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三葉草王

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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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RXMCDM: 金幣+1, 多謝應(yīng)助！ 2014-08-27 10:22:51
三葉草王: 金幣+25, 翻譯EPI+1, ★★★★★最佳答案 2014-08-27 15:49:49

3.1. 在NSCLC中DNMT3A和DNMT3B催化WIF-1的甲基化
用實(shí)時(shí)PCR和Western印跡檢測(cè)了30例（配對(duì)）小細(xì)胞肺癌和臨近非腫瘤性肺組織中WIF-1、DNMT1、DNMT3A和DNMT3B的表達(dá)水平。
在30配對(duì)非小細(xì)胞肺癌和鄰近非腫瘤性肺組織實(shí)時(shí)PCR和Western印跡法檢測(cè)。如圖1A和1E所示，與所配對(duì)的非腫瘤組織相比，腫瘤組織中WIF-1的表達(dá)水平明顯降低。與此相反，在肺癌組織中DNMTs（DNMT1、DNMT3A和DNMT3B）的表達(dá)被向上調(diào)節(jié)（圖1B-D和F-H）。（我們）用慢病毒載體介導(dǎo)的敲減（knockdown）方法使A549和H1299細(xì)胞中這些甲基轉(zhuǎn)移酶（即DNMTs）表達(dá)水平降低，以此驗(yàn)證了三種DNMTs在WIF-1基因甲基化中的作用，并研究了WIF-1在這些條件下的表達(dá)。Western印跡證實(shí)，細(xì)胞轉(zhuǎn)染DNMT3A或DNMT3B shRNA時(shí)，DNMT表達(dá)被下調(diào)，同時(shí)伴有WIF-1蛋白水平的顯著提高（圖1I和J）。這些結(jié)果表明，在NSCLC中，DNMT3A和DNMT3B在調(diào)節(jié)WIF-1基因表達(dá)中發(fā)揮一定作用。

3.2. 恢復(fù)miR-29s正調(diào)節(jié)WIF-1
為了研究DNMTs向上調(diào)節(jié)的機(jī)制，我們主要集中在miR-29家族的作用上，因?yàn)閙iR-29s在NSCLC中是向下調(diào)節(jié)的，而且是DNMT3A和DNMT3B的靶基因。為確定在NSCLC中WIF-1 mRNA表達(dá)水平是與miR-29s相關(guān)的，（我們）研究分析了30例NSCLC樣本中WIF-1 mRNA和miR-29s的（表達(dá)）水平。結(jié)果顯示，WIF-1 mRNA和miR-29a（p<0.01）、miR-29b（p<0.01）以及miR-29c（p<0.05）間有顯著相關(guān)性（圖2A-C）。為了證實(shí)miR-29s的表達(dá)可以引起WIF-1基因啟動(dòng)子的甲基化降低，我們用MSP檢測(cè)了WIF-1的甲基化狀態(tài)。如圖2D和E所示，在A549和H1299細(xì)胞中，轉(zhuǎn)染miR-29a、miR-29b或miR-29c后均出現(xiàn)WIF-1甲基化降低。而且，Western印跡分析證實(shí)，強(qiáng)使這些miRNAs在A549和H1299細(xì)胞中表達(dá)可以提高WIF-1的蛋白水平。

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2樓2014-08-27 07:57:31