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
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
他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因...他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2,NDRG2)在肿瘤发生发展中发挥重要作用,但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现,NDRG 2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调,且与耐药能力负相关(P<0.001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实,在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC 50和软琼脂克隆形成率(P<0.001),逆转耐药表型。分子机制上,X-box结合蛋白1(X-box binding protein 1,XBP1)mRNA剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation,ERAD)报告蛋白的结果显示,过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3ε-YFP表达(P<0.001),引发耐药细胞内质网强应激反应;免疫印迹检测结果显示,过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1,IRE1α)的磷酸化水平及其下游因子,例如内质网EIP辅助因子(endoplasmic reticulum-localized DnaJ 4,ERdj4)、PKR蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase,P58 IPK)、α甘露糖苷酶样应激蛋白(er degradation enhancingαmannosidase likeprotein,EDEM)和蛋白质二硫键异构酶家族A成员5(protein disulfide isomerase family a member 5,PDIA5)的表达水平(P<0.001)。小鼠异种移植瘤研究进一步证实,在耐药细胞中过表达NDRG2可增强TAM治疗效果,显著抑制耐药移植瘤生长(P<0.001)。以上研究结果表明,通过提高耐药细胞中NDRG2表达,增强TAM治疗引发的内质网强烈应激,可逆转ER+乳腺癌细胞耐药性,改善TAM治疗效果。研究结果为解决ER+乳腺癌TAM耐药问题提供了新的思路和有价值的潜在药物靶点。展开更多
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 ...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.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 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.
文摘他莫昔芬(tamoxifen,TAM)作为雌激素受体阳性(estrogen receptor,ER+)乳腺癌的一线化疗药物使大多数患者受益,但原发性和继发性耐药问题严重影响临床治疗效果。深入研究ER+乳腺癌TAM耐药机制,改善治疗效果是当前亟待解决的问题。抑癌因子NDRG2(N-myc downstream regulated gene 2,NDRG2)在肿瘤发生发展中发挥重要作用,但是否参与ER+乳腺癌TAM耐药尚不清楚。本研究旨在探明NDRG2在ER+乳腺癌TAM耐药中发挥的作用和机制。通过RT-PCR与免疫印迹分析对比TAM敏感型和耐药型ER+乳腺癌细胞发现,NDRG 2的mRNA转录水平和蛋白质翻译水平在TAM耐药细胞中表达显著下调,且与耐药能力负相关(P<0.001);CCK-8细胞毒性实验和软琼脂克隆形成实验证实,在耐药细胞中过表达NDRG2可显著降低TAM药物半抑制浓度IC 50和软琼脂克隆形成率(P<0.001),逆转耐药表型。分子机制上,X-box结合蛋白1(X-box binding protein 1,XBP1)mRNA剪切实验与内质网相关降解(endoplasmic-reticulum associated degradation,ERAD)报告蛋白的结果显示,过表达NDRG2可增强耐药细胞中剪切型XBP1s mRNA转录与ERAD报告蛋白CD3ε-YFP表达(P<0.001),引发耐药细胞内质网强应激反应;免疫印迹检测结果显示,过表达NDRG2可显著提高耐药细胞中内质网应激感受器肌醇需要激酶1α(inositol requiring enzyme 1,IRE1α)的磷酸化水平及其下游因子,例如内质网EIP辅助因子(endoplasmic reticulum-localized DnaJ 4,ERdj4)、PKR蛋白激酶的细胞抑制剂(cellular Inhibitor of the PKR protein kinase,P58 IPK)、α甘露糖苷酶样应激蛋白(er degradation enhancingαmannosidase likeprotein,EDEM)和蛋白质二硫键异构酶家族A成员5(protein disulfide isomerase family a member 5,PDIA5)的表达水平(P<0.001)。小鼠异种移植瘤研究进一步证实,在耐药细胞中过表达NDRG2可增强TAM治疗效果,显著抑制耐药移植瘤生长(P<0.001)。以上研究结果表明,通过提高耐药细胞中NDRG2表达,增强TAM治疗引发的内质网强烈应激,可逆转ER+乳腺癌细胞耐药性,改善TAM治疗效果。研究结果为解决ER+乳腺癌TAM耐药问题提供了新的思路和有价值的潜在药物靶点。
文摘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.
