The Regulation of circRNA RNF13/miRNA-1224-5p Axis Promotes the Malignant Evolution in Acute Myeloid Leukemia

Objective To study the biological function of circular RNA RNF13 (circRNF13) in acute myeloid leukemia (AML) and its relationship with prognosis. Methods We constructed stable AML cell lines with downregulated expression of circRNF13, and then, we explored the effect of downregulation of circRNF13 expression on the proliferation, migration, and invasion through qRT-PCR, MTT curve, colony formation, transwell migration and invasion experiment, cell cycle, apoptosis, Caspase 3/7 assay, and other experiments. We also studied the expression of C-myc and Tenascin-C by qRT-PCR to explore the role of circRNF13. Results When the expression of circRNF13 was downregulated, the proliferation rate of AML cells decreased significantly, the cell cycle was blocked to G1 phase, and apoptosis rate increased significantly. C-myc related to cell proliferation decreased significantly at RNA level. Furthermore, when the expression of circRNF13 was downregulated, the migration and invasion ability of AML cells was significantly reduced, and the expression of Tenascin-C related to migration and invasion also decreased significantly. The luciferase reporter assay system confirmed that miRNA-1224-5p was the direct target of circRNF13. Conclusion CircRNF13 inhibited the proliferation, migration, and invasion of AML cells by regulating the expression of miRNA-1224-5p. This study provides some clues for the diagnosis and treatment of AML.


Introduction
Acute myeloid leukemia (AML) is a kind of hematopoiesis system malignant disease with strong heterogeneity, often accompanied by a variety of genetic and genetic abnormalities [1][2][3]. AML is mainly characterized by uncontrolled proliferation, differentiation, and apoptosis of leukemia cells. FAB (French-American-British classification) is divided into M0-M7 type according to its morphological and histochemical characteristics [4]. At present, the treatment of AML is mainly chemotherapy. Most of the first diagnosed patients can get complete remission (CR) after combined chemotherapy, but the recurrence rate is as high as 70% [5]. The drug resistance, recurrence, and side effects during chemotherapy keep the mortality at a high peak. With the development of molecular genetic technology, various biological indicators of AML have been found gradually, so that we can deeply understand the pathogenesis and progress of AML from the molecular level. So far, a variety of cytogenetic and molecular indicators have been widely recognized in clinical practice and become a powerful tool to assist diagnosis, guide treatment, and prognosis stratification. In recent years, a large number of researches focusing on DNA mutation, micro-RNA, and lncRNA are widely carried out, aiming to find more biological indicators of AML and improve the understanding of the characteristics of leukemia, so as to help monitor minimal residual lesions, improve the ability of early warning, and develop new targeted therapeutic drugs [6][7][8].
Circular RNAs are a kind of single-strand noncoding RNAs that widely exist in organisms [9]. They were first found in plant viruses in 1970s and then successively found in hepatitis D virus and yeast mitochondria [10]. In the early studies, circRNA was thought to be formed from the wrong splicing of exon transcripts. As a random product, it does not have a biological function, so the relevant reports are relatively rare. More and more evidences show that circRNA is not produced by chance. It has high abundance, stable structure, and special expression in time and space [11][12][13]. Cir-cRNA is more stable than homologous linear RNA because it has a closed-loop structure and is not easy to be degraded.
According to the construction sequence, circRNA can be divided into three categories: intron circRNA, exon circRNA, and exon-intron circRNA. All kinds of circRNA are produced in different ways, with different sequences and structures, so their biological functions are different [14,15]. At present, its physiological function and mechanism are still unclear, and what role it plays in various diseases is rarely reported. According to the results of previous literature, some circRNAs can be used as miRNA "sponge," which can be used as translation templates to encode proteins or participate in the transcription of regulatory genes. CIRS-7 (CDT1as) is the first circular RNA to regulate miRNA and play a competitive adsorption mechanism to participate in tumor progression [16], which is upregulated in human malignant solid tumor cells. Further analysis showed that there were nearly 70 miRNA binding sites on CIRS-7 [17]. CIRS-7 can play the role of miRNA adsorbing sponge, competitively binding miR-7, which leads to the increase of target gene expression level downstream of miR-7 and promotes the growth and proliferation of malignant solid tumor cells [18]. Based on this, the researchers further pointed out that the discovery of CIRS-7 changed the concept of miRNA regulatory mechanism, made the mechanism of noncoding RNA regulatory miRNA more complex, and also provided a direction for further research of tumor molecular network and the development of therapeutic targets. The above phenomena suggest that the mechanism of microRNA adsorbing sponge may be a common biological phenomenon in cells and also indicate the possibility of the above mechanism in AML research. However, its expression and function in the process of AML are not clear.
Circular RNA hsa_circ_0001346 is produced from the RING finger protein 13 (RNF13) at chromosome 3q25.1. Some results showed that circRNF13 was downregulated nearly 2.98 times in lung adenocarcinoma [19]. However, whether circRNF13 can regulate the development of AML through the mechanism of ceRNA has not been reported. Therefore, in this study, we conducted a series of experiments to study the expression, function, and molecular mechanism of circRNF13 in AML.

