目的分析MAGE A6在胃癌中的表达及其意义和对胃癌细胞生物学作用的影响。方法收集2009年12月-2010年6月医院样本库中经手术切除的90例胃癌组织芯片样本,利用免疫组化检测胃癌组织芯片中MAGEA6的表达情况,分析MAGEA6表达与胃癌临床病理...目的分析MAGE A6在胃癌中的表达及其意义和对胃癌细胞生物学作用的影响。方法收集2009年12月-2010年6月医院样本库中经手术切除的90例胃癌组织芯片样本,利用免疫组化检测胃癌组织芯片中MAGEA6的表达情况,分析MAGEA6表达与胃癌临床病理特征的关系,Kaplan-Meier在线分析MAGEA6表达与胃癌预后的关系。体外培养BGC-823胃癌细胞系,设置si-MAGEA6组(转染si-MAGEA6)与si-Ctrl组(转染空载体对照)。采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡情况,Western blotting检测MAGEA6、凋亡相关蛋白[B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)]、自噬相关蛋白[微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、p62蛋白(p62)、自噬相关基因5(Atg-5)蛋白、酵母ATG6同源物(Beclin 1)]、Akt/m TOR信号通路相关蛋白[蛋白激酶B (A k t)、磷酸化蛋白激酶B(p-Akt)、哺乳动物雷帕霉素靶蛋白(m TOR)、磷酸化哺乳动物雷帕霉素靶蛋白(p-m TOR)]的表达,扫描电镜和激光共聚焦显微镜观察自噬小体形成情况和自噬流的变化。结果 MAGEA6在胃癌组织中的免疫组化评分高于癌旁组织[(3.77±1.50)分vs.(2.58±1.11)分,P<0.05]。MAGEA6高表达与年龄、TNM分期有关(P<0.05)。si-MAGEA6组胃癌细胞活力低于si-Ctrl组(P<0.05),细胞凋亡率高于si-Ctrl组(14.97%±0.86%vs.4.63%±0.55%,P<0.05)。si-M AGE A6组M AGE A6、Bcl-2、p62、p-Akt和p-m TOR表达水平低于si-Ctrl组,Bax、Atg-5、Beclin 1表达水平以及LC3-Ⅱ/LC3-Ⅰ高于si-Ctrl组(P<0.05)。si-MAGE A6组胃癌细胞胞质内自噬小体明显增多,且自噬小体与溶酶体形成自噬溶酶体。结论 MAGEA6在胃癌组织中呈高表达,沉默MAGEA6的表达可抑制胃癌细胞增殖。MAGEA6可能是胃癌有效的生物标志物及潜在的治疗靶点。展开更多
Objective: To explore the role of miR-448 in regulating MAGEA6/AMPK signaling pathway in the biological study of hepatocellular carcinoma (HCC) tumor stem cells. Methods: Using the database, the hepatocellular carcino...Objective: To explore the role of miR-448 in regulating MAGEA6/AMPK signaling pathway in the biological study of hepatocellular carcinoma (HCC) tumor stem cells. Methods: Using the database, the hepatocellular carcinoma related expression chips were obtained and the regulatory mirnas of candidate genes were predicted, and the predicted results were analyzed. The effects of miR-448 and MAGEA6 on the pellet formation rate and clone formation rate of hepatocellular carcinoma stem cells were detected by immunofluorescence identification of stem cell markers and light microscope counting method. The effects of miR-448 and MAGEA6 on migration and invasion of hepatocellular carcinoma stem cells were detected by scratch and Transwell assay. Dual luciferase reporter assay to verify whether miR-448 targets MAGEA6. The expression and influence of miR-448 on MAGEA6 and AMPK pathway were detected by qRT-PCR and Western blot. Results: It was found that miR-448 may directly regulate the expression of MAGEA6. Overexpression of miR-448 inhibited the characteristics, proliferation, migration, and invasion of hepatocellular carcinoma stem cells in vitro, as well as the ability of xenograft tumor formation in vivo. However, inhibition of miR-448 showed opposite results. In addition, miR-448 directly targets MAGEA6 and regulates AMPK signaling. Silencing MAGEA6 and adding AMPK activator further verified that miR-448 activated AMPK signaling pathway by targeting MAGEA6, thus affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. Conclusions: Our results reveal that miR-448 activates AMPK signaling pathway by targeting MAGEA6, thereby affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. It is suggested that overexpression of miR-448 may be a new therapeutic strategy for hepatocellular carcinoma.展开更多
