CircFN1 promotes acute myeloid leukemia cell proliferation and invasion but refrains apoptosis via miR‐1294/ARHGEF10L axis

Previous studies have proved circFN1 is highly expressed in acute myeloid leukemia (AML) patients and AML cell lines. This study aims to investigate the impact of circFN1 on AML and its mechanism. Via real‐time quantitative PCR to detect circFN1, miR‐1294, ARHGEF10L expressions in clinical plasma samples and AML cell lines, AML cells were cultured in vitro and transfected with si‐circFN1, pcDNA3.1‐circFN1, and si‐ARHGEF10L, respectively, or co‐transfected pcDNA3.1‐circFN1 + miR‐1294 mimic and pcDNA3.1‐circFN1 + si‐ARHGEF10L. Using dual luciferase reporter experiment to detect the relationship between circFN1 and miR‐1294, as well as miR‐1294 and ARHGEF10L. CCK‐8 was used to detect cell proliferation, Transwell to cell invasion, TUNEL staining and flow cytometry to detect cell apoptosis, RT‐qPCR to circFN1 RNA, miR‐1294, and ARHGEF10L expression levels in HL‐60 cells, and western blot to ARHGEF10L protein expression level in HL‐60 cells. We found highly expressed circFN1 and ARHGEF10L, as well as low‐expressed miR‐1294 in AML patients and AML cell lines. In contrast to si‐NC group, si‐circFN1 group could signally inhibit HL‐60 cell proliferation and migration, but promote cell apoptosis; compared with mimic NC group, miR‐1294 mimic group could visually inhibit HL‐60 cell proliferation and migration, but promote cell apoptosis. miR‐1294 was the target of circFN1, and ARHGEF10L was the target of miR‐1294. Over‐expressing miR‐1294 or silencing ARHGEF10L could signally inhibit circFN1 promoting HL‐60 cell proliferation and migration and repressing cell apoptosis. circFN1 promotes proliferation and invasion of AML cell and represses cell apoptosis via regulating miR‐1294/ARHGEF10L axis, which provides new insight for molecular targeted‐treatment for AML.


| INTRODUCTION
Acute myeloid leukemia (AML) is a heterogeneous leukemia occurring in the myeloid, erythroid, megakaryocyte, and monocyte precursors.
AML is defined as a devastating and rapidly developing disease, features rapid immature myeloid progenitor cell proliferation, which was blocked at various stages of myeloid differentiation.At the molecular level, AML is a heterogeneous genetic disease resulting from different mutations and translocations.These genetic changes usually result in transcription factor imbalance, which is essential for controlling the expansion and lineage determination of myeloid progenitor cells. 1 Clonal chromosome rearrangement is present in approximately 55% of AML patients, including t (8; 21), t (15; 17), and t (16; 16), resulting in fusion genes RUNX1/RUNX1T1, PML-RARα, and CBFB-MyH11, respectively. 2Some of these mutations are clinically associated with diagnostic or prognostic markers and potential therapeutic targets. 3According to the French-American-British system, there are 8 types of AML (M0-M7), with specific morphological characteristics and differentiation stages. 4The more recent World Health Organization classification is based on a combination of clinical symptoms, cell morphology, immunophenotype, and genetic abnormalities. 5In most cases, the disease involves the bone marrow, and malignant cells or extramedullary immersion can also be detected in peripheral blood.APL is a variant of AML that has specific genetic abnormalities of leukemic cells and severe clotting disorders, and its treatment differs from other types of AMLP1.For the vast majority of patients, AML mainly affects the bone marrow, and at least 20% of the nucleated bone marrow cells are immature or undifferentiated leukemic mother cells.AML can be divided into neotype, secondary and AML according to its cause. 6The incidence of AML increases with age, and because leukemia stem cells cannot be eradicated, resulting in a high rate of AML recurrence, the treatment outcome and prognosis of AML remain poor.Recently, the treatment of AML has taken on a new dimension due to the emergence of many new therapies such as targeted therapies. 7But, most AML patients will relapse within 3 years after diagnosis. 8Therefore, the search for new anti-cancer target drugs is essential for AML targeted therapy, recurrence, metastasis, and prognosis improvement.
Circular RNA (circRNA), a conserved single-stranded RNA molecule, stems from exon or intron sequence through reverse shear by precursor mRNA. 9Unlike standard linear RNA, circRNA forms a covalently closed, continuous stable loop, with no 5 0 end cap and 3 0 end poly (A) tail, therefore, it is resistant to exonuclease digestion. 10,11st circRNAs, abundant and conserved in different species, has specific expression in tissues or developmental stages. 93][14][15] CircRNA is out of control in cancer and leukemia, [16][17][18][19] and available to promote disease pathogenesis through affecting the characteristics of cancer. 19CircFN1 is highly expressed in gastric cancer cells with the impact of inhibiting cell apoptosis and elevating cell resistance. 20CircFN1 has the potential to be used as a drug target for treating AML.Most circRNAs can be used as miRNA sponges to regulate miRNA function through inhibiting the impact of ceRNA. 213][24] However, the role of miR-1294 in AML is still not clear.Starbase database analysis found that CircFN1 has a direct binding site with miR-1294.The downstream mRNA of miR-1294 was predicted through online prediction software microT, PicTar, PITA, and RNA22.Using Venn diagram and GEPIA online database analysis, it was found that ARHGEF10L and SULF2 were highly expressed in AML.ARHGEF10L is available to promote liver cancer occurrence, 25 but the mechanism and role of ARHGEF10L in AML have not been reported yet.
Our research aims to demonstrate the mechanism and role of circFN1 in AML, which will expand them in AML, with a view to finding new targets for treating AML from a genetic perspective.

