N-myc downstream-regulated gene 2 promotes proliferation of HO-8910 ovarian cancer cells

Objective To investigate N-myc downstream-regulated gene 2(NDRG2) expression in ovarian cancer cells and its potential usefulness as a diagnostic marker and/or target for therapeutic intervention.Methods Human NDRG2 L/S gene was obtained by revers-transcription polymerase chain reaction(RT-PCR). Sequence analysis confirmed the identity of NDRG2 L/S gene, which was then inserted into a eukaryotic vector p LNCX2, which was in turn transfected into NDRG2 gene-negative HO-8910 cells. Flow cytometry(FCM) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay were conducted to determine the proliferation rate of HO-8910 cells. Cisplatin resistance of HO-8910 cells transfected with p LNCX2-NDRG2 L/S was evaluated by FCM. Tumors were generated in female nude mice by subcutaneous injection of HO-8910 cells.Results NDRG2 gene was isolated and its expression vector was successfully constructed. NDRG2 expression positively correlated with the proliferation of HO-8910 cells. NDRG2 L/S promoted tumorigenicity in HO-8910 cells.Conclusion The present study identified a novel function of NDRG2 L/S gene and demonstrated its involvement in the promotion of ovarian cancer cell proliferation and enhancement of cisplatin resistance in HO-8910 cells. Future studies are warranted to determine the relationship between NDRG2 upregulation and ovarian cancer progression.

Prognostic and biological role of the N-Myc downstream-regulated gene family in hepatocellular carcinoma

BACKGROUND The N-Myc downstream-regulated gene(NDRG)family is comprised of four members(NDRG1-4)involved in various important biological processes.However,there is no systematic evaluation of the prognostic of the NDRG family in hepatocellular carcinoma(HCC).AIM To analyze comprehensively the biological role of the NDRG family in HCC.METHODS The NDRG family expression was explored using The Cancer Genome Atlas.DNA methylation interactive visualization database was used for methylation analysis of the NDRG family.The NDRG family genomic alteration was assessed using the cBioPortal.Single-sample Gene Set Enrichment Analysis was used to determine the degree of immune cell infiltration in tumors.RESULTS NDRG1 and NDRG3 were up-regulated in HCC,while NDRG2 was down-regulated.Consistent with expression patterns,high expression of NDRG1 and NDRG3 was associated with poor survival outcomes(P<0.05).High expression of NDRG2 was associated with favorable survival(P<0.005).An NDRG-based signature that statistically stratified the prognosis of the patients was constructed.The percentage of genetic alterations in the NDRG family varied from 0.3%to 11.0%,and the NDRG1 mutation rate was the highest.NDRG 1-3 expression was associated with various types of infiltrated immune cells.Gene ontology analysis revealed that organic acid catabolism was the most important biological process related to the NDRG family.Gene Set Enrichment Analysis showed that metabolic,proliferation,and immune-related gene sets were enriched during NDRG1 and NDRG3 high expression and NDRG2 low expression.CONCLUSION Overexpression of NDRG1 and NDRG3 and down-expression of NDRG2 are correlated with poor overall HCC prognosis.Our results may provide new insights into the indispensable role of NDRG1,2,and 3 in the development of HCC and guide a promising new strategy for treating HCC.

Expression and Biological Function of N-myc Down-regulated Gene 1 in Human Cervical Cancer

The expression of N-myc down-regulated gene 1 (NDRG1) has previously been reported to be involved in the proliferation,differentiation,invasion and metastasis of cancer cells,but its role in cervical cancer is still unclear.This study aimed to investigate the expression of NDRG1gene in human cervical cancer and its effect on aggressive tumor behaviors.The NDRG1 expression in cervical tissues and cells was detected by RT-PCR.Specific expression plasmid pEGFP-N1-NDRG1-GFP was used to enhance the expression of NDRG1 in human cervical cancer cell lines.The mRNA and protein level of NDRG1 was assessed by RT-PCR and Western blotting,respectively.Its effects on cell proliferation,migration,invasion,cell cycle and apoptosis were detected by MTT,transwell migration assay and flow cytometry (FCM),respectively.The results showed that the expression of NDRG1 in cervical cancer tissues and cells was significantly lower than in normal cervical tissues (P【0.001).After transfection with pEGFP-N1-NDRG1-GFP,the mRNA and protein expression of NDRG1 was up-regulated in Siha cells,which suppressed cell proliferation (P【0.001),induced cell cycle arrest (P【0.05),reduced invasion and migration of Siha cells (P【0.05),but caused no cell apoptosis.Moreover,vascular endothelial growth factor (VEGF),a tumor-induced angiogenesis factor,was markedly reduced and E-cadherin,a cell adhesion molecule,was increased in the cells transfected with pEGFP-N1-NDRG1-GFP.It was concluded that up-regulated NDRG1 may play a role in the suppression of malignant cell growth,invasion and metastasis of human cervical cancer.