文摘目的分析MAGE A6在胃癌中的表达及其意义和对胃癌细胞生物学作用的影响。方法收集2009年12月-2010年6月医院样本库中经手术切除的90例胃癌组织芯片样本,利用免疫组化检测胃癌组织芯片中MAGEA6的表达情况,分析MAGEA6表达与胃癌临床病理特征的关系,Kaplan-Meier在线分析MAGEA6表达与胃癌预后的关系。体外培养BGC-823胃癌细胞系,设置si-MAGEA6组(转染si-MAGEA6)与si-Ctrl组(转染空载体对照)。采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡情况,Western blotting检测MAGEA6、凋亡相关蛋白[B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)]、自噬相关蛋白[微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、p62蛋白(p62)、自噬相关基因5(Atg-5)蛋白、酵母ATG6同源物(Beclin 1)]、Akt/m TOR信号通路相关蛋白[蛋白激酶B (A k t)、磷酸化蛋白激酶B(p-Akt)、哺乳动物雷帕霉素靶蛋白(m TOR)、磷酸化哺乳动物雷帕霉素靶蛋白(p-m TOR)]的表达,扫描电镜和激光共聚焦显微镜观察自噬小体形成情况和自噬流的变化。结果 MAGEA6在胃癌组织中的免疫组化评分高于癌旁组织[(3.77±1.50)分vs.(2.58±1.11)分,P<0.05]。MAGEA6高表达与年龄、TNM分期有关(P<0.05)。si-MAGEA6组胃癌细胞活力低于si-Ctrl组(P<0.05),细胞凋亡率高于si-Ctrl组(14.97%±0.86%vs.4.63%±0.55%,P<0.05)。si-M AGE A6组M AGE A6、Bcl-2、p62、p-Akt和p-m TOR表达水平低于si-Ctrl组,Bax、Atg-5、Beclin 1表达水平以及LC3-Ⅱ/LC3-Ⅰ高于si-Ctrl组(P<0.05)。si-MAGE A6组胃癌细胞胞质内自噬小体明显增多,且自噬小体与溶酶体形成自噬溶酶体。结论 MAGEA6在胃癌组织中呈高表达,沉默MAGEA6的表达可抑制胃癌细胞增殖。MAGEA6可能是胃癌有效的生物标志物及潜在的治疗靶点。
文摘Objective: To explore the role of miR-448 in regulating MAGEA6/AMPK signaling pathway in the biological study of hepatocellular carcinoma (HCC) tumor stem cells. Methods: Using the database, the hepatocellular carcinoma related expression chips were obtained and the regulatory mirnas of candidate genes were predicted, and the predicted results were analyzed. The effects of miR-448 and MAGEA6 on the pellet formation rate and clone formation rate of hepatocellular carcinoma stem cells were detected by immunofluorescence identification of stem cell markers and light microscope counting method. The effects of miR-448 and MAGEA6 on migration and invasion of hepatocellular carcinoma stem cells were detected by scratch and Transwell assay. Dual luciferase reporter assay to verify whether miR-448 targets MAGEA6. The expression and influence of miR-448 on MAGEA6 and AMPK pathway were detected by qRT-PCR and Western blot. Results: It was found that miR-448 may directly regulate the expression of MAGEA6. Overexpression of miR-448 inhibited the characteristics, proliferation, migration, and invasion of hepatocellular carcinoma stem cells in vitro, as well as the ability of xenograft tumor formation in vivo. However, inhibition of miR-448 showed opposite results. In addition, miR-448 directly targets MAGEA6 and regulates AMPK signaling. Silencing MAGEA6 and adding AMPK activator further verified that miR-448 activated AMPK signaling pathway by targeting MAGEA6, thus affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. Conclusions: Our results reveal that miR-448 activates AMPK signaling pathway by targeting MAGEA6, thereby affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. It is suggested that overexpression of miR-448 may be a new therapeutic strategy for hepatocellular carcinoma.
