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
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 hu...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.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展开更多
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 u...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.展开更多
Background:Pulmonary hypertension(PH)represents a threatening pathophysiologic state that can be induced by chronic hypoxia and is characterized by extensive vascular remodeling.However,the mechanism underlying hypoxi...Background:Pulmonary hypertension(PH)represents a threatening pathophysiologic state that can be induced by chronic hypoxia and is characterized by extensive vascular remodeling.However,the mechanism underlying hypoxia-induced vascular remodeling is not fully elucidated.Methods and Results:By using quantitative polymerase chain reactions,western blotting,and immunohistochemistry,we demon-strate that the expression of N-myc downstream regulated gene-1(NDRG1)is markedly increased in hypoxia-stimulated endothelial cells in a time-dependent manner as well as in human and rat endothelium lesions.To determine the role of NDRG1 in endothelial dysfunction,we performed loss-of-function studies using NDRG1 short hairpin RNAs and NDRG1 over-expression plasmids.In vitro,silencing NDRG1 attenuated proliferation,migration,and tube formation of human pulmonary artery endothelial cells(HPAECs)un-der hypoxia,while NDRG1 over-expression promoted these behaviors of HPAECs.Mechanistically,NDRG1 can directly interact with TATA-box binding protein associated factor 15(TAF15)and promote its nuclear localization.Knockdown of TAF15 abrogated the effect of NDRG1 on the proliferation,migration and tube formation capacity of HPAECs.Bioinformatics studies found that TAF15 was involved in regulating PI3K-Akt,p53,and hypoxia-inducible factor 1(HIF-1)signaling pathways,which have been proved to be PH-related pathways.In addition,vascular remodeling and right ventricular hypertrophy induced by hypoxia were markedly alleviated in NDRG1 knock-down rats compared with their wild-type littermates.Conclusions:Taken together,our results indicate that hypoxia-induced upregulation of NDRG1 contributes to endothelial dysfunction through targeting TAF15,which ultimately contributes to the development of hypoxia-induced PH.展开更多
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...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.展开更多
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.
基金Supported by the National Natural Sciences Foundation of China(No.81072973)
文摘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.
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
基金supported by a grant from the Natural Sciences Foundation of Hubei Province(No.4-306)
文摘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.
基金supported by the National Natural Science Foundation of China(Grants No.81970048,82270058)starting fund for scientific research of Huashan Hospital Fudan University(Grant No.2017QD078).
文摘Background:Pulmonary hypertension(PH)represents a threatening pathophysiologic state that can be induced by chronic hypoxia and is characterized by extensive vascular remodeling.However,the mechanism underlying hypoxia-induced vascular remodeling is not fully elucidated.Methods and Results:By using quantitative polymerase chain reactions,western blotting,and immunohistochemistry,we demon-strate that the expression of N-myc downstream regulated gene-1(NDRG1)is markedly increased in hypoxia-stimulated endothelial cells in a time-dependent manner as well as in human and rat endothelium lesions.To determine the role of NDRG1 in endothelial dysfunction,we performed loss-of-function studies using NDRG1 short hairpin RNAs and NDRG1 over-expression plasmids.In vitro,silencing NDRG1 attenuated proliferation,migration,and tube formation of human pulmonary artery endothelial cells(HPAECs)un-der hypoxia,while NDRG1 over-expression promoted these behaviors of HPAECs.Mechanistically,NDRG1 can directly interact with TATA-box binding protein associated factor 15(TAF15)and promote its nuclear localization.Knockdown of TAF15 abrogated the effect of NDRG1 on the proliferation,migration and tube formation capacity of HPAECs.Bioinformatics studies found that TAF15 was involved in regulating PI3K-Akt,p53,and hypoxia-inducible factor 1(HIF-1)signaling pathways,which have been proved to be PH-related pathways.In addition,vascular remodeling and right ventricular hypertrophy induced by hypoxia were markedly alleviated in NDRG1 knock-down rats compared with their wild-type littermates.Conclusions:Taken together,our results indicate that hypoxia-induced upregulation of NDRG1 contributes to endothelial dysfunction through targeting TAF15,which ultimately contributes to the development of hypoxia-induced PH.
文摘目的:研究N-MYC基因拷贝数在神经母细胞源性肿瘤(neuroblastic tumors,NTs)患者中的异常改变及其临床病理学意义。方法:收集483例NTs患儿肿瘤组织标本,其中包括神经母细胞瘤(neuroblastoma,NB)388例、节细胞神经母细胞瘤(ganglioneuroblastoma,GNB)89例、节细胞神经瘤(ganglioneuroma,GN)6例。运用荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测N-MYC基因拷贝数改变。考察N-MYC基因拷贝数改变与临床病理学特征的关系并进行生存分析。结果:483例NTs患儿N-MYC基因扩增率为12.4%。N-MYC基因扩增均发生在NB中,而在GNB及GN中未见其扩增(P<0.05)。N-MYC基因拷贝数改变更易发生在低分化程度的NB中(P=0.01),且随着分化程度降低,N-MYC基因扩增率增加。男性患儿N-MYC基因拷贝数改变的发生率多于女性患儿(P=0.05)。患儿年龄≤18个月者N-MYC基因扩增率有低于>18个月者的趋势(P=0.092)。生存分析显示:N-MYC基因扩增组患儿生存率明显低于获得组及正常组。结论:NTs患儿N-MYC基因扩增与NTs的类型、分化程度、性别、年龄及生存密切相关。本研究为NTs患者的诊断、治疗及预后提供可靠的参考和帮助。
文摘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.