| Plasma sample collection
A total of 16 plasma samples of patients with AML were collected received in Three Gorges Hospital Affiliated to Chongqing University from 2019 to 2020.The patients were pathologically diagnosed as AML.And 16 healthy examiners were collected as normal control group.A total of 3 mL venous blood was collected with EDTA anticoagulation tubes to centrifuge at 1000 g for 10 min to took upper plasma.EDTA-K2 was collected anticoagulated plasma of all participants to put into EP tubes without RNase and DNase, and frozen in

| Cell culture
Normal HS-5 cells and AML cell lines (HL-60, NB4, and Thp-1) purchased from ATCC were cultured in RPMI 1640 medium (Gibco) containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin, placed in constant temperature incubators at 37 C, with 5% CO 2 and 95% humidity.Applied cells in logarithmic growth phase for our experiment.The cell lines used in the study could not represented all AML status.

| Real-time quantitative PCR experiment
TRIzol Reagent (Life Technologies, USA) was used to extract total RNA.The concentration and purity of RNA were detected with an ultra-micro spectrophotometer NanoDrop2000 (Thermo), reverse transcription on total RNA was performed via Revert Aid First Strand cDNA Synthesis Kit (Thermo), cDNA template was synthesized by using PrimeScriptTM RT Master Mix Kit (TaKaRa, Japan) and reversed transcription reaction via poly-A method.The qPCR was performed on the StepOne™ Real-Time PCR System (Applied Biosystems) with a SYBR Premix Ex Taq Kit (TaKaRa, Japan) then performed through fluorescent RT-qPCR analyzer (CFX Connect, USA).β-actin was used as the internal control to standardize RNA expression.Human 18SrRNA was amplified as an endogenous control for circFN1.
Human U6 RNA was amplified as an endogenous control for miR-1294.All the reactions were performed in triplicate.According to the manufacturer's instructions, the PCRs were conducted at 95 C for 30 s, followed by 40 cycles of 95 C for 3 s, and 60 C for 30 s in the StepOne™ Real-Time PCR System (Applied Biosystems).Data analysis was performed through 2-ΔΔCt, 26 and the formula was in the following: See Table 1 for the amplified primer sequences of each gene and its primers.

| Western blotting
The cells were rinsed gently with pre-cooled PBS buffer for three times.Then added 150-250 μL of lysis solution (Solebo, China) in accordance with the number of cells per well of the 6-well plates, of which each 1 mL RIPA was added to 10 μL PMSF to make the final PMSF concentration at 1 mM.Centrifuged the lysed samples at 12,000 g for 5 min to took the supernatant, performed BCA protein quantification kit (Norwegian, China) to detect protein concentration of each sample, separated the protein extract (30 μg per channel) on 10% SDS-PAGE, transferred it to PVDF membrane (Millipore, MA), and later blocked it with 5% skim milk at room temperature for 1 h.
After that, the membrane was placed into the incubators with rabbit anti-human ARHGEF10L (ab121866, 1:1000, Abcam, USA), overnight at 4 C. later washed 3 times with TBST, 10 min each, with horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000, Solebo), incubated for 1 h at room temperature, and washed 3 times with TBST, 10 times each.Chemiluminescence imaging system (Shanghai Tianneng Technology Co., Ltd.) was applied for visualization and data analysis, β-actin (4970S, 1:1000, Cell Signaling Technology, USA) as the loading control.The experiment was carried out three times independently.

