overexpression represses glioblastoma cell proliferation in vitro (Deng et al., Int J Cancer 2003;106;342-7). To further address the role of NDRG2 as a candidate tumor suppressor in human gliomas, we analyzed 67 astrocytic tumors (10 diffuse astrocytomas, 11 anaplastic astrocytomas, 34 primary glioblastomas and 12 secondary glioblastomas) for NDRG2 gene mutation, promoter methylation and expression at the mRNA and protein levels. Using real-time reverse transcription PCR analysis, we found decreased NDRG2 mRNA levels in primary glioblastomas as compared to diffuse and anaplastic astrocytomas. Similarly, immunohistochemistry revealed low or absent NDRG2 protein expression in primary glioblastomas. Mutational analysis of the entire NDRG2 coding sequence did not reveal any tumor-associated DNA sequence alterations. However, sequencing of sodium bisulfite-modified DNA identified hypermethylation of the NDRG2 promoter region in 21 of 34 primary glioblastomas (62%). Moreover, NDRG2 promoter hypermethylation was associated with decreased NDRG2 mRNA expression. In contrast to primary glioblastomas, NDRG2 promoter hypermethylation was detected in only 1 of 11 anaplastic astrocytomas (9%) and was absent in 10 diffuse astrocytomas and 12 secondary glioblastomas. Taken together, our data support NDRG2 as a candidate tumor suppressor gene that is epigenetically silenced in the majority of primary glioblastomas, but not in lower grade astrocytomas and secondary glioblastomas.