EIF4A3-induced circular RNA MMP9 (circMMP9) acts as a sponge of miR-124 and promotes glioblastoma multiforme cell tumorigenesis
Authors: Haiqian Liang
Jianwei Li
Ruichao Jia
Sai Zhang
Nan Li
Yuanqing Pan
Xuyi Chen
Renjie Wang
Journal: Molecular Cancer (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Circular RNA MMP9; Eukaryotic initiation factor 4A3; Glioblastoma multiforme; Migration and invasion; microRNA-124.
Affiliations: Institute of Traumatic Brain Injury and Neurology, Characteristic Medical Center of Chinese People's Armed Police Force, Tianjin, 300162, China., Department of Neurosurgery, Characteristic Medical Center of Chinese People's Armed Police Force, Tianjin, 300162, China., Department of Basic Medicine, Tianjin Medical College, Tianjin, 300222, China., Institute of Traumatic Brain Injury and Neurology, Characteristic Medical Center of Chinese People's Armed Police Force, Tianjin, 300162, China. ., Department of Neurosurgery, Characteristic Medical Center of Chinese People's Armed Police Force, Tianjin, 300162, China. ., Chinese Glioma Cooperative Group (CGCG), Tianjin, China. .

Abstract:  Background: Circular RNAs (circRNAs) have been found to play critical roles in the development and progression of various cancers. However, little is known about the effects of the circular RNA network on glioblastoma multiforme (GBM). Methods: A microarray was used to screen circRNA expression in GBM. Quantitative real-time PCR was used to detect the expression of circMMP9. GBM cells were transfected with a circMMP9 overexpression vector or siRNA, and cell proliferation, migration and invasion, as well as tumorigenesis in nude mice, were assessed to examine the effect of circMMP9 in GBM. Biotin-coupled miRNA capture, fluorescence in situ hybridization and luciferase reporter assays were conducted to confirm the relationship between circMMP9 and miR-124. Results: In this study, we screened differentially expressed circRNAs and identified circMMP9 in GBM. We found that circMMP9 acted as an oncogene, was upregulated in GBM and promoted the proliferation, migration and invasion abilities of GBM cells. Next, we verified that circMMP9 served as a sponge that directly targeted miR-124; circMMP9 accelerated GBM cell proliferation, migration and invasion by targeting miR-124. Furthermore, we found that cyclin-dependent kinase 4 (CDK4) and aurora kinase A (AURKA) were involved in circMMP9/miR-124 axis-induced GBM tumorigenesis. Finally, we found that eukaryotic initiation factor 4A3 (eIF4A3), which binds to the MMP9 mRNA transcript, induced circMMP9 cyclization and increased circMMP9 expression in GBM. Conclusions: Our findings indicate that eIF4A3-induced circMMP9 is an important underlying mechanism in GBM cell proliferation, invasion and metastasis through modulation of the miR-124 signaling pathway, which could provide pivotal potential therapeutic targets for the treatment of GBM. 

Retracted: No

PubPeer comments:
 Figure 4H: two panels appear to show an overlap. Shown with pink boxes. Figure 5B: two panels appear to show an overlap. Shown with red boxes. ![file] ![file] , 
 Most primers in thsi study (Additional file 1) are not for the target genes. Neither the divergent primers nor convergent primers can amplify circMMP9 (hsa_circ_0001162), MMP9 or any other genes after checking the primers with circPrimer and Primer-BLAST in the National Center for Biotechnology Information (NCBI). The miR-124 primers are the primers for CDK6. The AURK primers are not for Homo sapiens but for dictyostelium fasciculatum. Most important is that the authors used the GAPDH sequence of danio rerio to design primers to normalize the expression of their genes. In the Figure 1C of their paper, the authors confirmed the presence of circMMP9 using Sanger sequencing and agarose gel electrophoresis. Sanger sequencing analysis showed that the splice junction is between 533 and 534 nucleotide (Figure 1C). However, the full length of circMMP9 is only 312 bp. Why the authors obtained a product longer than 540 bp. ![](http: ) , 
 From the Figure 1C, we noted that the convergent primers was designed on exons 12 (P1) and 13 (P2) of MMP9, thus the convergent primers must amplify circMMP9. No matter whether the RNA was treated with Ribonuclease R (RNase R) or not, both the convergent primers and divergent primers could amplify circMMP9. However, the authors failed to produce a band for the RNA treated with RNase R, when using the convergent primers. Since forward primer (P1) and reverse primer (P2) of the convergent primers was designed on exons 12 and 13, respectively, when gDNA was amplified using the primers, the product length must longer than that amplified from cDNA, because an intron with a length of 1775 bp is between exons 12 and 13. We argue that the authors may use wrong primers to perform the PCR. In the Figure S1C (Additional file 2), the authors amplified GAPDH using two paired different primers. However, the authors only listed one paired primers in Table S1. What are the first paired primers of GAPDH in the Figure S1C represent? According to the direction of the triangles, the first paired primers of GAPDH are divergent primers. Why and how this type primers were designed? In addition, the authors stated that circMMP9, with a length of 328 bp, was formed from exons 12 and 13 of MMP9. We found that the length of exons 12 and 13 is 104 bp and 312 bp, respectively. Therefore, if the circMMP9 was devised from exons 12 and 13, the length should be 416 bp. Actually, 91 bp 3u2019 end sequence of exon 12 and 237 bp 5u2019 end sequence of exon 13 construct circMMP9. , 
 Some expriments in Figure 6 are very problemic. Fig.6b showed a RIP-qPCR experiment. However, in the right panel it seems that the authors showed that they treated the cells with or without MMP9 mRNA, then see the blotting of eIF4a3, which looks more like a RNA pulldown experiments rather than a RIP. It is expected to see blotting in IgG and anti-eIF4a3 samples in a typical RIP experiment. It is also wierd to see the description in  methods about RIP experiment that  U87 cell lysates (100u2009u03bcl) were treated with RIP buffer and were incubated with Proteinase K and magnetic beads conjugated with anti- eIF4A3 antibody or control (IgG) . Usually Proteinase K treatment is after IP rather than before. Meanwhile, in the figure legend the authors said  intron 11 of MMP9 was used as the negative control . However, in Fig.6a it showed that  f denotes for intron 9 rather than intron 11. Fig.6c showed RNA pulldown experiments. However, in  methods , they authors didn;t mention how they made these RNA probes. Instead, they mentioned that  MMP9 expression vectors, which were truncated to varying degrees (a1-a5), were constructed. A biotinylated MMP9 **DNA probe** was designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). Fig.6d,e, the authors claimed that they kd or oe eif4a3 in the cells but didn t provide any data showing the kd and oe efficiency of eif4a3. In almost all microscopic images, the authors either didn t provide scale bar, or didn t define the scale bar, or the scale bar is too small to read. ![file] In Fig.7b,e, the authors showed bioluminescence imaging of mice. However, in the  methods , the author didn t declare that the cell lines expressing luciferase; instead, they said  Both the stable U87 and U251 cells contained a GFP marker. . Most interestingly, in the graphic abstract, it seems that the authors suggested an undescribed cytoplasmic splicing mechanism to generate circRNA. ![file] ,