NELFE promotes the migration and invasion of triple negative breast cancer cells by inhibiting NDRG2 expression / 西安交通大学学报(医学版)

【Objective】 To explore the effect of negative elongation factor E(NELFE) on the invasion and migration of triple-negative breast cancer(TNBC) cells and its mechanisms. 【Methods】 qRT-PCR was used to detect the expression of negative elongation factor E(NELFE) in triple-negative breast cancer(TNBC) tissues and adjacent tissues. NELFE expression was upregulated in MDA-MB-231 cell lines via lentivirus transfection. Transwell assay was used to observe the effect of elevated NELFE expression on the migration and invasion of MDA-MB-231 cells. The RNA stability assay was used to detect the influence of NELFE on the stability of the mRNA of N-myc downstream regulated gene2(NDRG2). Western blotting analysis was used to detect the regulatory function of NELFE on NDRG2 protein expression. 【Results】 NELFE expression was increased in TNBC tissues. After the overexpression of NELFE, the migration and invasion of MDA-MB-231 cells were significantly enhanced, NDRG2 mRNA stability was decreased, and NDRG2 protein expression level was downregulated. NDRG2 expression was upregulated in MDA-MB-231 cell lines with overexpressed NELFE; cell migration and invasion abilities were decreased. 【Conclusion】 NELFE inhibited NDRG2 expression by decreasing the stability of NDRG2 mRNA and promoted the migration and invasion of TNBC cells.

Lentivirus-mediated NDRG2 gene overexpression inhibits radioresistance of bladder cancer cells / 中国医师杂志

Objective:The purpose of this study was to investigate the expression and role of N-myc downstream regulatory gene 2 (NDRG2) in radiation resistance of bladder cancer cells.Methods:T24 cells were cultured in vitro and irradiated with different doses of X-ray (0, 2, 4, 8, 10 and 20 Gy). The best dose of X-ray was selected for subsequent treatment. The radioresistant BCa cell line T24/R was established. The cytotoxicity of T24/R cells was detected by counting kit-8 (CCK-8) method. The proliferation and invasion ability of T24/R cells and T24 cells were detected by flow cytometry and transwell, respectively. Western blot was used to detect the expression of epithelial mesenchymal transition (EMT) related proteins. The survival rate of T24/R group (control group) and T24/R-NDRG 2 group was detected, and the migration ability of T24/R-NDRG 2 cells was detected after 2 Gy treatment. Results:The cell viability was inhibited significantly when the dose of X-ray was ≥2 Gy X-ray, so 2 Gy X-ray irradiation was chosen as the best condition for BCa cytotoxicity and T24/R radiation resistance cell line was successfully established; Apoptosis test showed that the number of S-phase cells was increased in T24/R group, and the proportion of S-phase cells in T24/R vs T24 was (26.49±4.5)% vs (14±2.6)% ( P<0.05); Transwell test showed that T24/R cells showed stronger migration ability than control group ( P<0.05), but there was no significant difference in EMT related protein expression between the two groups ( P>0.05). Overexpression of NDRG2 can significantly decreased the activity and migration ability of radiation-resistant T24/R cells ( P<0.05) when the radiation dose was gradually increasing in both groups. Conclusions:The radiation resistance of BCa cells is one of the causes of local tumor recurrence. Up-regulation of NDRG2 expression can inhibit the radiation resistance of T24 cells, so it can be used as a candidate for treatment of radiation-resistant BCa patients.

The role of hypoxia-related molecules in the pathogenesis of osteoarthritis / 中华创伤骨科杂志

Osteoarthritis is one of the most common chronic diseases in orthopedics.With increasing populations of aging and obese people,its incidence has risen year by year and become a major public health problem.The hallmark of osteoarthritis is cartilage destruction,the main cause of which is degradation of extracellular matrix by catabolic enzymes and death of chondrocytes caused by apoptosis or autophagy.Articular cartilage is a hypoxic environment because it lacks blood supply and the joint cavity is relatively closed.A hypoxic environment induces chondrocytes to produce a series of hypoxia-related molecules which can regulate the expression of catabolic enzymes,autophagy and apoptosis of chondrocytes for osteoarthritis.This paper aims to review recent reports on the relationship between hypoxic-related molecules and pathogenesis of osteoarthritis and discuss the role of hypoxia-related molecules in the pathogenesis of osteoarthritis.

Anti-fibrotic Effects and Mechanism of Shengmai Injection () on Human Hepatic Stellate Cells LX-2 / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2.@*METHODS@#LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot.@*RESULTS@#When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL (P<0.05).@*CONCLUSION@#The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.

