EIF4A3-induced Circ_0001187 facilitates AML suppression through promoting ubiquitin-proteasomal degradation of METTL3 and decreasing m6A modification level mediated by miR-499a-5p/RNF113A pathway

Abstract

Aberrant expression of circRNAs has been proven to play a crucial role in the progression of acute myeloid leukemia (AML); however, its regulatory mechanism remains unclear. Herein, we identified a novel circRNA, Circ_0001187, which is downregulated in AML patients, and its low level contributes to a poor prognosis. We further validated their expression in large-scale samples and found that only the expression of Circ_0001187 was significantly decreased in newly diagnosed (ND) AML patients and increased in patients with hematological complete remission (HCR) compared with controls. Knockdown of Circ_0001187 significantly promoted proliferation and inhibited apoptosis of AML cells in vitro and in vivo, whereas overexpression of Circ _0001187 exerted the opposite effects. Interestingly, we found that Circ_0001187 decreases mRNA m⁶A modification in AML cells by enhancing METTL3 protein degradation. Mechanistically, Circ_0001187 sponges miR-499a-5p to enhance the expression of E3 ubiquitin ligase RNF113A, which mediates METTL3 ubiquitin/proteasome-dependent degradation via K48-linked polyubiquitin chains. Moreover, we found that the low expression of Circ _0001187 is regulated by promoter DNA methylation and histone acetylation. Collectively, our findings highlight the potential clinical implications of Circ _0001187 as a key tumor suppressor in AML via the miR-499a-5p/RNF113A/METTL3 pathway.

Keywords: Acute myeloid leukemia; Circ_0001187; METTL3; Progression; RNF113A; miR-499a-5p.

Fig. 1

Decreased Circ_0001187 in AML correlates with poor prognosis and characteristics of Circ_0001187 in AML cells. A-D Volcano plots of differentially expressed circRNAs between AML patients (favorable risk and poor risk) and healthy controls. E Heatmap of differentially expressed circRNAs between AML patients and healthy controls (blue = down-regulated, red = up-regulated). Fold change was listed above. F The expressions of Circ_0001187 in AML patients and healthy controls. G Circ_0001187 mRNA expression was measured in paired samples from five follow-up AML patients at ND and HCR stages. Data were analyzed using Wilcoxon test. H qRT-PCR analysis of Circ_0001187 expression in AML patients with favorable risk and poor risk. I Kaplan–Meier analysis of the overall survival time of AML patients with different circRNAs expression as indicated by Log-Rank test (n = 40). J-N The results of correlation between the expression level of Circ_0001187 and white blood cell, hemoglobin, platelets, BM% and PB% in ND AML patients. Data were analyzed using Pearson correlation. O Relative expression of Circ_0001187 in age was measured by qRT-PCR. P Diagnostic values of Circ_0001187 in AML patients. AUC: Area under the ROC curve. Q The sequence length of spliced mature Circ_0001187 derived from the linear DOPEY2 mRNA is 301 bp. And the results of Sanger sequencing from the head-to-tail splicing in the RT-PCR product of Circ_0001187. R Expression levels of Circ_0001187 and linear DOPEY2 mRNA in THP-1 AML cells treated with actinomycin D by qRT-PCR. S The qRT-PCR expression results of Circ_0001187 and DOPEY2 using random primer and Oligo dT primer in THP-1 AML cells. T Theagarose gel electrophoresis results of Circ_0001187 PCR products of cDNA or gDNA using convergent and divergent primers in THP-1 and Molm-13 AML cells. U RNA FISH analysis for Circ_0001187 in THP-1 and Molm-13 AML cells. Scale bars = 10 μm. *p < 0.05; ****p < 0.0001. HCR: HematologicalComplete remission; Ctrl: Control; ND: Newly diagnosed; R/R: relapsed-refractory; ns: Not significant; qRT-PCR: Quantitative reverse transcription PCR

