目的:评估不同阶段结肠腺癌患者中CDC样激酶2 (CLK2)基因的表达,以及CLK2在结肠腺癌细胞中的表达变化和CLK2所介导的细胞功能。方法:在TIMER数据库和GEPIA数据库分析CLK2在健康与不同肿瘤组织中的表达水平,找到差异性表达显著的肿瘤类...目的:评估不同阶段结肠腺癌患者中CDC样激酶2 (CLK2)基因的表达,以及CLK2在结肠腺癌细胞中的表达变化和CLK2所介导的细胞功能。方法:在TIMER数据库和GEPIA数据库分析CLK2在健康与不同肿瘤组织中的表达水平,找到差异性表达显著的肿瘤类型。分析TCGA数据库中各个阶段结肠腺癌患者组织中CLK2的表达水平变化。使用RT-qPCR和western blot方法检测了CLK2基因在肿瘤组织和细胞系中的表达变化。在敲低CLK2的条件下,使用western blot方法检测细胞增殖标志物和细胞周期标志物的表达。最后,使用STRING工具分析与CLK2基因相关性较强的多个关键基因。结果:结肠腺癌组织中的CLK2基因表达显著升高(p Objective: To assess CDC-like kinase 2 (CLK2) gene expression in patients with different stages of colon adenocarcinoma, as well as changes in CLK2 expression in colon adenocarcinoma cells and cellular functions mediated by CLK2. Methods: Screening for CLK2 differentially expressed tumor types in the TIMER database and the GEPIA database. Detection of CLK2 gene changes with colon adenocarcinoma tumor stage in the TCGA database. Changes in CLK2 gene expression in tumor tissues and cell lines were detected using RT-qPCR and western blot methods. The expressions of cell proliferation markers and cell cycle markers were detected using western blot method under the condition of knocking down CLK2. Finally, multiple key genes with strong correlation with CLK2 gene were analyzed using STRING tool. Results: CLK2 gene expression was significantly elevated in colon adenocarcinoma tissues (p < 0.001), and the high expression of CLK2 gene was significantly correlated with the clinical features of colon adenocarcinoma (pathological stage, T stage) (p < 0.05). CLK2 gene expression is elevated in colon adenocarcinoma cells, and knockdown of CLK2 expression can effectively inhibit cell proliferation and cell cycle. Finally, CLK3, CLK1, SRSF12 and other genes are potential binding targets of CLK2 gene. Conclusion: The expression level of the CLK2 gene is significantly correlated with the progression of colon adenocarcinoma, and decreasing the expression of the CLK2 gene is expected to be a new approach to the treatment of patients with colon adenocarcinoma.展开更多
目的:探究m6A去甲基化酶FTO及其下游基因CLK2对3T3-L1细胞成脂分化的影响,以及FTO影响CLK2表达水平的分子机制。方法:(1)通过成脂分化诱导和后续的油红染色以及油红定量探究FTO和CLK2对3T3-L1细胞成脂分化的影响;(2)通过蛋白免疫印迹和...目的:探究m6A去甲基化酶FTO及其下游基因CLK2对3T3-L1细胞成脂分化的影响,以及FTO影响CLK2表达水平的分子机制。方法:(1)通过成脂分化诱导和后续的油红染色以及油红定量探究FTO和CLK2对3T3-L1细胞成脂分化的影响;(2)通过蛋白免疫印迹和实时荧光定量PCR测定FTO和CLK2蛋白水平和m RNA水平的改变;(3)通过生物信息学分析筛选不同分化时期的3T3-L1细胞差异化m6A修饰位点;(4)通过MeRIP-qPCR测定CLK2 m RNA上的m6A修饰水平;(5)通过放线菌素D抑制新生转录本合成探究CLK2 m RNA的降解速率;(6)通过胰岛素刺激探究3T3-L1细胞Insulin-AKT通路激活情况。结果:(1)3T3-L1细胞的成脂分化依赖FTO的去甲基化酶活性和CLK2的激酶活性;(2)CLK25'UTR区域存在可被FTO去甲基化的m6A修饰,且该位点m6A修饰提高CLK2 m RNA的降解速率;(3)CLK2表达水平与FTO表达水平存在正相关,且CLK2和FTO抑制剂均抑制3T3-L1细胞Insulin-AKT通路的激活。结论:FTO通过降低CLK25'UTR区域的m6A修饰水平从而抑制CLK2 m RNA的降解,促进CLK2的蛋白表达,CLK2进而通过维持Insulin-AKT通路活性促进3T3-L1细胞成脂分化。展开更多