Methods
2.1. Tissue Samples. The blood of ten cases of AML and ten healthy volunteers was taken from the patients admitted to our hospital, and the specimens were immediately stored in liquid nitrogen. Our study was approved by the Ethical Committee and Institutional Review Board of our Hospital. All participants provided written informed consent.

Cell Transfection.
At 24 h before transfection, the same number of cells was inoculated in each well of the 6-well plate, and the cell saturation was 80%~90% at the time of transfection. The required volume of each plasmid is calculated according to the concentration of plasmid 2 g per plasmid. Four microliters of Lipofectamine 2000 transfection reagent was added to each tube. DMEM medium without FBS was added and diluted to 50 μl, respectively. Plasmid solution was mixed with Lipofectamine 2000 solution (total volume of 100 μl). After 48 hours of transfection, cells were collected to extract total RNA and protein.
2.4. RNA Extraction and qRT-PCR Detection. The total RNA was extracted using TRIzol Reagent (Invitrogen) and the cDNA was obtained using the PrimerScript™ Reagent Kit (Takara, Dalian, China). Fluorescence quantitative PCR detection was carried out according to the instructions. The reaction system was 10 μl, and the reaction procedure was 2.5. CCK8 Assay for Cell Proliferation. After the cells were digested by trypsin, they were suspended in DMEM medium and then counted by cell counting gun. The 96-well plate was inoculated with 100 μl (3000 cells) per well. Each group was cultured for 7 days. The number of cells needed was calculated 4-5 times. The cell suspension was prepared with a DMEM medium containing 10% FBS. After repeated blowing and mixing, the cells were inoculated on a 96-well plate. 12-24 h after the plate laying, when the cell adhered to the wall completely, suck out the complete medium in the first hole measured and add 100 μl DMEM medium containing 10% CCK8 into each hole. After incubation at 37°C for 1 h, remove 90 μl from each hole to a new 96-well plate, and the optical density (wavelength at 450 nm) was measured by enzyme scale. After that, the data were measured and recorded at a fixed time every day [20]. During the experiment, the fluid was changed every two days. After six days, the data were statistically analyzed.
2.6. Colony Forming Experiment. After cell count, cells were diluted with DMEM medium containing 10% FBS, and cell 2 BioMed Research International suspension was inoculated in a 6 cm cell culture dish. After shaking evenly, culture in a 37°C 5% CO 2 incubator and change the complete culture medium every 3-4 days. When the visible colony was formed in the cell culture dish, absorbed the culture medium, rinsed PBS twice, fixed it with 4% paraformaldehyde for 30min, then dyed it with methanol solution containing 0.5% crystal violet for 30min, rinsed it with clear water and dried it.
2.7. Dual-Luciferase Reporter Assay. Luciferase assay was divided into four groups: mutant group, NC group, miRNA-1224-5p group, and miRNA-1224-5p+circRNA RNF13 group. On the next day, when about 70% of the cells were fused, the luciferase plasmid containing miRNA-1224-5p mimic or inhibitor was cotransfected with lipofectamine 2000. After 48 h of conventional culture, it was measured and analyzed using the Dual-Luciferase Reporter Assay System (Promega). All experiments were repeated in independent triplicate.