| CCK-8 for cell proliferation detection
Cells were seeded in each group into a 96-well plate 24 h after transfection.100 μL of diluted cell suspension (1 Â 10 5 cells/ml) was added in each well, and 3 replicates were set for each sample.After incubating them in incubators for 24, 48, 72, and 96 h respectively, 10 μL of CCK-8 reagent (Dojindo, Japan) was added to each well.
After incubation for 2 h in incubators, the absorbance was measured at 450 nm.Name of primer Sequences

| Transwell experiment
from three random fields and count the number of TUNEL positive cells.The apoptosis index was used to measure the degree of apoptosis.

| circFN1 could negatively regulated miR-1294
circRNA can act as a sponge of miRNAs to regulate gene expression in many cancers, thereby exerting biological functions. 13In order to probe into the mechanism, we applied Starbase online database (http://starbase.sysu.edu.cn/index.php)to predict circFN1 target gene, finding that circFN1 and miR-1294 had direct binding sites.
Therefore, we speculate that circFN1 negatively regulates miR-1294 by targeting to promote AML cell proliferation, migration, and invasion.Through designing the mutation sites in circFN13 0 -UTR region and miR-1294 (Figure 2A) to conduct dual luciferase reporter gene experiment, we found that luciferase activity in miR-1294 mimic + MT-circFN1 group was signally elevated (P < 0.01) in comparison with miR-1294 mimic + WT-circFN1 group; in contrast to miR-1294 inhibitor + WT-circFN1 group, luciferase activity in miR-1294 inhibitor + MT-circFN1 group was significantly decreased (P < 0.01) (Figure 2B).This indicates that miR-1294 and circFN1 directly bind to each other.RT-qPCR revealed that compared to si-NC group, miR-1294 expression in si-circFN1 group was significantly elevated (Figure 2C, P < 0.01).These indicate that circFN1 negatively regulates miR-1294 through targeting.

| DISCUSSION
AML is a malignant tumor with short survival and poor prognosis. 27It is necessary to probe into new and effective AML diagnostic and prognostic indicators.Increasing evidences describe circRNA dysregulation in various cancers, 28 and the role of circRNA in AML has been continuously elucidated.CircFN1, highly expressed in gastric cancer cells, can inhibit cell apoptosis and increasing cell resistance. 20Here, in accordance with the GEPIA online database and clinical sample analysis, it was found that circFN1 in plasma samples of AML patients was signally up-regulated in comparison with healthy controls, indicating that circ-PTK2 may be an oncogene in AML.Through silencing circFN1, we found that circFN1 promoted AML cell proliferation and invasion, but refrained apoptosis.
CircRNA participates in human cancer occurrence via its miRNA sponge activity. 29For example, circ_0072995 accelerates breast cancer cell movement through acting as a sponge for miR-30c-2-3p. 303][24] We verified through bioinformatics analysis and dual luciferase experiment that miR-1294 could be a direct target of circ FN1.Through silencing circFN1, our work found that miR-1294 is negatively regulated by circFN1 in AML cells.
CircFN1 promotes AML cell proliferation and invasion, but refrains apoptosis through down-regulating miR-1294.At present, studies have found that MiRNA-mRNA regulation axis is involved in regulating cell functions.For example, miR-214 inhibits liver cancer cell proliferation and migration through reducing FOXM1 levels. 31miR-370 exerts a tumor suppressor impact through targeting FOXM1 in AML cells. 32The direct interaction between miR-1294 and ARHGEF10L has been verified via dual luciferase reporter experiment.Based on our experiments, miR-1294 exerts biological functions through targeting ARHGEF10L, and circFN1 releases ARHGEF10L through chelating miR-1294, thereby up-regulating ARHGEF10L level in AML cells.
5][36][37] ARHGEF10L acts through RhoA-ROCK1-ERM signal conduction (an essential pathway in tumorigenesis) and stimulates EMT expression in liver and gastric cancer cells. 25,38Abnormal ROR1 expression appears in many malignant tumors and is related to Rho-GTPase activation; high ROR1 expression level in leukemia cells is related to poor prognosis. 39Abnormal ARHGEF10L expression appears in various malignant tumors and is also related to Rho-GTPase activation.Therefore, we reasonably speculate that ARH-GEF10L may be involved in various cancer occurrences through RhoA/Rho kinase signaling pathway.