Fig. 2

Circ_0001187 suppresses AML progression in vitro and in vivo. A-B The effect of Circ_0001187 knockdown or overexpression on the proliferation of THP-1 and Molm-13 AML cells by CCK-8 assay. C, D The effect of Circ_0001187 knockdown or overexpression on the apoptosis of THP-1 and Molm-13 AML cells treated with Ara-c(1uM, 24 h) or not by flow cytometry. E THP-1 and Molm-13 AML cells were as sensitive to Circ_0001187 knockdown or overexpression in terms of cell differentiation. F Western blot results of P21, MDM2 and P53 in THP-1 cells with knockdown or overexpressed Circ_0001187. G-I The results of spleen and liver weight of Circ_0001187 knockdown AML mice. J Kaplan–Meier analysis of the overall survival of mice treated with sh-Circ or NC. K The results of GFP⁺ leukemia cell frequencies in spleen, bone marrow and peripheral blood of mice treated with sh-circRNA or NC. L H&E staining showing infiltration of leukemic cells in the spleen and liver of mice engrafted with sh-Circ AML cells compared with that in control mice. And the results of Ki67 staining in the spleen and liver of mice engrafted with sh-Circ AML cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

Circ_0001187 facilitates AML suppression by promoting ubiquitin-proteasomal degradation of METTL3 and decreasing m6A modification level. A AGO2 RIP assay using AGO2 antibody in THP-1 cells lysates. B-C The results of the GO enrichment and pathways analysis from RNA pull-down in THP-1 cells. D Western blot results of METTL3 in THP-1 and Molm-13 AML cells treated by sh-Circ compared with negative control. E The results of m.⁶A dot blot in THP-1 and Molm-13 cells treated by sh-Circ compared with negative control. F Western blot results of METTL3 in 293 T cell treated with MG132 (10 μM, 6 h) or chloroquine (100 μM, 6 h). G Western blot results of METTL3 in THP-1 cells with Circ_ 0001187 knockdown treated with 20 μg/ml CHX at different times. H Western blot results of METTL3 in THP-1 cells with Circ_ 0001187 knockdown or overexpression treated by DMSO or MG132 (10 μM, 10 h). I-J IP results of METTL3 ubiquitination in 293 T cells. K-M The effect of Circ_0001187 knockdown or Circ_0001187/ METTL3 inhibitor STM2457 on the proliferation or apoptosis of THP-1 and Molm-13 cells treated with Ara-c (1 μM, 24 h) or not. Data were analyzed using unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

RNF113A functions as an E3 ubiquitin ligase to mediate Circ_0001187-induced METTL3 degradation. A Western blot results of METTL3 in THP-1 cells transduced with the siRNA of potential E3 ligases respectively compared with negative control. B The expression levels of 9 potential E3 ligases in THP-1 cells after downregulating Circ_0001187 with siRNA through qRT-PCR. C Expression levels of RNF113A in AML patients and healthy controls through qRT-PCR. D The results of correlation between the expression level of RNF113A and Circ_0001187. E The effect of si-Circ or si-Circ/oe-RNF113A on the apoptosis of THP-1 and Molm-13 cells cultured with Ara-C (1 μM, 24 h). F IF assays showing the co-localization of RNF113A with METTL3 in THP-1 and Molm-13 AML cells. Scale bar, 20 μm. G Co-IP showed the binding of METTL3 with RNF113A in 293 T cells. H Western blot results of METTL3 in 293 T cells with the RNF113A knockdown or overexpression treated with DMSO or MG132 (10 μM, 6 h). I The results of METTL3 ubiquitination in 293 T cells expressed GFP-RNF113A, Flag-METTL3 and HA-ubiquitin and treatment with or without MG132 (10 μM, 6 h). J Western blot results of investigate the form of polyubiquitin chains linked to METTL3. K The results of RNF113A-induced METTL3 ubiquitination detected in WT HA-UB and K48 transfected cells. L The effect of RNF113A overexpression or RNF113A/METTL3 overexpression on the proliferation of THP-1 and Molm-13 cells. M The effect of RNF113A overexpression or RNF113A/METTL3 overexpression on the apoptosis of THP-1 and Molm-13 cells cultured with Ara-C (1 μM, 24 h). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: Not significant

