CircNFIX knockdown inhibited AML tumorigenicity by the miR-876-3p/TRIM31 axis

ABSTRACT Background Acute myeloid leukemia (AML) is one of the most common malignant myeloid diseases in adults with a dismal prognosis. We aimed to explore the effects of circNFIX on the proliferation and apoptosis of AML cells. Methods The expressions of circNFIX, miR-876-3p and tripartite motif (TRIM) 31 in the bone marrow specimens of AML patients and AML cell lines were detected by qRT-PCR or western blot. Cell proliferation was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-ethynyl-29-deoxyuridine (EdU) assays. Cell cycle and apoptosis were analyzed by flow cytometry. Western blot was used to detect protein expression. The relationship between miR-876-3p and circNFIX or TRIM31 was identified by dual-luciferase reporter assay or RNA pull-down assay. Results The expression level of circNFIX was significantly increased in the bone marrow samples of AML patients and AML cells when compared with normal controls. CircNFIX silencing inhibited AML cell proliferation and promoted apoptosis. Inhibition of miR-876-3p reversed the effect of circNFIX knockdown on AML cell progression. In addition, circNFIX indirectly regulated TRIM31 through miR-876-3p. Further, TRIM31 overexpression counteracted the effect of circNFIX silencing on AML cell proliferation and apoptosis. Conclusion CircNFIX knockdown could suppress the proliferation and induce the apoptosis of AML cells by targeting the miR-876-3p/TRIM31 axis.


Introduction
Acute myeloid leukemia (AML) is the most usual hematological malignancy in adults with deviant myeloid hematopoietic progenitor cell proliferation, differentiation and maturation [1,2]. Although much progress has been achieved in chemotherapy and allogeneic stem cell transplantation, the prognosis of most patients with AML is still poor with a 5-year survival rate of 24% due to tumor relapse and drug resistance [3]. Thus, developing new therapies against AML is critical to improve the prognosis. Circular RNAs (circRNAs) belong to non-coding RNAs (ncRNAs) and are generated by back-splicing without 5 ′ ∼3 ′ polarity and poly adenine tails, and they act key modulatory roles in cancer progression [4]. CircRNAs can act as miRNA sponges or combine with RNA-associated proteins [5,6]. Cheng et al. confirmed that circNFIX promoted pituitary adenoma development by mediating the miR-34a-5p/CCNB1 cascade [7]. A previous study has shown that circNFIX facilitates glioma development by modulating the miR-378e/RPN2 cascade [8]. Moreover, circNFIX aggravated non-small cell lung cancer (NSCLC) progression [9]. Nevertheless, there is no relevant study on the effect of circNFIX on AML.
MicroRNAs are a class of short ncRNA molecules that can modulate gene expression by combining with mRNAs [10]. Previous documents have indicated that miRNAs can modulate cancer cell proliferation, differentiation and apoptosis by targeting oncogenes or tumor suppressors, thus playing a significant role in cancer diagnosis and treatment [11]. MiR-876-3p was downregulated in GC tissues and served as a tumor suppressor in GC [12], NSCLC [13], and lung cancer [14]. Moreover, Mao et al. discovered that miR-876-3p was decreased in acute lymphoblastic leukemia and inhibited the growth of acute lymphocytic leukemia (ALL) cells [15]. But the impact and mechanism of miR-876-3p in AML remain unclear.
In this study, we evaluated the function of circNFIX in AML pathogenesis. In addition, we assembled the cir-cNFIX/miR-876-3p/TRIM31 pathway to determine the molecular mechanism of circNFIX in this process with the hope of providing therapeutic targets for AML.

Clinical assay
Bone marrow samples were acquired from 47 AML cases and 40 normal patients at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. This study was approved by the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Ethics Committee. All participants provided the informed consent.
Quantitative real-time polymerase chain reaction (qRT-PCR) Trizol (Invitrogen) was used to prepare RNA. Complementary DNA (cDNA) was synthesized using the RevertAid First Strand cDNA kit (Thermo Fisher Scientific, Foster City, CA, USA) and miScript reverse transcription kit (Qiagen, Hilden, Germany). Then, amplification reaction was implemented with SYBR Mix (Applied Biosystems, Foster City, CA, USA). Primer sequences are shown in Table 1. Relative gene level was evaluated by the formula of 2 −ΔΔCt with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 as a housekeeping gene.

