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...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.展开更多
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 ...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.展开更多
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 ...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.展开更多
Objective:the N-myc downstream regulatory gene 4(NDRG4)is involved in cell growth,cell proliferation,cell survival and tumor invasion.In this paper,the role of NDRG4 in glioma was explored.Method:the expression of NDR...Objective:the N-myc downstream regulatory gene 4(NDRG4)is involved in cell growth,cell proliferation,cell survival and tumor invasion.In this paper,the role of NDRG4 in glioma was explored.Method:the expression of NDRG4 in glioma clinical specimens and its relationship with the prognosis of glioma patients were analyzed by the Cancer Genome Atlas(TCGA)and the Chinese Glioma Genome Atlas(CGGA),and the expression of NDRG4 protein and mRNA in glioma cell lines were tested and verified by Western blot and quantitative real-time fluorescence polymerase chain reaction(qRT-PCR).Result:it showed that the expression of NDRG4 in glioma tissues and cell lines is closely related to the prognosis of glioma patients.Conclusion:NDRG4 is a highly potential target gene for glioma therapy.展开更多
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...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.展开更多
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 lu...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.展开更多
Objective: To study the correlation of LSD1 and NDRG1 gene expression in ovarian cancer tissue with cancer cell migration and invasion. Methods: Patients with ovarian cancer who underwent surgical resection in Fufeng ...Objective: To study the correlation of LSD1 and NDRG1 gene expression in ovarian cancer tissue with cancer cell migration and invasion. Methods: Patients with ovarian cancer who underwent surgical resection in Fufeng County People's Hospital between March 2014 and July 2017 were selected as the research subjects, and the ovarian cancer tissue and adjacent tissue were collected after surgical resection to determine the expression of LSD1, NDRG1, migration genes and invasion genes. Results: LSD1, YKL40, COX2, Twist, IFITM1, CatL, CTHRC1, MMP2 and FUNDC1 mRNA expression in ovarian cancer tissue were significantly higher than those in adjacent tissue whereas NDRG1, E-cadherin and Wnt5a mRNA expression were significantly lower than those in adjacent tissue;YKL40, COX2, Twist, IFITM1, CatL, CTHRC1, MMP2 and FUNDC1 mRNA expression in ovarian cancer tissue with high LSD1 expression were significantly higher than those in ovarian tissue with low LSD1 expression whereas E-cadherin and Wnt5a gene mRNA expression were significantly lower than those in ovarian tissue with low LSD1 expression. Conclusion: The high LSD1 expression and low NDRG1 expression in ovarian cancer tissue can promote the migration and invasion of cancer cells.展开更多
基金the National Natural Science Foundation of China,No.81260361Incubation Project of Mianyang Central Hospital,No.2020FH05.
文摘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.
基金Supported by the Fund for Key Technologies R and D Pro-gramme of Hubei Province(2006AA301A03 )
文摘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.
基金Supported by Deutsche Forschungsgemeinschaft DFG,VO 871/2-3,to Vordermark Dthe IZKF Würzburg,B25,to Hagemann C
文摘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.
文摘Objective:the N-myc downstream regulatory gene 4(NDRG4)is involved in cell growth,cell proliferation,cell survival and tumor invasion.In this paper,the role of NDRG4 in glioma was explored.Method:the expression of NDRG4 in glioma clinical specimens and its relationship with the prognosis of glioma patients were analyzed by the Cancer Genome Atlas(TCGA)and the Chinese Glioma Genome Atlas(CGGA),and the expression of NDRG4 protein and mRNA in glioma cell lines were tested and verified by Western blot and quantitative real-time fluorescence polymerase chain reaction(qRT-PCR).Result:it showed that the expression of NDRG4 in glioma tissues and cell lines is closely related to the prognosis of glioma patients.Conclusion:NDRG4 is a highly potential target gene for glioma therapy.
基金Supported by the National Natural Science Foundation of China(No.81272490 and 81100764)
文摘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.
文摘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.
文摘Objective: To study the correlation of LSD1 and NDRG1 gene expression in ovarian cancer tissue with cancer cell migration and invasion. Methods: Patients with ovarian cancer who underwent surgical resection in Fufeng County People's Hospital between March 2014 and July 2017 were selected as the research subjects, and the ovarian cancer tissue and adjacent tissue were collected after surgical resection to determine the expression of LSD1, NDRG1, migration genes and invasion genes. Results: LSD1, YKL40, COX2, Twist, IFITM1, CatL, CTHRC1, MMP2 and FUNDC1 mRNA expression in ovarian cancer tissue were significantly higher than those in adjacent tissue whereas NDRG1, E-cadherin and Wnt5a mRNA expression were significantly lower than those in adjacent tissue;YKL40, COX2, Twist, IFITM1, CatL, CTHRC1, MMP2 and FUNDC1 mRNA expression in ovarian cancer tissue with high LSD1 expression were significantly higher than those in ovarian tissue with low LSD1 expression whereas E-cadherin and Wnt5a gene mRNA expression were significantly lower than those in ovarian tissue with low LSD1 expression. Conclusion: The high LSD1 expression and low NDRG1 expression in ovarian cancer tissue can promote the migration and invasion of cancer cells.