Detection of Cell
Cycle by Flow Cytometry. The cells were fixed overnight with glacial ethanol at 4°C. After washing the cells with phosphate buffer, 0.5 ml phosphate buffer and 50 μl ethidium bromide were added to each sample; then, 100 μg/ml RNaseA and 0.2% Triton X-100 were added. After incubated at 4°C for 30 min, flow analysis was conducted by flow cytometer (FACSCailbur; BD Biosciences). All experiments were repeated in independent triplicate.
2.9. Detection of Caspase-3/7 Activation Form. According to the instructions of Caspase-Glo 3/7 Assay kit, the logarithmic growth phase cells were selected and inoculated. The blank reaction group, negative control group, si-NC group, and si-RNF13 group were set in each experiment: the blank reaction group was cultured with a medium without cells; the si-NC group and si-RNF13 group were transfected with si-NC and si-RNF13, respectively. Three multiple holes were set in each group. After 48 hours of transfection, the 96-well plates of the cells in the si-NC group and si-RNF13 group were taken out from the 37°C incubator and balanced to room temperature. Caspase-Glo 3/7 reagent of the same volume was added with the culture medium into each pore, shook for 30 s, mixed well, and incubated for 1 hour in the dark at room temperature. Then, transfer the liquid from each hole to the opaque 96-well white plate and read the fluorescence value of each hole with Bertold Centro LB 960 microplate light detector.

Transwell Experiment.
After cell counting, cell suspension was diluted with DMEM medium, and cell concentration was controlled at 4 × 10 3 cells/ml. 800 μl DMEM medium containing 20% FBS was added to the 24-well cell culture plate, and the cell was gently placed in the 24-well plate. Slowly add the cell suspension to the chamber, incubate at room temperature for 15 min, and then transfer it to a CO 2 incubator for 40 h. After 30 min of fixation and staining, rinse and gently wipe off the extra cells in the chamber with cotton swabs. Then, took photos with a microscope and count the number of cells passing through the chamber.
2.11. Statistical Analysis. The SPSS16.0 software was used for data analysis. T-test was used for the normal distribution of data between the two groups, and ANOVA was used for the comparison of the two groups in case of more groups.
The difference was statistically significant as p < 0:05.

Expression of circRNF13 in the Blood of AML Patients.
We first measured the expression of circRNF13 in the blood of AML patients (10 cases) and healthy people (10 cases). It was found that in AML patients' blood samples, the expression of circRNF13 was at least 2 times higher than the average value of the expression in the blood of healthy people, which was significantly different from that in the blood of healthy people (Figure 1(a)). Therefore, we believed that circRNF13 may exist as an oncogene in the blood of AML patients.

Effect of Downregulation of circRNF13 on the
Proliferation of AML Cells. Subsequently, we selected the cell lines with high expression of circRNF13 as experimental materials for the RNA interference experiment. Endogenous circRNF13 in the existing AML cell lines Kasumi-1 and HL60 was detected. As seen from the figure (Figure 1(b)), the expression of circRNF13 in HL60 cells is significantly higher than that in Kasumi-1 cells. Therefore, we chose HL60 cells for RNA interference to construct low expression cell lines. As seen from Figure 1(c), the expression of shRNA-1 circRNF13 in the low expression cell line decreased by about 35%, and the expression of circRNF13 in shRNA-2 decreased by more than 50%. Therefore, it can be concluded that the stable cell line with low expression of circRNF13 was constructed successfully, and the inhibition effect of the expression of circRNF13 was significant (Figure 1(c)).
In order to study whether the cell lines with low expression of circRNF13 have an effect on the proliferation of AML cells, we used the obtained control cell lines shRNA-NC and the low expression cell lines shRNA-1 and shRNA-2 to conduct MTT proliferation experiments. The proliferation rate of shRNA-1 as well as shRNA-2 decreased significantly 48 h (Figure 1(d)). Therefore, we can conclude that the downregulation of circRNF13 can inhibit the proliferation of AML cells. Furthermore, the number of clones in the low expression cell lines shRNA-1 and shRNA-2 was significantly reduced, and the number of colonies formed was significantly smaller (Figure 1(e)). Again, it showed that the downregulation of circRNF13 can inhibit cell proliferation, which was consistent with the results of the MTT proliferation experiment.

Effect of Downregulation of circRNF13 Expression on Cell
Cycle and Apoptosis of HL60 Cells. Later, the cell cycle of AML cells after the downregulation of circRNF13 expression was detected. It can be seen from the figure that the cells in the G0/G1 phase enhanced evidently, while in the S phase did not change significantly and that in the G2/M phase suppressed (Figure 2(a)). It can be concluded that when the expression of circRNF13 was 3 BioMed Research International downregulated, the cell cycle was blocked at G0/G1 and the cell proliferation rate was reduced.
In the previous experiment, we have detected that the expression of circRNF13 in AML increased significantly. In order to verify its effect on apoptosis, we carried out Annexin V/PI double staining experiment. It can be seen from the result chart that the number of early apoptosis cells in the experimental group was 3-4 times that in the control group (Figure 2(b)). So we concluded that the downregulation of circRNF13 can promote the early apoptosis of AML cells. It was found that the relative activity of Caspase 3/7 in the experimental group was significantly higher  C-myc is an important regulator of cell proliferation. Therefore, we detected the expression of C-myc at the molecular level. As shown in Figure 2(d), the expression of shRNA-1 and shRNA-2 in C-myc low expression cell lines was lower. Therefore, we suggested that circRNF13 downregulated the expression of C-myc at the mRNA level.