| CONCLUSION
To sum up, we clarified the carcinogenic impact of circFN1 in AML.
CircFN1 promotes AML cell proliferation and invasion, but restrains apoptosis.Additionally, through bioinformatics analysis, miR-1294 and ARHGEF10L were found as circFN1 downstream genes.CircFN1 up-regulates ARHGEF10L level through its miR-1294 sponge activity, thereby promoting AML cell proliferation and invasion, and restraining apoptosis.
Cell migration and invasion experiments were proceeded in 8 μmpore-size polyethylene terephthalate (PET)-based migration chamber and BD Matrigel gel invasion chamber.A serum-free cell suspension was prepared to perform cell count, and 2 Â 10 4 cells were taken to put in 0.6 mL sterile EP tubes.DMEM was added up to 200 μL.Later 800 μL of DMEM containing 15% FBS was added to the 24-well plates, the Transwell chamber was placed on top, and gently 200 μL of cell suspension was added to the upper chamber to make the cells evenly distributed in the wells.After incubating them for 48 h, the Transwell cell was taken out, the culture medium was discarded in wells, washed twice with PBS, fixed with 100% methanol for 10 min, then discarded the methanol.An appropriate amount of Giemsa staining solution was added to stain for over 40 min, and gently washed off the staining solution in water, and non-migrated cells were gently wiped off in upper layer with cotton swabs.After natural drying, inverted microscopes (Zeiss Axioskop 2) was applied to count at 200 times magnification.AML cell migration and invasion ability was calculated in reference to the average number of cells in all regions, and expressed as the relative ratio to the control cells.The experiment was carried out three times independently.

2. 8 |
Terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling (TUNEL) TUNEL Apoptosis Detection Kit was applied for cell apoptosis detection according to the manufacturer's manual (Thermo Fisher Scientific, Waltham, MA, USA).Confocal microscopy (Olympus BX51TRF; Olympus Corporation) (magnification, Â100) was applied to scan the signal T A B L E 1 Primer sequence.

Flow
cytometry was used for cell apoptosis detection.Centrifuge sample and aspirate supernatant.Resuspend cell pellets (10 6 cell each tube) 0.5 mL of Hanks balanced salt solution (HBSS), then add 1.5 mL of ethanol.Incubate for 1 h at 4 C. Centrifuge samples at 800 g for 10 min and aspirate supernatant.Add to the pellet 250 μL HBSS, 250 μL RNAse and 500 μL propidium iodide.Incubate the mixture for 15 min at room temperature and then maintain it at 4 C in the dark until flow cytometric analysis.Each experiment was repeated three times.

