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三叶草王铁虫 (正式写手)
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3.1. DNMT3A and DNMT3B catalyze the methylation of WIF-1 in NSCLC The expression level of WIF-1, DNMT1, DNMT3A and DNMT3B was detected in 30 paired NSCLC and adjacent non-neoplastic lung tissues by real-time PCR and Western blotting. As shown inFig. 1A and E, WIF-1 was expressed at significantly lower levels in tumor tissues than in matched non-tumor tissues. In contrast, the expression of DNMTs (DNMT1, DNMT3A and DNMT3B) was up-regulated in cancerous lung tissues (Fig. 1B–D and F–H). The role of the three DNMTs in the methylation of the WIF-1 gene was verified by lentiviral vector mediated knockdown of these methyltransferases in A549 and H1299 cells and assessment of WIF-1 expression. The results of Western blotting confirmed the downregulation of DNMT expression and showed a significant increase in WIF-1 protein levels in cells transfected with DNMT3A or DNMT3B shRNA (Fig. 1I and J). These results indicated that DNMT3A and DNMT3B play a role in the regulation of WIF-1 gene expression in NSCLC. 3.2. Restoration of miR-29s positively regulates WIF-1 To examine the mechanism of up-regulation if DNMTs, we focused on the effect of the miR-29 family because miR-29s are down-regulated in NSCLC and target DNMT3A and -3B. To determine whether WIF-1 mRNA expression is correlated with the levels of miR-29s in NSCLC tissues, the mRNA levels of WIF-1 and miR-29s were analyzed in 30 NSCLC samples. The results showed statistically significant positive correlations (Fig. 2A–C) between WIF-1 mRNA and miR-29a (p< 0.01), miR-29b (p< 0.01) and miR-29c (p< 0.05). To confirm that expression of miR-29s contributes to the reduction of promoter methylation of the WIF-1 gene, we examined the methylation status of WIF-1 using MSP. As shown inFig 2D and E, reduced methylation of WIF-1 was observed in A549 and H1299 cells transfected with miR-29a, -29b, or -29c. Furthermore, Western blot analysis confirmed that enforced expression of these miRNAs increased the protein level of WIF-1 in A549 and H1299 cells. |
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至尊木虫 (知名作家)
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【答案】应助回帖
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RXMCDM: 金币+1, 多谢应助！ 2014-08-27 10:22:51
三叶草王: 金币+25, 翻译EPI+1, ★★★★★最佳答案 2014-08-27 15:49:49
RXMCDM: 金币+1, 多谢应助！ 2014-08-27 10:22:51
三叶草王: 金币+25, 翻译EPI+1, ★★★★★最佳答案 2014-08-27 15:49:49
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3.1. 在NSCLC中DNMT3A和DNMT3B催化WIF-1的甲基化 用实时PCR和Western印迹检测了30例（配对）小细胞肺癌和临近非肿瘤性肺组织中WIF-1、DNMT1、DNMT3A和DNMT3B的表达水平。 在30配对非小细胞肺癌和邻近非肿瘤性肺组织实时PCR和Western印迹法检测。如图1A和1E所示，与所配对的非肿瘤组织相比，肿瘤组织中WIF-1的表达水平明显降低。与此相反，在肺癌组织中DNMTs（DNMT1、DNMT3A和DNMT3B）的表达被向上调节（图1B-D和F-H）。（我们）用慢病毒载体介导的敲减（knockdown）方法使A549和H1299细胞中这些甲基转移酶（即DNMTs）表达水平降低，以此验证了三种DNMTs在WIF-1基因甲基化中的作用，并研究了WIF-1在这些条件下的表达。Western印迹证实，细胞转染DNMT3A或DNMT3B shRNA时，DNMT表达被下调，同时伴有WIF-1蛋白水平的显著提高（图1I和J）。这些结果表明，在NSCLC中，DNMT3A和DNMT3B在调节WIF-1基因表达中发挥一定作用。 3.2. 恢复miR-29s正调节WIF-1 为了研究DNMTs向上调节的机制，我们主要集中在miR-29家族的作用上，因为miR-29s在NSCLC中是向下调节的，而且是DNMT3A和DNMT3B的靶基因。为确定在NSCLC中WIF-1 mRNA表达水平是与miR-29s相关的，（我们）研究分析了30例NSCLC样本中WIF-1 mRNA和miR-29s的（表达）水平。结果显示，WIF-1 mRNA和miR-29a（p<0.01）、miR-29b（p<0.01）以及miR-29c（p<0.05）间有显著相关性（图2A-C）。为了证实miR-29s的表达可以引起WIF-1基因启动子的甲基化降低，我们用MSP检测了WIF-1的甲基化状态。如图2D和E所示，在A549和H1299细胞中，转染miR-29a、miR-29b或miR-29c后均出现WIF-1甲基化降低。而且，Western印迹分析证实，强使这些miRNAs在A549和H1299细胞中表达可以提高WIF-1的蛋白水平。 |
2楼2014-08-27 07:57:31
