circRNA circAF4 functions as an oncogene to regulate MLL-AF4 fusion protein expression and inhibit MLL leukemia progression
Authors: Yu-Meng Sun
Xue-Qun Luo
Ke Fang
Yue-Qin Chen
Zhen-Hua Chen
Cai Han
Tian-Qi Chen
Lin-Yu Sun
Wen-Tao Wang
Zhan-Cheng Zeng
Qi Pan
Qian-Qian Yang
Wei Huang
Journal: Journal of hematology & oncology (2019)
Discipline: CLINICAL MEDICINE
Keywords: Circular RNA; Leukemia progression; MLL-AF4 fusion protein.
Affiliations: MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Science, Sun Yat-sen University, Guangzhou, 510275, People's Republic of China., The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, People's Republic of China., MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Science, Sun Yat-sen University, Guangzhou, 510275, People's Republic of China. .

Abstract:  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. 

Retracted: No

PubPeer comments:
 There are some concerning issues with the flow cytometry plots in this paper as there are signs of some post-acquisition editing. In the heatmap plots, white boxes have been inserted to obfuscate the original text in a semi-transparent box (in places the semi-transparent box is viewable, however not the original text). There are also multiple signs of the grid lines having been edited (red circles). ![file] These things would not be overly concerning if it were not for the fact that the counts do not add up. 5 events in the upper left quadrant suggests 500 total events, which is too few for that plot. Moreover it is as if there is a blank rectangle added in in the lower right quadrant cutting right through the data. ![file] Could the authors show the original, unedited, flow cytometry plots, or perhaps explain how the counts in these plots have been calculated? Finally, these survival plots cannot have been obtained using five mice per group, as stated. You can never have 50 % survival with five mice. ![file] , 
 #1 Dear colleague: First of all, we very much appreciate the concerns you raised, following are our explanationsuff1a As shown in Figure 4f and Figure 2g below, the font sizes in the original flow cytometry plots were very small, so we inserted white boxes with enlarged number size to replace the original ones to make the data more clear. The white boxes indeed covered some dots and Y axis. As for a blank rectangle inserted in the lower right quadrant, the label B4 build-in white boxes covered the dots in the scatter plots. Hope that these explanations are clear enough. Belows are original flow cytometry plots and raw data as. All of the counts in these plots have been automatically calculated by the flow cytometry machine. ![file] ![file] These are very good comments for us, thank you. Regarding to the mice used for the survival plots, it was a typo in the figre legend, the number of mice was 6 not 5. Thank you very much leting us know this, we will contact the editor for sending a correction. Finally, thank you again. Wen-Tao Wang and Yue-Qin Chen , 
 I uploaded a figure twice, here is another one. ![file] , 
 I uploaded a figure twice, here is another one.![file] , 
 Dear Drs. Wang and Chen, Thank you for responding so quickly to this and to your commitment to transparency. These explanations are, indeed, clear. If I may come with two suggestions for the future: 1) The font sizes and box positions can be changed in the software (the heatmaps look like they were made in FloJo) before exporting the images. This way you do not have to do post-processing on the images, which can cause odd discrepancies, as was the case here. 2) The background fluorescence noise causing the counts pressed up against the Y-axis can be gated out (using e.g., a threshold). This would improve your data a bit - although it would, of course, not significantly change the end results. Thank you again and the best of luck in your future endeavors. , 
 Your suggestions are appreciated, thanks a lot. Wen-Tao Wang and Yue-Qin Chen ,