3 | RESULTS 3 . 1 |
circFN1 promoted AML cell proliferation, migration, and invasion hsa_circ_0058124 (located at chr2:216270960-216274462 and derived from gene FN1 exons 15-19, with a spliced mature sequence length of 864 bp) has been termed as "circFN1" (http:// www.circbase.org/cgi-bin/singlerecord.cgi?id=hsa_circ_0058124; http://circnet.mbc.nctu.edu.tw/, by searching circFN1).CircFN1, highly expressed in gastric cancer cells, has impact of inhibiting cell apoptosis and increasing cell resistance.20To ascertain the impact of circFN1 on AML, clinical samples were collected and performed via RT-qPCR experiments.We found that circFN1 was highly expressed in plasma in AML group (Figure1A, P < 0.0001) in comparison with the Normal group.After detecting circFN1 expression level in human bone marrow stromal HS-5 cells and three AML cell lines (HL-60, NB4, and Thp-1), we found that in comparison with HS-5 cells, circFN1 was up-regulated in AML cell lines (Figure1B, P < 0.01, P < 0.001), and HL-60 was obviously proliferated.Therefore, we speculate that circFN1 promotes AML cell proliferation, migration, and invasion.Based on RT-qPCR, circFN1 mRNA content in si-circFN1 group was signally reduced in contrast to si-NC group (Figure1C, P < 0.01), indicating that circFN1 was successfully silenced.CCK-8 experiment showed in contrast to si-NC group, cell proliferation ability in si-circFN1 group was visually reduced (Figure1D, P < 0.01), indicating that circFN1 promoted cell proliferation.Based on Transwell experiment, we found that in comparison with si-NC group, the cell invasion ability in si-circFN1 group was signally reduced (Figure1E, P < 0.01), indicating that circFN1 promoted cell invasion.TUNEL assay and Flow cytometry detection showed the apoptosis ability of si-circFN1 group was apparently enhanced versus si-NC group (Figure1F, G, P < 0.01), indicating that circFN1 repressed apoptosis.There was no significant difference between control group and si-NC group in each detection index.In summary, circFN1 promotes AML cell proliferation and invasion but represses apoptosis.
available to inhibit AML cell proliferation, migration, and invasion.RT-qPCR experiment found that miR-1294 was down-expressed in AML patients (Figure3A, P < 0.0001); in comparison with HS-5 group, miR-1294 expression in HL-60 group was signally reduced (Figure3B, P < 0.01), indicating that miR-1294 was under-expressed in AML cells.F I G U R E 1 circFN1 promoted acute myeloid leukemia (AML) cell proliferation and invasion, while repressed apoptosis.(A) RT-qPCR to detect circFN1 expression level in the plasma of each group; (B) RT-qPCR to test circFN1 expression level in cells of each group; (C) RT-qPCR to detect circFN1 expression level in HL-60 cells of each group; (D) CCK-8 experiment to detect HL-60 cell proliferation ability of each group; (E) Transwell experiment to detect HL-60 cell invasion ability of each group; (F) TUNEL staining to detect cell apoptosis; Scale bar: 40 μm; (G) Flow cytometry to detect cell apoptosis.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.F I G U R E 2 circFN1 negatively regulated miR-1294.(A) Biology online software Starbase to predict the design of the targeting sites of circFN1 and miR-1294 as well as the mutation sites of LINC00852; (B) Dual luciferase reporter gene experiment to calculate the relative activity of each plasmid luciferase; (C) RT-qPCR to detect miR-1294 expression level in HL-60 cells of each group.**P < 0.01.miR-1294 expression in miR-1294 mimic group was obviously higher than that in mimic NC group (Figure 3C, P < 0.01), indicating that miR-1294 was successfully over-expressed.CCK-8 experiment revealed that compared to mimic NC group, the cell proliferation ability in miR-1294 mimic group was signally decreased (Figure 3D, P < 0.01), indicating that miR-1294 inhibited cell proliferation.Transwell experiment revealed that cell invasion ability in miR-1294 mimic group was signally reduced in contrast to mimic NC group (Figure 3E, P < 0.01), indicating that miR-1294 inhibited cell invasion.Data of TUNEL staining and flow cytometry implied that in contrast to mimic NC group, the miR-1294 mimic group had visually elevated cell apoptosis ability (Figure 3F, G, P < 0.01), indicating that miR-1294 accelerated cell apoptosis.The control group and mimic NC group showed no obvious differences in detection indicators.In summary, miR-1294 inhibits AML cell proliferation and invasion but accelerates apoptosis.

F
I G U R E 3 miR-1294 inhibited acute myeloid leukemia (AML) cell proliferation, migration and invasion.(A) RT-qPCR to detect miR-1294 expression level in plasma of each group; (B) RT-qPCR to examine miR-1294 expression level in cells of each group; (C) RT-qPCR to detect miR-1294 expression level in HL-60 cells of each group; (D) CCK-8 experiment to test HL-60 cell proliferation ability in each group; (E) Transwell experiment to detect HL-60 cell invasion ability in each group; (F) TUNEL staining to detect cell apoptosis.Scale bar: 40 μm; (G) Flow cytometry to detect cell apoptosis.**P < 0.01, ****P < 0.0001.Therefore, our work designed the mutation sites in ARHGEF10L3 0 -UTR region and miR-1294 (Figure 4F), as well as performed dual luciferase reporter gene experiment.It was found that in comparison with miR-1294 mimic + WT-ARHGEF10L group, luciferase activity of miR-1294 mimic + MT-ARHGEF10L group was significantly reduced (P < 0.01); in contrast to miR-1294 inhibitor + WT-ARHGEF10L group, luciferase activity of miR-1294 inhibitor + WT-ARHGEF10L group was signally elevated (P < 0.01) (Figure 4G, P < 0.01), indicating that miR-1294 and ARHGEF10L directly bound to each other.RT-qPCR and western blot experiments revealed that ARHGEF10L expression in miR-1294 mimic group was signally reduced in contrast to mimic NC group (Figure 4H, I, P < 0.01).These data indicate that miR-1294 negatively regulates ARHGEF10L.