Effect of Downregulation of circRNF13 Expression on the Migration and Invasion of AML Cells.
In order to investigate the effect of low expression of circRNF13 on the migration ability of AML cells, we used Transwell cell for migration experiments. As shown in Figure 3(a), compared with the control cell line shRNA-NC, the number of cells passing through the basement membrane of the chamber in the low expression cell lines shRNA-1 and shRNA-2 decreased significantly. Therefore, the low expression of circRNF13 can inhibit the migration of AML cells. Compared with the control cell lines shRNA-NC, the number of cells with low expression shRNA-1 and shRNA-2 passing through Transwell cells decreased significantly (Figure 3(b)). Therefore, we believed that the low expression of circRNF13 can also inhibit the invasion of AML cells, which was consistent with the migration results. In order to test Tenascin-C, an important signal molecule affecting cell migration and invasion, we used qRT-PCR to measure Tenascin-C mRNA. As shown in Figure 3(c), the downregulation of circRNF13 expression significantly reduced Tenascin-C mRNA. Therefore, we suggested that the downregulation of circRNF13 may attenuate the migration and invasion of AML cells by inhibiting the expression of Tenascin-C.

Confirmation of circRNF13 as Direct
Target of miRNA-1224-5p. In order to predict the miRNA interacting with circRNF13, we used bioinformatics to analyze it. When circRNF13 was the direct site of miRNA interaction, it can be used to identify the 3'UTR region or other sites for complete or incomplete complementary pairing, then further affecting the target gene. After preliminary screening, it was considered that miRNA 1224-5p was the interaction miRNA of circRNF13.
In order to confirm the correctness of the bioinformatics prediction results, we first transfected the stable cell line with the overexpression of circRNF13 into NC and miRNA-1224-5p, then carried out qRT-PCR 48 h later, and obtained the results as shown in Figure 4(a). In the stable cell line with overexpression of circRNF13, the expression of circRNF13   (Figure 4(b)). Therefore, we can preliminarily think that miRNA-1224-5p interacted with circRNF13, further inhibiting its expression.
As shown in Figure 4(c), the relative activity of firefly luciferase in the experimental group transfected with circRNF13-1, circRNF13-2, and circRNF13-3 plasmid recombinants was reduced. Therefore, we believed that the two predicted binding sites in circRNF13 can interact with miRNA-1224-5p and performed their functions. Therefore, we concluded that circRNF13 was the direct target of miRNA-1224-5p. The relative activity of firefly luciferase did not change significantly in the experimental group (Figure 4(c)). Therefore, we further proved that miRNA-1224-5p regulated the function of circRNF13 by recognizing specific sequences.
3.6. Effect of Overexpression of miRNA-1224-5p on the Proliferation of AML Cells. In order to investigate the effect of overexpression of miRNA-1224-5p on the proliferation of AML cells, we first carried out the MTT proliferation curve experiment. We found that overexpression of miRNA-1224-5p can significantly reduce the cell proliferation ability through the MTT proliferation experiment (Figures 5(a)  and 5(b)). Then, in order to further verify the inhibitory effect of miRNA-1224-5p on cell proliferation, we also carried out cell colony formation experiments. The same number of cells was inoculated in the six-well culture plate. After 10-14 days of culture, the colonies were counted and observed, and then, the colonies with a diameter of more than 100 μm were statistically analyzed with an inverted microscope. Overexpressed miRNA-1224-5p decreased significantly, and the number of formed colonies was smaller ( Figure 5(c)). The results of the clonogenesis experiment were consistent with the results of the MTT proliferation curve, so we thought that the overexpression of miRNA-1224-5p can attenuate the proliferation. In addition, the cell cycle was blocked in G0/G1 after the overexpression of miRNA-1224-5p, which led to the slowdown of cell 3.7. Effect of Overexpression of miRNA-1224-5p on the Migration and Invasion of AML Cells. In order to further study the effect of overexpression of miRNA-1224-5p on the metastasis and infiltration of AML cells, we used the Transwell cell to detect its migration, cultured in the cell for 24 h, stained with crystal violet, observed, and photographed under the microscope. As shown in Figure 6(a), compared with NC, the number of cells passing through Transwell's ependyma after overexpression of miRNA-1224-5p decreased significantly, which showed that the overexpression of miRNA-1224-5p can inhibit the migration of AML cells. Overexpression of miRNA-1224-5p also inhibited the invasion of AML cells, which was consistent with the migra-tion results (Figure 6(b)). In order to test Tenascin-C, an important signal molecule affecting cell migration and invasion, we used qRT-PCR to measure Tenascin-C mRNA. As shown in Figure 6(c), the overexpression of miRNA-1224-5p decreased the expression of Tenascin-C at mRNA level. It was suggested that overexpression of miRNA-1224-5p may inhibit the migration and invasion of AML cells by inhibiting the expression of Tenascin-C.

