目的:筛选上调尾型同源盒基因2(caudal type homeobox gene 2,CDX2)基因表达时人的胃癌细胞SGC-7901中差异表达的miRNA并预测其靶基因,并观察其对胃癌细胞生物学行为的影响.方法:提取瞬时转染48 h后空载体组(转染P E G F P-N1组)和转染...目的:筛选上调尾型同源盒基因2(caudal type homeobox gene 2,CDX2)基因表达时人的胃癌细胞SGC-7901中差异表达的miRNA并预测其靶基因,并观察其对胃癌细胞生物学行为的影响.方法:提取瞬时转染48 h后空载体组(转染P E G F P-N1组)和转染组(转染P E G F P-N1-C D X2组)细胞的总R N A,应用m i R N A芯片检测差异表达的miRNA,并通过Miranda、TargetScan、Mirtarget2软件预测其靶基因,通过Gene Ontology(GO)和KEGG pathway分析了解靶基因功能.CCK8法检测细胞增殖能力,划痕试验、Transwell小室、细胞黏附实验检测各组细胞体外迁移黏附能力.结果:PEGFP-N1-CDX2组较空载体组有59种差异表达的miRNA,其中25种miRNA发生2倍以上表达上调,34种miRNA发生2倍以上表达下调.通过Gene Ontology(GO)和KEGG pathway分析得到部分miRNA靶基因功能,这些靶基因参与了肿瘤的发生、发展、转移及预后.与对照组相比,PEGFP-N1-CDX2组的胃癌细胞生长、迁移和黏附能力均明显受到抑制(P<0.05).结论:上调CDX2基因表达可明显抑制人胃癌细胞SGC-7901的生长,抑制其迁移黏附能力,CDX2基因的抗肿瘤作用可能与miRNA有关.展开更多
AIM: To explore the role of CDX2 in the multi-drug resistance (MDR) process of gastric cancer in vitro and in vivo . METHODS: A cisplatin-resistant gastric cancer cell line with stable downregulation of CDX2 was estab...AIM: To explore the role of CDX2 in the multi-drug resistance (MDR) process of gastric cancer in vitro and in vivo . METHODS: A cisplatin-resistant gastric cancer cell line with stable downregulation of CDX2 was established. mRNA and protein expression levels of CDX2, survivin, cyclin D1, and c-Myc were detected by western blotting and semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The influence of downregulation of CDX2 on MDR was assessed by measuring IC50 of SGC7901/DDP cells to cisplatin, doxorubicin, and 5-fluorouracil, rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. In addition, we determined the in vivo effects of CDX2 small interfering RNA (siRNA) on tumor size, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining. RESULTS: CDX2 siRNA led to downregulation of endogenous CDX2 mRNA (0.31 ± 0.05 vs 1.10 ± 0.51, 0.31 ± 0.05 vs 1.05 ± 0.21, P = 0.003) and protein (0.12 ± 0.08 vs 0.51 ± 0.07, 0.12 ± 0.08 vs 0.55 ± 0.16, P = 2.57 × 10 -4) expression. It significantly promoted the sensitivity of SGC7901/DDP cells to cisplatin (0.12 ± 0.05 vs 0.33 ± 0.08, 0.12 ± 0.05 vs 0.39 ± 0.15, P = 0.001), doxorubicin (0.52 ± 0.13 vs 4.11 ± 1.25, 0.52 ± 0.13 vs 