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. 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. 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. 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.

中文翻译：

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circRNA circAF4作为癌基因，可调节MLL-AF4融合蛋白的表达并抑制MLL白血病的进展。

环状RNA（circRNA）代表一类内源性非编码RNA，它们是通过反向剪接事件产生的，并且有利于重复序列。最近的研究报道，与癌症相关的染色体易位可以并列遥远的互补重复内含子序列，导致circRNA的异常形成。然而，在报道的融合基因中，仅少数circRNA被发现在基因易位期间源自融合区。我们质疑circRNA是否也可以在基因易位期间源自融合伴侣。首先，我们设计了用于qRT-PCR的不同引物，以鉴定AF4基因中的circRNA circAF4，并研究了在不同类型的白血病样品中的表达模式。其次，我们设计了两个针对circAF4的反向剪接连接点的小干扰RNA，以进行功能研究。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表达之间的关系。我们发现了一种名为circAF4的circRNA，其源于AF4基因，该基因是MLL-AF4白血病中MLL融合基因的伴侣。我们表明，circAF4在MLL-AF4白血病中起致癌作用，并在体外和体内促进白血病的发生。更重要的是，敲除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未观察到任何作用。在机械上，circAF4可以充当miR-128-3p海绵，从而释放其对MLL-AF4表达的抑制作用。我们最终分析了大多数MLL融合基因基因座，发现许多circRNA可能源自这些伴侣，这表明起源于融合基因伴侣的circRNA（称为FP-circRNA）在具有染色体易位的白血病中的潜在作用。我们的发现表明，circAF4的异常升高的表达通过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，这表明circAF4可能是MLL-AF4白血病的一种新型治疗靶点。circAF4可以充当miR-128-3p海绵，从而释放其对MLL-AF4表达的抑制作用。我们最终分析了大多数MLL融合基因基因座，发现许多circRNA可能源自这些伴侣，这表明起源于融合基因伴侣的circRNA（称为FP-circRNA）在具有染色体易位的白血病中的潜在作用。我们的发现表明，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，这表明circAF4可能是MLL-AF4白血病的一种新型治疗靶点。circAF4可以充当miR-128-3p海绵，从而释放其对MLL-AF4表达的抑制作用。我们最终分析了大多数MLL融合基因基因座，发现许多circRNA可能源自这些伴侣，这表明起源于融合基因伴侣的circRNA（称为FP-circRNA）在具有染色体易位的白血病中的潜在作用。我们的发现表明，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，这表明circAF4可能是MLL-AF4白血病的一种新型治疗靶点。提示融合基因伴侣起源的circRNA（称为FP-circRNA）在具有染色体易位的白血病中的潜在作用。我们的发现表明，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，这表明circAF4可能是MLL-AF4白血病的一种新型治疗靶点。提示融合基因伴侣起源的circRNA（称为FP-circRNA）在具有染色体易位的白血病中的潜在作用。我们的发现表明，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，这表明circAF4可能是MLL-AF4白血病的一种新型治疗靶点。