Discussion
CircRNA is closely related to human diseases, especially in tumors. CircRNA can be secreted into body fluids, such as saliva, blood, and exosomes [21,22]. In clinical standard blood samples, hundreds of circRNA are more abundant than the corresponding linear mRNAs, so circRNA can be used as a new tumor marker in clinical detection [23]. In recent years, the high expression level of circRNAs in AML, including hsa_circ_0004277, hsa_circ_ 00750, has been screened by a gene chip [24,25]. It is found that the   BioMed Research International expression levels of these circRNAs in AML present corresponding dynamic changes with the evolution of AML. At the biological level, they have competitive or cleavage effects with homologous linear RNAs and have high potential as prognostic indicators of AML. It is predicted that this may be related to the absorption of miRNA as a "sponge." Therefore, the function of these circRNAs may be worth exploring in our future research projects. hsa_ circ_ RNF13, located in chr3:149563797-149639014, is derived from the ring finger protein 13 (RNF13) gene. It was found that the expression of circRNF13 in cancer tissue was 2.98 times lower than that in the surrounding normal lung tissue. In vitro, circRNF13 can inhibit the invasion and metastasis of the lung adenocarcinoma cell line. Bioinformatics analysis and RIP experiments showed that circRNF13 can interact with RNA binding protein Ago2 and act as a sponge of miR-93-5p, which provided a new way to further study the molecular function of circRNF13 [19]. The above data indicated that circRNF13 was a new potential LAC biomarker and therapeutic target.
In our study, we found that the downregulation of circRNF13 expression suppressed the proliferation, migration, and invasion of AML cells and studied its mechanism. Through literature review, it was found that miRNA and 3'UTR region or other parts of mRNA were complementary pairing and that miRNA and lncRNA could also be a completely complementary pairing or incomplete complementary pairing. Therefore, we used bioinformatics to predict the miRNA of the direct effect of circRNF13. After the prediction and literature search, we believed that miRNA-1224-5p regulated tumor cells with circRNF13 as the target.
At present, it has been found that miRNA-1224-5p is abnormally expressed in a variety of tumors, and it can be used as an oncogene or as an antioncogene and can be regulated at the protein expression levels of Caspase-3, Bcl-2, and Bax [26]. It was found that the expression of miRNA-1224-5p in lung cancer changed significantly and inhibited the formation of keloid fibroblasts [27,28].
Our study of circRNF13 is the first time to be studied in AML. With its involvement in the mechanism of action of AML gradually explored, circRNF13 may become a new target of targeted therapy. At the same time, this subject still needs further research, including the mechanism of a possible specific function, the molecules of interaction, the pathway involved, whether it is verified in animal experiments, and whether it can be easily detected in CSF and blood. Meanwhile, with the discovery of more and more circRNA, its function has been paid more and more attention by researchers. The function and mechanism of circRNA are diverse. circRNA can affect the life process, and its role in tumors is also concerned. However, at present, most researches focus on its formation mechanism, but the understanding of its molecular mechanism in the process of disease occurrence and development is still limited. More and more circRNA will be found and explained, and the mystery of its role in AML and other tumor diseases will be gradually revealed.

Conclusion
In conclusion, we concluded that circRNF13 was highly expressed in AML compared with normal brain tissue, the 9 BioMed Research International proliferation of AML cells was inhibited by cell cycle block and apoptosis induction after the downregulation of circRNF13 expression, and the migration and invasion of AML cells were inhibited significantly after the downregulation of circRNF13 expression. miRNA-1224-5p inhibits the proliferation, migration, and invasion of AML cells by regulating the expression of circRNF13. This study may provide some clues for the diagnosis and treatment of AML.

Data Availability
We can provide our data when others need it.

Conflicts of Interest
There is no conflict of interest.  BioMed Research International