4.05 ± 1.44, P = 2.81 × 10-4), and 5-fluorouracil (0.82 ± 0.13 vs 2.81 ± 0.51, 0.82 ± 0.13 vs 3.28 ± 1.03, P = 1.71 × 10-4). Flow cytometry confirmed that the percentage of apoptotic cells increased after CDX2 downregulation (32.15% ± 2.15% vs 17.63% ± 3.16%, 32.15% ± 2.15% vs 19.3% ± 2.25%, P = 1.73 × 10-6). This notion was further supported by the observation that downregulation of CDX2 blocked entry into the S-phase of the cell cycle (31.53% ± 3.78% vs 65.05% ± 7.25%, 31.53% ± 3.78% vs 62.27% ± 5.02%, P = 7.55 × 10-7). Furthermore, downregulation of CDX2 significantly increased intracellular accumulation of doxorubicin (0.21 ± 0.06 vs 0.41 ± 0.11, 0.21 ± 0.06 vs 0.40 ± 0.08, P = 0.003). In molecular studies, semiquantitative RT-PCR and western blotting revealed that CDX2 downregulation could inhibit expression of c-Myc, survivin and cyclin D1. CONCLUSION: CDX2 may be involved in regulating multiple signaling pathways in reversing MDR, suggesting that CDX2 may represent a novel target for gastric cancer therapy.展开更多
目的对孤立性心房颤动(AF)患者进行垂体同型框2(PITX2)基因的突变扫描,并分析突变患者的相应临床特点。方法对160例孤立性AF患者和200名健康者的PITX2基因进行突变扫描。采用聚合酶链反应(PCR)扩增PITX2基因,通过单链构象多态性技术筛...目的对孤立性心房颤动(AF)患者进行垂体同型框2(PITX2)基因的突变扫描,并分析突变患者的相应临床特点。方法对160例孤立性AF患者和200名健康者的PITX2基因进行突变扫描。采用聚合酶链反应(PCR)扩增PITX2基因,通过单链构象多态性技术筛查突变,对发现异常的个体采用双脱氧核苷链末端合成终止法进行直接测序。采用Sequence scanner v 1.0软件分析测序图。结果在2例永久性AF患者中发现位于内含子区域的新改变g.766C>A、位于5’UTR区域的新改变g.8310C>T(c.-150C>T)。这两例携带者对药物及电复律治疗的反应较差,接受环肺静脉消融手术后,随访1年未再复发。200名健康者中未发现此改变。结论首次在中国孤立性AF患者中发现影响肺静脉肌袖发育基因PITX2的非编码区改变,其可能为我们进一步理解肺静脉肌袖在AF的发病过程中的作用提供新的启示。展开更多
文摘目的:筛选上调尾型同源盒基因2(caudal type homeobox gene 2,CDX2)基因表达时人的胃癌细胞SGC-7901中差异表达的miRNA并预测其靶基因,并观察其对胃癌细胞生物学行为的影响.方法:提取瞬时转染48 h后空载体组(转染P E G F P-N1组)和转染组(转染P E G F P-N1-C D X2组)细胞的总R N A,应用m i R N A芯片检测差异表达的miRNA,并通过Miranda、TargetScan、Mirtarget2软件预测其靶基因,通过Gene Ontology(GO)和KEGG pathway分析了解靶基因功能.CCK8法检测细胞增殖能力,划痕试验、Transwell小室、细胞黏附实验检测各组细胞体外迁移黏附能力.结果:PEGFP-N1-CDX2组较空载体组有59种差异表达的miRNA,其中25种miRNA发生2倍以上表达上调,34种miRNA发生2倍以上表达下调.通过Gene Ontology(GO)和KEGG pathway分析得到部分miRNA靶基因功能,这些靶基因参与了肿瘤的发生、发展、转移及预后.与对照组相比,PEGFP-N1-CDX2组的胃癌细胞生长、迁移和黏附能力均明显受到抑制(P<0.05).结论:上调CDX2基因表达可明显抑制人胃癌细胞SGC-7901的生长,抑制其迁移黏附能力,CDX2基因的抗肿瘤作用可能与miRNA有关.