N-myc downstream regulated gene 1 inhibition of tumor progression in Caco2 cells

BACKGROUND Invasion and migration are the irreversible stages of colorectal cancer(CRC).The key is to find a sensitive,reliable molecular marker that can predict the migration of CRC at an early stage.N-myc downstream regulated gene 1(NDRG1)is a multifunctional gene that has been tentatively reported to have a strong relationship with tumor invasion and migration,however the current molecular role of NDRG1 in CRC remains unknown.AIM To explore the role of NDRG1 in the development of CRC.METHODS NDRG1 stably over-expressed Caco2 cell line was established by lentiviral infection and NDRG1 knock-out Caco2 cell line was established by CRISPR/Cas9.Furthermore,the mRNA and protein levels of NDRG1 in Caco2 cells after NDRG1 over-expression and knockout were detected by real-time polymerase chain reaction and western blot.The cell proliferation rate was measured by the cell counting kit-8 method;cell cycle and apoptosis were detected by flow cytometry;invasion and migration ability were detected by the 24-transwell method.RESULTS NDRG1 over-expression inhibited Caco2 proliferation and the cell cycle could be arrested at the G1/S phase when NDRG1 was over-expressed,while the number of cells in the G2 phase was significantly increased when NDRG1 was knocked out.This suggests that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase.Our data also demonstrated that NDRG1 promotes early cell apoptosis.Invasion and migration of cells were extensively inhibited when NDRG1 was over-expressed.CONCLUSION NDRG1 inhibits tumor progression in Caco2 cells which may represent a potential novel therapeutic strategy for the treatment of CRC.

Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

AIM: To study short ds RNA oligonucleotides(si RNA)as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1(NDRG1) gene induced under different physiological conditions(Normoxia and hypoxia) modulating NDRG1 transcription, m RNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The p SUPERNDRG1 vectors were designed, two sequences were selected from the human NDRG1 c DNA(5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor(HIF)-1α m RNA sequences in Gen Bank. NDRG1 m RNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia(P < 0.05 was considered significant).RESULTS: si RNA- and iodoacetate(IAA)-mediated downregulation of NDRG1 m RNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it canrepresent a potential target for tumor treatment in human glioblastoma. The si RNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays.

Regulation of HIF-1 α to Expression of N-myc Downstream Regulated Gene 1 in Colorectal Carcinoma

Plasmid expressing small interfering RNA （siRNA） against HIF-1α （pSilence-2.1-U6-siRNA） was constructed and transfected into LS174T cells in hypoxia condition.After expression of siRNA against HIF-1 α in LS174T cells, expressions of HIF-1 α and N-myc downstream regulated gene 1 （NDRG1） gene were inhibited significantly. HIF-1 cta transcripts were positive in 67.7% （42/62） and 44.4% （8/18） of colorectal adenocarcinoma and adenoma, re- spectively. The mean percentage of cells with positive hybridization of HIF-1 α mRNA increases with the development from Duke stage A to stage C＋D （p〈 0.05）. The positive staining rate of NDRG1 protein was significant higher in than that in colorectal adenoma colorectal adenocarcinoma group group （p〈 0.05）. The level of HIF-1 a transcripts was positively correlated with the level of NDRG1 protein （p 〈 0.05） during colorectal tumor progression. HIF-1α and its down stream gene NDRG1 may play roles in tumor progression of human colorectal carcinoma.

Lipoxin A4 Ameliorates Lipopolysaccharide-lnduced A549 Cell Injury through Upregulation of N-myc Downstream-Regulated Gene-1

Background： Lipoxin A4 （LXA4） can alleviate lipopolysaccharide （LPS）-induced acute lung injury （ALl） 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. Methods： 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 ofA549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis ofA549 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. Results： The A549 cell models of ALl were constrticted and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream- regulated gent- 1 （NDRG 1 ） was validated by real-time-PCR and Western blot. NDRG 1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG 1 expression induced by LXA4. NDRG I 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 NDRG 1 （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 I 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 NDRG 1 expression induced by LXA4. Conclusion： 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.

分化相关基因NDRG1在消化系统肿瘤中的研究进展

N-myc下游调节基因1（N-myc downstream regulated gene 1,NDRG1）是近年来发现的一种分化相关基因,多种生理或病理因素的刺激可诱导NDRG1的表达,但其确切的生物学功能尚不完全清楚。

Anti-fibrotic Effects of Salvia miltiorrhiza and Ligustrazine Injection on LX-2 Cells Involved with Increased N-myc Downstream-Regulated Gene 2 Expression

Objective: 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）. Methods: 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 2.50×10;cells/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. Results: 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. Conclusion: SML inhibit LX-2 cell proliferation in a concentration-dependent manner, and the mechanism may be associated with NDRG2 over-expression.