Lipoxin A4 Ameliorates Lipopolysaccharide-Induced A549 Cell Injury through Upregulation of N-myc Downstream-Regulated Gene-1 / 中华医学杂志(英文版)

<p><b>Background</b>Lipoxin A4 (LXA4) can alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI) and acute respiratory distress syndrome through promoting epithelial sodium channel (ENaC) expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4.</p><p><b>Methods</b>A549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing of A549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis of A549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor (ALX) inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression.</p><p><b>Results</b>The A549 cell models of ALI were constructed and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream-regulated gene-1 (NDRG1) was validated by real-time-PCR and Western blot. NDRG1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG1 expression induced by LXA4. NDRG1 siRNA suppressed viability of LPS-treated A549 cells (treatment vs. control, 0.605 ± 0.063 vs. 0.878 ± 0.083, P = 0.040) and ENaC-α expression (treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008). LY294002 inhibited NDRG1 (treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P = 0.001) and ENaC-α (treatment vs. control, 0.236 ± 0.021 vs. 0.814 ± 0.025, P < 0.001) expressions and serum- and glucocorticoid-inducible kinase 1 phosphorylation (treatment vs. control, 0.442 ± 0.024 vs. 1.046 ± 0.082, P = 0.002), indicating the PI3K signaling pathway was involved in regulating NDRG1 expression induced by LXA4.</p><p><b>Conclusion</b>Our research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.</p>

The expression and significance of N-myc downstream regulated gene-2 in rat kidney after ureteral obstruction / 国际儿科学杂志

Objective To investigate the expression of N-myc downstream regulated gene-2 (NDRG2) on renal fibrosis in unilateral ureteral obstruction(UUO) rat model and the mechanism of renal fibrosis.Methods Forty-eight male Wistar rats (120-150g)were randomly divided into two groups:the sham-operation group (n =24)underwent the left ureteral dissection,the UUO group (n =24)underwent the left ureteral ligation.At the 3,7,14,21day after the operation,6 rats from each of the group were sacrificed and the obstructive kidneys were collected.The histopathological changes were observed through HE and Masson staining.E-cadherin and α-SMA were detected by Western blot and immunohistochemistry.NDRG2 were detected by Western blot,immunohistochemistry and real-time PCR.Results There is not diffenrence in the sham group at anytime.Compared with the sham-group,the fibrosis was obvious in UUO group.The protein of E-cadherin decreased(P ＜ 0.05) and α-SMA increased(P ＜0.05).The expression of NDRG2 in UUO group was lower than that in the sham group.The OD of immunohistochemistry was detected by ImagePro Plus 6.0.OD of NDRG2 was the lowest[(14.33 ± 2.45) x 10-3] in 21d group.The western blot showed that the ratio of NDRG2 to β-actin was the lowest(0.03 ± 0.01) in 21d group.The mRNA of NDRG2 decreased obverously in UUO group (P ＜ 0.05).Conclusion NDRG2 decreases in UUO group and NDRG2 might be involved in the pathologic process of renal fibrosis.

Anti-fibrotic effects of Salvia miltiorrhiza and Ligustrazine Injection on LX-2 cells involved with increased N-myc downstream-regulated gene 2 expression / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Salvia miltiorrhiza and Ligustrazine Injection (SML) on proliferation and apoptosis of human hepatic stellate cell LX-2 and the expression of N-myc downstreamregulated gene 2 (NDRG2, a tumor suppressor gene).</p><p><b>METHODS</b>HSCs from the LX-2 cell line were cultured in vitro. The proliferative state of different initial LX-2 cell numbers was measured using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. LX-2 cells were plated in 96-well plates at an approximate density of 2.50×10cells/mL and cultured for 24 h followed by the application of different concentrations of SML (1, 2, 4 and 8 μL/mL). Cell proliferation was measured using the MTT assay at 24 and 48 h. Apoptosis was detected by flow cytometry at 24 h. LX-2 cells were treated with different concentrations of SML and extracted with protein lysis buffer. The levels of NDRG2 and β-catenin were measured by Western blot.</p><p><b>RESULTS</b>With the exception of the 1 and 2 μL/mL concentrations, 4 and 8 μL/mL SML inhibited cell proliferation in a concentration-dependent manner at 24 and 48 h (P<0.05). With the exception of the 1 and 2 μL/mL concentrations, the NDRG2 expression level was greatly increased in a concentration-dependent manner. However, the level of β-catenin was unaffected.</p><p><b>CONCLUSION</b>SML inhibit LX-2 cell proliferation in a concentration-dependent manner, and the mechanism may be associated with NDRG2 over-expression.</p>

N-myc downstream regulated gene 2 promotes apoptosis of bladder cancer cells through signal transducer and activator of transcription 3 signaling pathway / 中华泌尿外科杂志