Fig. 5

Circ_0001187 acts as a sponge to enhance RNF113A expression via Circ_0001187/ miR-499a-5p/ RNF113A axis. A Heatmap for differentially expressed miRNAs via RNA pulldown assay by probe-Circ_0001187. B RNA FISH showing colocalization of Circ_0001187 and miR-499a-5p in THP-1 and Molm-13 cells. Scale bars, 20um. C RNF113A contains conserved target sites of miR-499a-5p by using the Target Scan and its mutations. D The results of dual-luciferase reporter assay for miR-499a-5p and RNF113A. E The expression levels of RNF113A in THP-1 and Molm-13 cells transfected with miR-499a-5p mimics or mimics/inhibitor respectively through qRT-PCR. F The expression levels of RNF113A in THP-1 and Molm-13 cells transfected with si-Circ or si-Circ/miR-499a-5p inhibitor through qRT-PCR. G Western blot results of RNF113A and METTL3 in THP-1 and Molm-13 cells transfected with si-Circ or si-Circ/miR-499a-5p inhibitor. H The effect of si-Circ or si-Circ/miR-499a-5p inhibitor on the proliferation of THP-1 and Molm-13 cells. I, K The effect of si-Circ or si-Circ/miR-499a-5p inhibitor on the apoptosis of THP-1 and Molm-13 cells. J, L The effect of si-Circ or si-Circ/miR-499a-5p inhibitor on the apoptosis of THP-1 and Molm-13 cells cultured with Ara-C (1 μM, 24 h). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: Not significant

Fig. 6

Circ_0001187 is epigenetically regulated, and combining chidamide and 5-azacytidine with METTL3 inhibitor synergistically suppresses AML cells. A The expression results of DOPEY2 in AML patients and healthy controls through qRT-PCR. B The expression results of DOPEY2 in paired samples from six follow-up AML patients at ND and HCR stage. C The results of correlation between the expression levels of Circ_0001187 and DOPEY2. D The expression levels of Circ_0001187 in AML cells cultured with 5-azacytidine (2.5, 5 and 10 μM, 24 h) or not by qRT-PCR. E The results of MSP by Agarose gel electrophoresis. F The expression levels of Circ_0001187 in AML cells cultured with chidamide (0.5, 1 and 2 μM, 24 h) or not by qRT-PCR. G ChIP‒qPCR showing the effect of chidamide on the histone acetylation levels of Circ_0001187 by promoter-1. H The expression levels of Circ_0001187 in AML cells cultured with CHi or 5-Aza or combination by qRT-PCR. I The effect of oe-Circ_0001187 on the apoptosis of AML cells cultured with METTL3 inhibitor, CHi or 5-Araz or combination. J-K The expression levels of Circ_0001187 in THP-1 cells transfected with si1/si2-EIF4A3 or oe-EIF4A3. L RIP assay to verify the physical interaction between EIF4A3 and the pre-mRNA of Circ_0001187 and Circ_0001947. M The results of RNA pull-down assays. N The expression level of EIF4A3 in AML patients and healthy controls through qRT-PCR. O The results of correlation between the expression levels of Circ_0001187 and EIF4A3. P-Q The results of correlation between the expression levels of RNF113A and EIF4A3. HCR: Hematological Complete remission; Ctrl: Control; ND: Newly diagnosed; R/R: relapsed-refractory; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: Not significant

Fig. 7

Proposed working model of EIF4A3-induced Circ_0001187 facilitates AML suppression through promoting ubiquitin-proteasomal degradation of METTL3 and decreasing m⁶A modification level mediated by miR-499a-5p/RNF113A pathway