NDRG2 gene copy number is not altered in colorectal carcinoma

AIM To 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).METHODS The following were investigated in the human colorectal cancer cell lines DLD-1, Lo Vo and SW-480: NDRG2 mRNA expression levels using quantitative reverse transcriptionpolymerase 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.RESULTS As 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.CONCLUSION Our 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.

d-limonene prevents ultraviolet irradiation:Induced cyclobutane pyrimidine dimers in Skh1 mouse skin

AIM: To establish whether d-limonene can protect against induction of cyclobutane pyrimidine dimers(CPDs) and sunburn in ultraviolet irradiation(UVR) irradiated mouse skin. METHODS: The d-limonene was given in 4 daily oral 20 μL aliquots at different concentrations as follows: 100%, 10% or 1% in liponate and 100% liponate as control. One day after the final d-limonene treatment, the mice were anesthetized with i.p. sodium pentobarbital and placed in boxes to allow a rectangular(2 cm × 4 cm) region of dorsal skin to be irradiated with a single, ultraviolet radiation dose of 1.5 kJ /m2. Skin samples from UVR irradiated area were obtained at 5 min after UVR exposure for CPD detection, at 6 d after UVR exposure, skin samples were obtained for in situ analysis for N-myc downstream regulating gene 1(NDRG1)(a stress response gene), proliferating cell nuclear antigen(PCNA)(an S-phase marker) and filaggrin(a barrier integrity gene). Based on immunohistochemistry staining, the number of CPD, NDRG1 and PCNA positive cells, as well as unstained cells was counted in 3 different individually selected areas and percentage of positive cells was established. RESULTS: CPD reduction occurred as follows: liponate only-none; 1% d-limonene-54.3% reduction of CPDs; 10% d-limonene-73.4% reduction of CPDs; 100% d-limonene-86.1% reduction of CPDs, the latter equivalent to a UV dose of only 0.21 k J/m2. Sunburn was also dose-dependently reduced by d-limonene. The NDRG1 protein was strongly induced by UVR(70.0% ± 10.4% positive cells), but 1% d-limonene reduced the response to 64.6% ± 9.2%, 10% d-limonene reduced the response to 16.2% ± 3.4% and 100% d-limonene reduced the response to 6.3% ± 1.7%. Similarly, PCNA was 52.4% ± 9.9% positive in UVR exposed skin, and 1% d-limonene reduced it to 42.9% ± 8.1%, 10% d-limonene reduced it to 36.2% ± 6.7% and 100% d-limonene reduce it to 13.8% ± 3.4%. NDRG1 and PCNA were increased by d-limonene or UVR separately, but combined they produced less than either agent separately owing to the protective effect of pre-exposure to d-limonene. CONCLUSION: Overall d-limonene acted to protect against ultraviolet B-induced DNA photodamage and sunburn in UVR exposed skin.

The delivery of N-myc downstream-regulated gene 2(NDRG2)self-amplifying mRNA via modified lipid nanoparticles as a potential treatment for drug-resistant and metastatic cancers

The protein,N-myc downstream-regulated gene 2(NDRG2),a tumor suppressor,is significantly decreased or absent in many types of cancer.There is a significant negative correlation between the levels of NDRG2 and the development and progression of cancer tumor recurrence and tumor invasion,in different cancers.In contrast,the in vitro and in vivo overexpression of the NDRG2 protein decreases the proliferation,growth,adhesion and migration of many types of cancer cells.The in vitro overexpression of NDRG2 increases the efficacy of certain anticancer drugs in specific types of cancer cells.We hypothesize that the delivery of the mRNA of the NDRG2 protein,encapsulated by lipid nanoparticles,could represent a potential treatment of metastatic and drug-resistant cancers.This would be accomplished using a self-amplifying mRNA that encodes the NDRG2 protein and an RNA-dependent-RNA polymerase,obtained from an in vitrotranscribed(IVT)mRNA.The IVT mRNA would be encapsulated in a lipid nanoformulation.The efficacy of the nanoformulation would be determined in cultured cancer cells and if the results are positive,nude mice transplanted with either drug-resistant or metastatic drug-resistant cancer cells,would be treated with the nanoformulation and monitored for efficacy and adverse effects.If the appropriate preclinical studies indicate this formulation is efficacious and safe,it is possible it could be evaluated in clinical trials.

NDRG2截短体真核表达载体的构建

目的：构建稳定表达N-MYC下游调节基因2（NDRG2）截短体的真核表达载体。方法分别用PCR方法扩增NDRG2的截短体NDRG21～178aa和NDRG21～257aa ，以BamHⅠ和EcoRⅠ酶切后，以SolutionⅠ连接酶反应体系将NDRG2截短体片段装载入pCDNA4．0表达载体中，并通过蛋白免疫印迹（Western blotting）验证载体的表达。结果 NDRG2截短体在pCDNA4．0表达载体中正确表达。结论成功构建了NDRG2截短体的真核表达载体。

佛波酯对食管癌细胞分化相关基因NDRG1表达影响的研究

分化相关基因（N-myc downstream regulated gene 1，NDRG1）定位于人染色体8q24.3，NDRG1 mRNA全长约3kb,编码蛋白产物含394个氨基酸，

Anti-fibrotic Effects and Mechanism of Shengmai Injection(生脉注射液)on Human Hepatic Stellate Cells LX-2

Objective: To investigate the effects of Shengmai Injection(生脉注射液, 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 2.5×10~4 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% confluence, apoptosis was detected by flow 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/m L 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). Conclusions: 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.