Objective To investigate the effect of N-myc downstream regulated gene 2 (NDRG2) on the apoptosis of bladder cancer cells by regulating the signal transducer and activator of transcription 3(STAT3) signaling pathway.Methods The expression level of NDRG2 in human bladder cancer cell BIU-87 and immortalized cell SV-HUC-1 was detected by Western blot.NDRG2 over expression vector and empty vector control (pcDNA3.1),siRNA-NDRG2,siRNA control were transfected into BIU-87 cells.After transfected 48 h,the expression level of NDRG2,Cleaved caspase 3,STAT3,p-STAT3,JAK2,p-JAK2 were detected by Western blot,the cell proliferation and apoptosis were measured by MTT and flow cytometry.After adding inhibitor AG490 of 45 μmol/L in cultured BIU-87 cells,MTT assay was used to detect cell proliferation and flow cytometry was used to detect the cell apoptosis,Western blot to detect the expression level of Cleaved caspase 3,STAT3,p-STAT3,JAK2,p-JAK2.Results The expression level of NDRG2 in bladder cancer cells was higher than that in bladder epithelial cells.The cell survival rate of pcDNA3.1/NDRG2 group was lower than that of pcDNA3.1 group,the difference was statistically significant (P ＜ 0.01).The cell survival rate of siRNA-NDRG2 group was higher than that of siRNA control group (P＜ 0.01).The apoptosis rate of pcDNA3.1/NDRG2 group was higher than that of pcDNA3.1 group (P ＜ 0.01).The apoptosis rate of NDRG2 siRNA group was lower than that of siRNA control group (P ＜ 0.01).The level of p-STAT3 and p-JAK2 in pcDNA3.1/NDRG2 group was lower than that in pcDNA3.1 group (P＜ 0.01).The survival rate and apoptosis rate of BIU-87 cells cultured with AG490 were in agreement with the trend of pcDNA3.1/NDRG2 after transfection.Conclusions NDRG2 could promote the apoptosis of bladder cancer cells,and its mechanism may be related to STAT3 signaling pathway.

NDRG2 inhibits the proliferation of breast cancer cells via regulating β-catenin expression and nuclear translocation / 中国癌症杂志

Background and purpose:Breast cancer is one of the most common malignant diseases in women and its malignant proliferation is the major cause of death. To investigate the effects of N-myc downstream regulated gene 2 (NDRG2) on proliferation of breast cancer cells by using two parallel cell lines (MCF-7 and LM-MCF-7) with different metastatic abilities.Methods:The expression level of NDRG2 in breast cancer cells was detected by Western blot. The effects of overexpressing (or down-regulating) NDRG2 on proliferation of breast cancer cells were investigat-ed by lfow cytometry. The expression and location of β-catenin were detected by Western blot and immunolfuorescence respectively. NDRG2 blocking the transcription activity of β-catenin was investigated via co-transfecting MCF-7 cells with NDRG2 siRNA and pCMV-Tcfδ (lacking the portion responsible for the protein binding to DNA).Results:The expression level of NDRG2 was negatively related to the proliferation ability of breast cancer cells. Over-expressing NDRG2 (or down-regulating) via transfecting LM-MCF-7 (or MCF-7) cells with pCMV-NDRG2 (or NDRG2 siRNA) could inhibit (or promote) cell proliferation. Interestingly, the results of Western blot, immunolfuorescence and lfow cytometry revealed that down-regulation of NDRG2 resulted from the down-regulation of β-catenin and blocking its nuclear translocation, which led to losing control of the proliferation of breast cancer cells.Conclusion:NDRG2 inhibit the proliferation of breast cancer cells via down-regulating the expression of β-catenin and blocking its nuclear translo-cation, which is signiifcant for exploring the molecular mechanism of proliferation of breast cancer cells.

Preliminary study of NDRG1 gene promoter methylation status in prostate cancer / 中华泌尿外科杂志

