Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2.
Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2.
复制标题
DOI：
10.1371/journal.ppat.1005031
复制
发表时间：
2015-07
期刊：
影响因子：
6.7
通讯作者：
Allday MJ
中科院分区：
文献类型：
--
作者：
Bazot Q;Paschos K;Skalska L;Kalchschmidt JS;Parker GA;Allday MJ
我们发现，两个主机编码的初级RNA（pri-miR）和相应的microRNA（miR）簇-广泛报道具有细胞转化相关的活动-由EBNA 3A和EBNA 3C调节。利用携带敲除、回复突变或条件性EBV重组体的多种EBV转化的淋巴母细胞系（LCL），可以明确证明EBNA 3A和EBNA 3C都是致癌miR-221/miR-222簇的反式激活所需的，所述致癌miR-221/miR-222簇在多种人类肿瘤（包括淋巴瘤/白血病）中以高水平表达。ChIP、ChIP-seq和染色体构象捕获分析表明，这种激活是由于两种EBV蛋白直接靶向miR-221/miR-222基因组位点的染色质，以及通过增强子元件与位于miR序列上游28 kb的长非编码pri-miR的转录起始位点之间的长程相互作用而激活。通过EBNA 3A或EBNA 3C的失活或缺失产生的miR-221/miR-222水平的降低导致细胞周期蛋白依赖性激酶抑制剂p57 KIP 2的表达增加，p57 KIP 2是miR-221/miR-222的一个公认的靶点。miR-221/miR-222阻断实验证实miR-221/miR-222靶向LCL中的p57 KIP 2表达。相比之下，EBNA 3A和EBNA 3C对于沉默肿瘤抑制基因簇miR-143/miR-145是必要的，但ChIP-seq表明这种抑制可能是间接的。该miR簇在人类癌症中经常下调或缺失，然而，B细胞中的靶点是未知的。这些数据一起表明，EBNA 3A和EBNA 3C通过抑制多种肿瘤抑制蛋白而促进B细胞转化，不仅通过直接抑制蛋白质编码基因，而且通过操纵宿主长的非编码pri-miR和miR。对人微小RNA（miR）的相对无偏筛选揭示，在EBV转化的B细胞中，只有当病毒核蛋白EBNA 3 A和EBNA 3 C都表达时，潜伏EBV才诱导在癌症中频繁上调的miR簇miR-221/miR-222。相同的两种EBV蛋白沉默肿瘤抑制miR簇miR-143/miR-145。miR-221/miR-222的诱导是由长的非编码一级RNA（pri-miR）通过结合EBNA 3A和EBNA 3C的增强子元件与pri-miR的转录起始位点之间的长距离染色质环的激活引起的。miR-221/miR-222的一个公认的靶点是细胞周期蛋白依赖性激酶（CDK）抑制剂p57 KIP 2，由于它可以灭活各种CDK，因此可以抑制细胞增殖，但可能在B细胞中具有其他功能。由于EBNA 3A和EBNA 3C还协同抑制至少两种其他CDK抑制剂（p16 INK 4a和p15 INK 4 b）的表达，这意味着潜伏性EB病毒对细胞周期检查点的失调存在一定程度的功能冗余。这项研究首次表明，这种减少多种细胞周期抑制剂表达的能力不仅来自蛋白质编码基因的直接抑制，而且还来自长的非编码RNA和致癌miR簇的激活。
We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters – widely reported to have cell transformation-associated activity – are regulated by EBNA3A and EBNA3C. Utilising a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours – including lymphoma/leukemia. ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28kb upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in increased expression of the cyclin-dependent kinase inhibitor p57KIP2, a well-established target of miR-221/miR-222. MiR blocking experiments confirmed that miR-221/miR-222 target p57KIP2 expression in LCLs. In contrast, EBNA3A and EBNA3C are necessary to silence the tumour suppressor cluster miR-143/miR-145, but here ChIP-seq suggests that repression is probably indirect. This miR cluster is frequently down-regulated or deleted in human cancer, however, the targets in B cells are unknown. Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs. A relatively unbiased screen of human microRNAs (miRs) revealed that in EBV-transformed B cells, a miR cluster, miR-221/miR-222, that is frequently up-regulated in cancer, is induced by the latent EBV only if the viral nuclear proteins EBNA3A and EBNA3C are both expressed. The same two EBV proteins silence a tumour-suppressor miR cluster miR-143/miR-145. The induction of miR-221/miR-222 results from the activation of a long non-coding primary RNA (pri-miR) via long-range chromatin looping between enhancer elements that bind EBNA3A and EBNA3C and the transcription start site of the pri-miR. A well-established target of miR-221/miR-222 is the cyclin-dependent kinase (CDK) inhibitor p57KIP2, which, because it can inactivate various CDKs, can inhibit cell proliferation—but might have additional functions in B cells. Since EBNA3A and EBNA3C also cooperate to repress the expression of at least two other inhibitors of CDKs (p16INK4a and p15INK4b), this implies a degree of functional redundancy in the deregulation of cell cycle checkpoints by latent EBV. This study has shown for the first time that this capacity to reduce expression of multiple cell cycle inhibitors results not only from direct repression of protein-encoding genes, but also the activation of a long non-coding RNA and cluster of oncogenic miRs.
登录
查看更多内容
影响因子：
6.7
作者：
Banerjee S;Lu J;Cai Q;Sun Z;Jha HC;Robertson ES
通讯作者：
Robertson ES
影响因子：
5.4
作者：
Cameron, Jennifer E.;Yin, Qinyan;Flemington, Erik K.
通讯作者：
Flemington, Erik K.
DOI：
10.1006/bbrc.1997.7124
发表时间：
1997-08-28
影响因子：
3.1
作者：
Feil, R;Wagner, J;Chambon, P
通讯作者：
Chambon, P
影响因子：
8.8
作者：
Chen, H-C;Chen, G-H;Chen, Y-H;Liao, W-L;Liu, C-Y;Chang, K-P;Chang, Y-S;Chen, S-J
通讯作者：
Chen, S-J
影响因子：
6.4
作者：
Akao, Y.;Nakagawa, Y.;Naoe, T.
通讯作者：
Naoe, T.