3. 5 |
circFN1 promoted AML cell proliferation, migration, and invasion through miR-1294/ ARHGEF10L axis circFN1 is available to promote AML cell proliferation and invasion, but repress apoptosis through inhibiting miR-1294.miR-1294 inhibited expression of ARHGEF10L.In liver cancer, ARHGEF10L is available to promote tumorigenesis.25 Our work found that in contrast to si-NC group, ARHGEF10L expression in si-ARHGEF10L group was signally reduced (Figure5A, B, P < 0.01), indicating that ARHGEF10L was successfully silenced.RT-qPCR detection revealed that circFN1 expression in pcDNA3.1-circFN1 group was significantly higher than that in pcDNA3.1 group, indicating that circFN1 was successfully overexpressed (Figure5C, P < 0.01); in comparison with pcDNA3.1-circFN1F I G U R E 4 miR-1294 negatively regulated ARHGEF10L.(A) Online software RNA22, PicTar, PITA, and microT to predict miR-1294 downstream mRNA, combined with Venn diagram analysis; (B) GEPIA2 online database analysis to predict gene expression in acute myeloid leukemia (AML); (C) RT-qPCR to detect ARHGEF10L mRNA expression level in plasma of each group; (D) RT-qPCR to examine ARHGEF10L mRNA expression level in cells of each group; (E) Western blot to detect ARHGEF10L protein expression level in cells of each group; (F) Designing the mutation sites in ARHGEF10L 3 0 -UTR region and miR-1294; (G) Conducting dual luciferase reporter gene experiment to calculate each plasmid luciferase relative activity; (H) RT-qPCR to detect ARHGEF10L mRNA expression level in HL-60 cells of each group; (I) Western blot to detect ARHGEF10L protein expression level in HL-60 cells.*P < 0.05, **P < 0.01, ****P < 0.0001.+ mimic NC group, miR-1294 content in pcDNA3.1-circFN1+ miR-1294-mimic group was visually increased (Figure 5D, P < 0.01); in contrast to pcDNA3.1-circFN1+ si-NC group, ARHGEF10L content in pcDNA3.1-circFN1+ si-ARHGEF10L group was significantly reduced (Figure 5E, P < 0.01).Western blot experiment showed that in comparison with pcDNA3.1-circFN1+ si-NC group, ARHGEF10L content in pcDNA3.1-circFN1+ si-ARHGEF10L group was obviously decreased (Figure 5F, P < 0.05).In contrast to pcDNA3.1 group, pcDNA3.1-circFN1 group had signally higher cell proliferation and migration capabilities, and apparently reduced apoptosis capability; in F I G U R E 5 circFN1 promoted acute myeloid leukemia (AML) cell proliferation, migration and invasion via miR-1294/ARHGEF10L axis.(A) RT-qPCR to detect ARHGEF10L mRNA expression levels in HL-60 cells of each group; (B) Western blot to detect ARHGEF10L protein expression level in HL-60 cells of each group; (C) RT-qPCR to detect circFN1 expression level in HL-60 cells of each group; (D) RT-qPCR to examine miR-1294 expression level in HL-60 cells of each group: (E) RT-qPCR to test ARHGEF10L mRNA expression level in HL-60 cells of each group; (F) Western blot to detect ARHGEF10L protein expression level in HL-60 cells of each group; (G) CCK-8 experiment to examine HL-60 cell proliferation ability of each group; (H) Transwell experiment to test HL-60 cell invasion ability of each group; (I) TUNEL staining to detect cell apoptosis, Scale bar: 40 μm; (G) Flow cytometry to detect cell apoptosis.*P < 0.05, **P < 0.01.contrast to pcDNA3.1-circFN1+ mimic NC group, proliferation and migration capabilities of pcDNA3.1-circFN1+ miR-1294-mimic group cell were significantly reduced, but cell apoptosis capability was clearly enhanced; in comparison with pcDNA3.1-circFN1+ si-NC group, cell proliferation and migration capabilities of pcDNA3.1-circFN1+ si-ARHGEF10L group were signally reduced, but cell apoptosis capability was clearly enhanced (Figure 5G-J, P < 0.05, P < 0.01), indicating that over-expressing miR-1294 or silencing ARHGEF10L can inhibit the promotion role of circFN1 in AML cell proliferation and invasion, as well as the repression in cell apoptosis.There was no obvious difference in detection indexes between Control group and pcDNA3.1 group.CircFN1 can negatively regulate miR-1294 through targeting with ARH-GEF10L acting as the target of miR-1294, which reveals that circFN1 promotes AML cell proliferation and invasion, and refrains cell apoptosis via miR-1294/ARHGEF10L axis.