基金Supported by Grants from the Natural Science Foundation of China, No. 81060201Natural Science Foundation of Guangxi,No. 2011GXNSFA018273 and No. 2013GXNSFAA019163the Key Health Science Foundation of Guangxi, No. 1298003-2-6
文摘AIM: To explore the role of CDX2 in the multi-drug resistance (MDR) process of gastric cancer in vitro and in vivo . METHODS: A cisplatin-resistant gastric cancer cell line with stable downregulation of CDX2 was established. mRNA and protein expression levels of CDX2, survivin, cyclin D1, and c-Myc were detected by western blotting and semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The influence of downregulation of CDX2 on MDR was assessed by measuring IC50 of SGC7901/DDP cells to cisplatin, doxorubicin, and 5-fluorouracil, rate of doxorubicin efflux, apoptosis, and cell cycle progression detected by flow cytometry. In addition, we determined the in vivo effects of CDX2 small interfering RNA (siRNA) on tumor size, and apoptotic cells in tumor tissues were detected by deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and hematoxylin and eosin staining. RESULTS: CDX2 siRNA led to downregulation of endogenous CDX2 mRNA (0.31 ± 0.05 vs 1.10 ± 0.51, 0.31 ± 0.05 vs 1.05 ± 0.21, P = 0.003) and protein (0.12 ± 0.08 vs 0.51 ± 0.07, 0.12 ± 0.08 vs 0.55 ± 0.16, P = 2.57 × 10 -4) expression. It significantly promoted the sensitivity of SGC7901/DDP cells to cisplatin (0.12 ± 0.05 vs 0.33 ± 0.08, 0.12 ± 0.05 vs 0.39 ± 0.15, P = 0.001), doxorubicin (0.52 ± 0.13 vs 4.11 ± 1.25, 0.52 ± 0.13 vs 4.05 ± 1.44, P = 2.81 × 10-4), and 5-fluorouracil (0.82 ± 0.13 vs 2.81 ± 0.51, 0.82 ± 0.13 vs 3.28 ± 1.03, P = 1.71 × 10-4). Flow cytometry confirmed that the percentage of apoptotic cells increased after CDX2 downregulation (32.15% ± 2.15% vs 17.63% ± 3.16%, 32.15% ± 2.15% vs 19.3% ± 2.25%, P = 1.73 × 10-6). This notion was further supported by the observation that downregulation of CDX2 blocked entry into the S-phase of the cell cycle (31.53% ± 3.78% vs 65.05% ± 7.25%, 31.53% ± 3.78% vs 62.27% ± 5.02%, P = 7.55 × 10-7). Furthermore, downregulation of CDX2 significantly increased intracellular accumulation of doxorubicin (0.21 ± 0.06 vs 0.41 ± 0.11, 0.21 ± 0.06 vs 0.40 ± 0.08, P = 0.003). In molecular studies, semiquantitative RT-PCR and western blotting revealed that CDX2 downregulation could inhibit expression of c-Myc, survivin and cyclin D1. CONCLUSION: CDX2 may be involved in regulating multiple signaling pathways in reversing MDR, suggesting that CDX2 may represent a novel target for gastric cancer therapy.
文摘目的对孤立性心房颤动(AF)患者进行垂体同型框2(PITX2)基因的突变扫描,并分析突变患者的相应临床特点。方法对160例孤立性AF患者和200名健康者的PITX2基因进行突变扫描。采用聚合酶链反应(PCR)扩增PITX2基因,通过单链构象多态性技术筛查突变,对发现异常的个体采用双脱氧核苷链末端合成终止法进行直接测序。采用Sequence scanner v 1.0软件分析测序图。结果在2例永久性AF患者中发现位于内含子区域的新改变g.766C>A、位于5’UTR区域的新改变g.8310C>T(c.-150C>T)。这两例携带者对药物及电复律治疗的反应较差,接受环肺静脉消融手术后,随访1年未再复发。200名健康者中未发现此改变。结论首次在中国孤立性AF患者中发现影响肺静脉肌袖发育基因PITX2的非编码区改变,其可能为我们进一步理解肺静脉肌袖在AF的发病过程中的作用提供新的启示。