文摘目的:评估不同阶段结肠腺癌患者中CDC样激酶2 (CLK2)基因的表达,以及CLK2在结肠腺癌细胞中的表达变化和CLK2所介导的细胞功能。方法:在TIMER数据库和GEPIA数据库分析CLK2在健康与不同肿瘤组织中的表达水平,找到差异性表达显著的肿瘤类型。分析TCGA数据库中各个阶段结肠腺癌患者组织中CLK2的表达水平变化。使用RT-qPCR和western blot方法检测了CLK2基因在肿瘤组织和细胞系中的表达变化。在敲低CLK2的条件下,使用western blot方法检测细胞增殖标志物和细胞周期标志物的表达。最后,使用STRING工具分析与CLK2基因相关性较强的多个关键基因。结果:结肠腺癌组织中的CLK2基因表达显著升高(p Objective: To assess CDC-like kinase 2 (CLK2) gene expression in patients with different stages of colon adenocarcinoma, as well as changes in CLK2 expression in colon adenocarcinoma cells and cellular functions mediated by CLK2. Methods: Screening for CLK2 differentially expressed tumor types in the TIMER database and the GEPIA database. Detection of CLK2 gene changes with colon adenocarcinoma tumor stage in the TCGA database. Changes in CLK2 gene expression in tumor tissues and cell lines were detected using RT-qPCR and western blot methods. The expressions of cell proliferation markers and cell cycle markers were detected using western blot method under the condition of knocking down CLK2. Finally, multiple key genes with strong correlation with CLK2 gene were analyzed using STRING tool. Results: CLK2 gene expression was significantly elevated in colon adenocarcinoma tissues (p < 0.001), and the high expression of CLK2 gene was significantly correlated with the clinical features of colon adenocarcinoma (pathological stage, T stage) (p < 0.05). CLK2 gene expression is elevated in colon adenocarcinoma cells, and knockdown of CLK2 expression can effectively inhibit cell proliferation and cell cycle. Finally, CLK3, CLK1, SRSF12 and other genes are potential binding targets of CLK2 gene. Conclusion: The expression level of the CLK2 gene is significantly correlated with the progression of colon adenocarcinoma, and decreasing the expression of the CLK2 gene is expected to be a new approach to the treatment of patients with colon adenocarcinoma.
文摘目的:探究m6A去甲基化酶FTO及其下游基因CLK2对3T3-L1细胞成脂分化的影响,以及FTO影响CLK2表达水平的分子机制。方法:(1)通过成脂分化诱导和后续的油红染色以及油红定量探究FTO和CLK2对3T3-L1细胞成脂分化的影响;(2)通过蛋白免疫印迹和实时荧光定量PCR测定FTO和CLK2蛋白水平和m RNA水平的改变;(3)通过生物信息学分析筛选不同分化时期的3T3-L1细胞差异化m6A修饰位点;(4)通过MeRIP-qPCR测定CLK2 m RNA上的m6A修饰水平;(5)通过放线菌素D抑制新生转录本合成探究CLK2 m RNA的降解速率;(6)通过胰岛素刺激探究3T3-L1细胞Insulin-AKT通路激活情况。结果:(1)3T3-L1细胞的成脂分化依赖FTO的去甲基化酶活性和CLK2的激酶活性;(2)CLK25'UTR区域存在可被FTO去甲基化的m6A修饰,且该位点m6A修饰提高CLK2 m RNA的降解速率;(3)CLK2表达水平与FTO表达水平存在正相关,且CLK2和FTO抑制剂均抑制3T3-L1细胞Insulin-AKT通路的激活。结论:FTO通过降低CLK25'UTR区域的m6A修饰水平从而抑制CLK2 m RNA的降解,促进CLK2的蛋白表达,CLK2进而通过维持Insulin-AKT通路活性促进3T3-L1细胞成脂分化。