Objective To evaluate the methylation status of prostate cancer NDRG1 gene promoter region,and to explore the influence of methylation inhibitor 5-azacytidine on NDRG1 gene's mRNA expression in prostate cancer cells and its effects on cell proliferation.Methods Bisulfite-sequencing PCR (BSP) were used to detect the NDRG1 gene promoter methylation status in prostate cancer and BPH tissue,prostate cancer cell lines (PC3,22RV1,LNCaP and DU145) and human normal prostate cell line's RWPE-1.After 10 μmol/L 5-azacytidine were used on LNCaP and DU145 cells for 72 h,5-azacytidine's influence on cell proliferation was analyzed with MTT,two prostate cancer cell lines NDRG1 mRNA expressions were detected with RT-PCR.Results The methylation rates of NDRG1 gene in prostate cancer cell lines PC-3,22RV1,LNCaP and DU145 were (24.8 ± 3.3) ％,(36.2 ± 2.5) ％,(48.6 ± 2.8) ％ and (69.5 ± 1.7) ％,respectively.Methylation rate of Human normal prostate cell lines RWPE-1 was (4.8 ± 4.5) ％;prostate carcinoma was (48.6 ± 5.3) ％,BPH tissue was (4.3 ± 2.1) ％.The differences between groups were statistically significant.After 10 μmol/L 5-azacytidine added on LNCaP and DU145 cells for 72 h,NDRG1 gene demethylation occurred in both cells,its mRNA expression enhanced 8-9 times compared with previous and its cell growth was inhibited (P ＜ 0.05).Conclusions NDRG1 gene promoter region's hypermethylation is one of the reasons of its aberrant expression in prostate cancer.5-azacytidine can reverse NDRG1 gene promoter methylation status,regulate the expression of the gene and can inhibit prostate cancer cell proliferation.

Expression and distribution of N-myc downstream regulated gene 2 in pancreatic diseases / 中华内分泌代谢杂志

The pancreatic tissues from patients with islet cell hyperplasia,insulinoma,and pancreatic adenocarcinoma,as well as normal pancreatic tissues were embedded with paraffin,serial sections were cut and mounted on glass slides.Immunohistochemical staining was carried out with N-myc down-stream regulated gene 2 (Ndrg2) monoclonal antibody by means of ABC method,and Western blotting was carried out to detect the expression and distribution of Ndrg2.The results showed that Ndrg2 positive immunoreactivity was mainly localized in the cytoplasm of islet cell,being similar to the localization of insulin positive immunoreactivity.The number and volume of pancreatic islets were increased in the patients with islet cell hyperplasia,and Ndrg2 expression was also increased.Western blotting results showed that the expression of Ndrg2 in the pancreas of patients with islet cell hyperplasia was increased compared with normal group.The above results suggest that Ndrg2 may play an important role in performing physiological function of islet cells.

Avaliação do papel funcional do gene NDRG4 na tumorigênese de mama / Evaluation of the functional role of gene NDRG4 in breast tumorigenesis

Em trabalho anterior realizado pelo nosso grupo, descrevemos que o gene NDRG4 se encontra silenciado em linhagens tumorais de mama devido a presença de metilação na sua região promotora. Neste trabalho, exploramos o papel do silenciamento do gene NDRG4 na tumorigênese da mama. Em um primeiro momento, investigamos a associação entre a presença de metilação na região promotora do gene NDRG4 em 61 amostras de tumores de mama e os dados clínico-patológicos das pacientes. Observamos uma associação estatisticamente significativa entre a presença de metilação do DNA na região promotora do gene NDRG4 e fatores de pior prognóstico, tais como: número de linfonodos positivos (p=0,025), níveis elevados da proteína p53 (p=0,014) e o tamanho do tumor (p=0,036); bem como com uma menor taxa de sobrevida livre de metástase em 10 anos (p=0,001). Em análise multivariada, a presença de metilação do DNA na região promotora do gene NDRG4 se mostrou um fator independente de prognóstico para sobrevida livre de mestástase (HR=5.5 e p=0.006). Paralelamente, realizamos o silenciamento do gene NDRG4 na linhagem de tumor de mama MCF7 utilizando a metodologia de shRNA. Variantes celulares, silenciadas para o gene NDRG4, apresentaram uma redução significativa na taxa de proliferação e na capacidade de formação de colônias isoladas e um aumento significativo na capacidade de migração. No entanto, não foram observadas diferenças significativas na capacidade de adesão e na susceptibilidade a taxanos dos clones silenciados

Expression of N-myc downstream regulated gene 1 in breast invasive duct carcinoma and its relationship with tumor metastasis and invasive potential / 上海第二医科大学学报

Objective To study the correlation of expression of N-myc downstream regulated gene 1(NDRG1) with invasion of breast invasive duct carcinoma and lymph node metastasis. Methods A total of 71 specimens including 26 case of primary breast invasive duct carcinoma with lymphnode metastasis,45 case of nonmetastasis breast invasive duct carcinoma were observed.NDRG1 was detected by immunohistochemistry in formalin-fixed and paraffin-embedded sections.At the same time,the correlations of NDRG1 with E-cad,MMP2,MMP9,and TIMP2 were investigated. Results The expression of NDRG1 in breast cancer with metastasis of lymph nodes(9/26) was lower than that of non-metastasis of lymph nodes(32/45)(P