Mazzoccoli Luciano, Robaina Marcela Cristina, Apa Alexandre Gustavo, Bonamino Martin, Pinto Luciana Wernersbach, Queiroga Eduardo, Bacchi Carlos E, Klumb Claudete Esteves
Programa de Pesquisa em Hemato-Oncologia Molecular, Instituto Nacional de Câncer, Rio de Janeiro, Brazil.
Serviço de Hematologia, Instituto Nacional de Câncer, Rio de Janeiro, Brazil.
J Cancer Res Clin Oncol. 2018 Mar;144(3):483-497. doi: 10.1007/s00432-017-2575-3. Epub 2018 Jan 9.
Burkitt lymphoma (BL) is a B-cell lymphoma frequently diagnosed in children. It is characterized by MYC translocations, which lead to the constitutive expression of the MYC oncogene. MYC contributes to miR-29 repression through an E-box MYC binding site on the miR-29b-1/miR-29a promoter region. We evaluated the role of miR-29a/b/c and their predicted targets in BL pathogenesis.
Mature sequences of miR-29a/b/c were transfected to the BL cell lines BL41 and Raji, and evaluated for DNMT3B, MCL1, BIM, CDK6, AKT and TCL1 protein expression as well as for MCL-1 and CDK6 mRNA expression. BL cells were treated with 5-aza-2'-deoxycytidine (decitabine) and evaluated for miR29 expressions and methylation status. DNMT3B inhibition was performed by DNMT3B siRNA.
Ectopic expression of miR-29s in BL cells decreased CDK6, DNMT3B, TCL1 and MCL-1 protein levels, but CDK6 and MCL-1 mRNA expression was unaffected by miR-29. Decitabine enhanced miR-29 expression levels and decreased CDK6 protein expression. Additionally, inhibition of DNMT3B by siRNA increased miR-29a/b expression. Notably, the miR-29a/b1 and miR-29b2/c promoter genes showed methylated CpG sequences that were demethylated after decitabine treatments. Furthermore, MYC-negative tumours had higher levels of miR-29 expression compared with MYC-translocated cases, suggesting that MYC regulates miR-29 in BL tumours.
Our results suggest a significant role for miR-29s in BL pathogenesis in altering the expression of targets involved in critical cancer pathways, such as cell cycle control, apoptosis inhibition and DNA methylation. Moreover, methylation-mediated miR-29 epigenetic silencing may occur during BL development.
伯基特淋巴瘤（BL）是一种常见于儿童的B细胞淋巴瘤。其特征为MYC基因易位，导致MYC癌基因的组成性表达。MYC通过miR - 29b - 1/miR - 29a启动子区域的E盒MYC结合位点促进miR - 29的抑制。我们评估了miR - 29a/b/c及其预测靶点在BL发病机制中的作用。
将miR - 29a/b/c的成熟序列转染至BL细胞系BL41和Raji，并评估DNMT3B、MCL1、BIM、CDK6、AKT和TCL1蛋白表达以及MCL - 1和CDK6 mRNA表达。用5 - 氮杂 - 2'-脱氧胞苷（地西他滨）处理BL细胞，并评估miR29表达和甲基化状态。通过DNMT3B siRNA进行DNMT3B抑制。
BL细胞中miR - 29的异位表达降低了CDK6、DNMT3B、TCL1和MCL - 1蛋白水平，但miR - 29对CDK6和MCL - 1 mRNA表达无影响。地西他滨提高了miR - 29表达水平并降低了CDK6蛋白表达。此外，siRNA抑制DNMT3B增加了miR - 29a/b表达。值得注意的是，miR - 29a/b1和miR - 29b2/c启动子基因显示出甲基化的CpG序列，在地西他滨处理后去甲基化。此外，与MYC易位病例相比，MYC阴性肿瘤的miR - 29表达水平更高，表明MYC在BL肿瘤中调节miR - 29。
我们的结果表明miR - 29在BL发病机制中具有重要作用，可改变参与关键癌症途径（如细胞周期控制、凋亡抑制和DNA甲基化）的靶点表达。此外，甲基化介导的miR - 29表观遗传沉默可能在BL发展过程中发生。