Flow cytometry
An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze cell apoptosis. After 48 h of transfection, AML cells were harvested and incubated with Annexin V-FITC and PI in darkness for 15 min. After centrifugation, the cells were re-suspended by PBS and analyzed using flow cytometry.
To analyze cell cycle progression, cells were cultured in 24-well plates. After 48 of transfection, the cells were collected and fixed with 70% iced ethanol at 4°C overnight. The cells were resuspended in 400 μL binding buffer and then incubated with 50 μL PI (Sigma) for 30 min under dark conditions. Cell cycle was detected by flow cytometry.

Western blot
Radio Immunoprecipitation Assay (RIPA) lysis buffer (EpiZyme, Shanghai, China) was utilized to prepare proteins. Protein quantification was carried out with BCA kit (Beyotime, Shanghai, China). Then, the proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with TBST containing 0.05% Tween 20 and 2.5% skim milk for 1 h and then labeled with primary antibodies at 4°C overnight. The primary antibodies include anti-CyclinD1 (ab16663;

RNA pull-down assay
All processes were conducted under RNase-free condition. The wild-type biotin-coupled miR-876-3p probe (Bio-miR-876-3p-WT) was mixed with C-1 magnetic beads (Life Technologies, Carlsbad, CA, USA) for 4 h to obtain probe-coated beads. Bio-miR-NC and Bio-miR-876-3p-MUT were used as the controls. Subsequently, cell extracts were mixed with the probecoated beads at 4°C overnight. QRT-PCR was implemented to analyze circNFIX enrichment.

Statistical analysis
All results were processed by GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA) and the data were expressed as mean ± standard deviation. The differences were evaluated by Student's t-test or one-way analysis of variance (ANOVA). P < 0.05 was deemed as statistically significant.

Results
CircNFIX was up-regulated in AML specimens and cells As shown in Figure 1A, circNFIX was overexpressed in AML samples when compared with normal samples. To evaluate the potential diagnostic value of circNFIX, the ROC curve of serum circNFIX levels in AML patients was generated. We found that the area under ROC curve (AUC) was 0.927, the sensitivity was 83%, and the specificity was 97.5% ( Figure 1B). Next, the level of cir-cNFIX in AML cell lines was monitored. As shown in Figure 1C, circNFIX expression was up-regulated in HL-60 and MOLM-13 cells when compared with HS-5 cells. These results suggested that circNFIX might be involved in AML progression.

Repression of circNFIX impeded AML cell growth
The results showed that sh-circNFIX markedly reduced the abundance of circNFIX, which suggested the high transfection efficiency of sh-circNFIX ( Figure  2A). MTT assay revealed that circNFIX knockdown suppressed the proliferation of AML cells ( Figure 2B, C) but did not affect HS-5 cell proliferation ( Figure  S1). EdU assay showed that circNFIX silencing reduced the percentage of EdU positive cells when compared with the sh-NC group ( Figure 2D,E), further suggesting that circNFIX knockdown restrained AML cell proliferation. CircNFIX silencing resulted in cell cycle arrest at G0/G1 phase ( Figure  2F,G). In addition, circNFIX knockdown increased the apoptosis rate of HL-60 and MOLM-13 cells ( Figure 2H). Accordingly, in HL-60 and MOLM-13 cells transfected with sh-circNFIX, the levels of CyclinD1 and Bcl2 were decreased, while the expression of apoptosis-related protein C-casp3 was increased ( Figure 2I,J). These results indicated that cir-cNFIX silencing suppressed the proliferation and induced the apoptosis of AML cells.

