circRNA circAF4 functions as an oncogene to regulate MLL-AF4 fusion protein expression and inhibit MLL leukemia progression
circRNA circAF4 functions as an oncogene to regulate MLL-AF4 fusion protein expression and inhibit MLL leukemia progression
复制标题
circRNA circAF4 作为癌基因调节 MLL-AF4 融合蛋白表达并抑制 MLL 白血病进展
DOI：
10.1186/s13045-019-0800-z
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发表时间：
2019-10-17
影响因子：
28.5
通讯作者：
Chen, Yue-Qin
中科院分区：
文献类型：
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作者：
Huang, Wei;Fang, Ke;Chen, Yue-Qin
背景
环状 RNA (circRNA) 代表一类内源性非编码 RNA，由反向剪接事件产生并有利于重复序列。最近的研究报告称，癌症相关的染色体易位可能会并置远处互补的重复内含子序列，导致 circRNA 的异常形成。然而，在已报道的融合基因中，只有少数circRNA被发现源自基因易位过程中的融合区域。我们质疑 circRNA 是否也可能源自基因易位过程中的融合伙伴。
方法
首先，我们设计了不同的qRT-PCR引物来鉴定AF4基因中的circRNA circAF4，并研究了不同类型白血病样本中的表达模式。其次，我们设计了两个专门针对 circAF4 反向剪接连接点的小干扰 RNA，用于功能研究。进行CCK8细胞增殖和细胞周期测定，并使用NOD-SCID小鼠模型研究circAF4在白血病发生中的作用。最后，进行荧光素酶报告基因测定、AGO2 RNA 免疫沉淀 (RIP) 和 RNA 荧光原位杂交 (FISH) 以确认 miR-128-3p、circAF4 和 MLL-AF4 表达的关系。
结果
我们发现了一种名为circAF4的circRNA，源自AF4基因，AF4基因是MLL-AF4白血病中MLL融合基因的伴侣。我们发现 circAF4 在 MLL-AF4 白血病中发挥致癌作用，并在体外和体内促进白血病发生。更重要的是，敲低circAF4会增加MLL-AF4白血病细胞中的白血病细胞凋亡率，而在不携带MLL-AF4易位的白血病细胞中则没有观察到任何影响。从机制上讲，circAF4可以充当miR-128-3p海绵，从而释放其对MLL-AF4表达的抑制。最后，我们分析了大部分 MLL 融合基因位点，发现许多 circRNA 可能源自这些伙伴，这表明源自融合基因伙伴的 circRNA（称为 FP-circRNA）在染色体易位白血病中的潜在作用。
结论
我们的研究结果表明，circAF4 的异常升高表达通过 circAF4/miR-128-3p/MLL-AF4 轴调节细胞生长，这可能有助于白血病的发生，表明 circAF4 可能是 MLL-AF4 白血病的新治疗靶点。
Background
Circular RNAs (circRNAs) represent a type of endogenous noncoding RNAs that are generated by back-splicing events and favor repetitive sequences. Recent studies have reported that cancer-associated chromosomal translocations could juxtapose distant complementary repetitive intronic sequences, resulting in the aberrant formation of circRNAs. However, among the reported fusion genes, only a small number of circRNAs were found to originate from fusion regions during gene translocation. We question if circRNAs could also originate from fusion partners during gene translocation.
Methods
Firstly, we designed divergent primers for qRT-PCR to identify a circRNA circAF4 in AF4 gene and investigated the expression pattern in different types of leukemia samples. Secondly, we designed two small interfering RNAs specially targeting the back-spliced junction point of circAF4 for functional studies. CCK8 cell proliferation and cell cycle assay were performed, and a NOD-SCID mouse model was used to investigate the contribution of circAF4 in leukemogenesis. Finally, luciferase reporter assay, AGO2 RNA immunoprecipitation (RIP), and RNA Fluorescent in Situ Hybridization (FISH) were performed to confirm the relationship of miR-128-3p, circAF4, and MLL-AF4 expression.
Results
We discovered a circRNA, named circAF4, originating from the AF4 gene, a partner of the MLL fusion gene in MLL-AF4 leukemia. We showed that circAF4 plays an oncogenic role in MLL-AF4 leukemia and promotes leukemogenesis in vitro and in vivo. More importantly, knockdown of circAF4 increases the leukemic cell apoptosis rate in MLL-AF4 leukemia cells, while no effect was observed in leukemia cells that do not carry the MLL-AF4 translocation. Mechanically, circAF4 can act as a miR-128-3p sponge, thereby releasing its inhibition on MLL-AF4 expression. We finally analyzed most of the MLL fusion genes loci and found that a number of circRNAs could originate from these partners, suggesting the potential roles of fusion gene partner-originating circRNAs (named as FP-circRNAs) in leukemia with chromosomal translocations.
Conclusion
Our findings demonstrate that the abnormal elevated expression of circAF4 regulates the cell growth via the circAF4/miR-128-3p/MLL-AF4 axis, which could contribute to leukemogenesis, suggesting that circAF4 may be a novel therapeutic target of MLL-AF4 leukemia.