Silencing of miR-876-3p attenuated the effect of circNFIX deletion on the progression of AML cells
MiR-876-3p inhibitor down-regulated the level of miR-876-3p in HL-60 and MOLM-13 cells ( Figure 4A). The results of MTT and EdU assays together showed that circNFIX knockdown-induced suppressive effect on  (Figure 4G,H). Meanwhile, the effects of sh-circNFIX on CyclinD1, Bcl2 and C-casp3 expression were overturned after the addition of in-miR-876-3p ( Figure 4I). These data demonstrated that circNFIX knockdown suppressed AML progression by up-regulating miR-876-3p in vitro.
Ectopic TRIM31 expression rescued the effect of circNFIX silencing on AML cell progression TRIM31 expression was dramatically increased in HL-60 and MOLM-13 cells after transfection with TRIM31 ( Figure 7A). MTT and EdU assays displayed that the proliferation of HL-60 and MOLM-13 cells was reduced after transfection with sh-circNFIX, while this effect was reversed in the sh-circNFIX + TRIM31 group ( Figure 7B-D). Sh-circNFIX induced cell cycle arrest in G0/G1 phase, but overexpression of TRIM31 eliminated this effect in HL-60 and MOLM-13 cells ( Figure 7E). In addition, TRIM31 addition abolished the increase in HL-60 and MOLM-13 cell apoptosis induced by sh-circNFIX transfection ( Figure 7F).
Meanwhile, the effects of sh-circNFIX on CyclinD1, Bcl2, and C-casp3 expression were significantly reversed by TRIM31 overexpression (Figure 7G,H). Overall, these data suggested that circNFIX knockdown restrained AML progression largely by downregulating TRIM31 in vitro.

Discussion
For the past few years, molecular targeted therapy, immunotherapy and hematopoietic stem cell transplantation for AML have made great progress, but many patients still die from disease progression every year due to recurrence and drug resistance [22]. Therefore, it is particularly urgent to find novel molecular targets and biomarkers for AML patients.
CircRNA is a special endogenous ncRNA that can administrate gene expression [23]. It is highly conserved in evolution and has better stability when compared with linear mRNA [24]. CircRNA is involved in multiple stages of life activities and can be used not only as a biomarker for diagnosing human diseases, but also as a proxy for disease activity or severity [25,26]. CircRNA can regulate many biological processes by working as a molecular sponge of miRNA [27,28]. For example, circ_0000526 repressed the development of BC by sponging miR-492 [29]. Hsa_circ_0000515 could absorb miR-326 to expedite cervical cancer progression by increasing ELK1 abundance [30]. We observed a significant increase in circNFIX in AML. Further analysis exhibited that cir-cNFIX knockdown hampered AML cell proliferation and facilitated cell apoptosis. Moreover, circNFIX silencing decreased CyclinD1 and Bcl2 protein expression and increased cleaved caspase 3 production. The above evidence suggested that circNFIX might be an oncogene in AML progression.
We also analyzed the possible mechanism by which circNFIX silencing inhibited AML cell growth. MiRNA is an endogenous small ncRNA that is widely involved in the regulation of growth and apoptosis of organisms and is associated with the pathogenesis of a variety of disorders. Accumulating evidence has suggested that circRNAs can regulate cell biological behaviors by sponging miRNAs [31,32]. In this study, bioinformatics screening showed that circNFIX targeted miR-876-3p. Dual-luciferase and RNA pull-down tests identified the interaction between circNFIX and miR-876-3p. In addition, miR-876-3p expression was downregulated in AML tissues and AML cells. MiR-876-3p silencing attenuated circNFIX knockdown-mediated   A and B) The expression of TRIM31 in HL-60 and MOLM-13 cells transfected with sh-circNFIX, sh-circNFIX + in-miR-876-3p or corresponding controls (sh-NC or sh-circNFIX + in-miR-NC) was detected by qRT-PCR and western blot assays. **P < 0.01, ***P < 0.001. effects in AML cells, which suggested that circNFIX knockdown suppressed AML cell malignant potential largely by enhancing miR-876-3p.
Our study was the first one to discover that circNFIX was augmented in the bone marrow samples of AML patients and AML cells. CircNFIX promoted AML progression through the miR-876-3p/TRIM31 axis. Cir-cNFIX may be an effective target for future AML therapy.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.