Regulatory function and mechanism research for m6A modification WTAP via SUCLG2-AS1- miR-17-5p-JAK1 axis in AML

Abstract

Acute myeloid leukemia (AML), characterized by the abnormal accumulation of immature marrow cells in the bone marrow, is a malignant tumor of the blood system. Currently, the pathogenesis of AML is not yet clear. Therefore, this study aims to explore the mechanisms underlying the development of AML. Firstly, we identified a competing endogenous RNA (ceRNA) SUCLG2-AS1-miR-17-5p-JAK1 axis through bioinformatics analysis. Overexpression of SUCLG2-AS1 inhibits proliferation, migration and invasion and promotes apoptosis of AML cells. Secondly, luciferase reporter assay and RIP assay validated that SUCLG2-AS1 functioned as ceRNA for sponging miR-17-5p, further leading to JAK1 underexpression. Additionally, the results of MeRIP-qPCR and m6A RNA methylation quantification indicted that SUCLG2-AS1(lncRNA) had higher levels of m6A RNA methylation compared with controls, and SUCLG2-AS1 is regulated by m6A modification of WTAP in AML cells. WTAP, one of the main regulatory components of m6A methyltransferase complexes, proved to be highly expressed in AML and elevated WTAP is associated with poor prognosis of AML patients. Taken together, the WTAP-SUCLG2-AS1-miR-17-5p-JAK1 axis played essential roles in the process of AML development, which provided a novel therapeutic target for AML.

Keywords: AML; JAK1; WTAP; lncRNA SUCLG2-AS1; miR-17-5p.

Fig. 1

Flowchart of this study

Fig. 2

Hierarchical heatmaps and volcano plots presenting differentially expressed lncRNAs, miRNAs, and mRNAs. Left panels, heatmaps for all differentially expressed A lncRNAs, B miRNAs, and C mRNAs in AML; Right panels, volcano plots showing D lncRNAs, E miRNAs, and F mRNAs with fold change ≥ 1 (P < 0.05). Green, downregulated; red, upregulated; black, not differentially expressed. lncRNA: long noncoding RNA; miRNA: microRNA

Fig. 3

Screening key genes from ceRNA network and functional analysis. A Construction of a lncRNA-miRNA-mRNA ceRNA network for AML. In the ceRNA network, the blue and red nodes show decreased and increased expression of RNAs, respectively, while the colors are related to the absolute value of the fold change. Diamonds represent lncRNAs, ellipses represent miRNAs, rectangles represent mRNAs, and gray edges represent interactions among the lncRNAs-miRNAs and mRNAs. B PPI network of ceRNA network-related DEmRNAs. The nodes denote DEmRNAs (confidence score > 0.4) and the size of the nodes represents the degree of each node. C Top ten genes from the sub-network modularized by the plug-in cytoHubba. D, E Top 20 GO terms (P < 0.05) of the ceRNA-related DEmRNAs, respectively. F, G Top 20 KEGG pathways (P < 0.05) of the ceRNA-related DEmRNAs, respectively. Interactions and overlap among the important KEGG pathways. ceRNA: competing endogenous RNA;GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes

Fig. 4

The expression of key genes by qRT-PCR. A SUCLG2-AS1. B miR-17-5p. C JAK1

Fig. 5

Overexpression of SUCLG2-AS1 inhibits proliferation, migration and invasion and promotes apoptosis of AML cells. A Validation of SUCLG2-AS1 overexpression in AML cells by qRT-PCR. B The viability was detected after overexpression of SUCLG2-AS1 by CCK-8 assay. C The cell proliferation was detected after overexpression of SUCLG2-AS1 by EdU assay. D Detection of cell migration and invasion after overexpression of SUCLG2-AS1 by Transwell assay. E Detection of cell invasion after overexpression of SUCLG2-AS1 by EMT assay. F Detection of cell apoptosis after overexpression of SUCLG2-AS1 by flow cytometry

Fig. 6

SUCLG2-AS1 functioned via negatively regulating miR-17-5p expression in AML cells. A The regulatory function of SUCLG2-AS1 on the expression level of miR-17-5p in AML cells. B The luciferase reporter assay validated the relationships between SUCLG2-AS1 and miR-17-5p. C Ago2 RNA-binding protein immunoprecipitation assay to verify SUCLG2-AS1 binding with miR-17-5p. D, E SUCLG2-AS1 can affect the proliferation of AML cells through miR-17-5p by CCK-8 assay and EdU assay. F SUCLG2-AS1 can affect the migration and invasion of AML cells through miR-17-5p by Transwell assay. G SUCLG2-AS1 can regulate the apoptosis of AML cells through miR-17-5p by flow cytometry

Fig. 7

SUCLG2-AS1 regulates the expression of JAK1 through competitive binding of miR-17-5p. A The validation of JAK1 expression in AML cells and normal cells by WB. B The luciferase reporter assay validated the relationships between miR-17-5p and JAK1. C Ago2 RNA-binding protein immunoprecipitation assay to verify miR-17-5p binding with JAK1. D The regulation of SUCLG2-AS1 on the expression level of JAK1 through miR-17-5p in AML cells by qRT-PCR. E The regulation of SUCLG2-AS1 on the expression level of JAK1 through miR-17-5p in AML cells by WB

Fig. 8

miR-17-5p regulates the occurrence and development of AML through JAK1. A The negative regulatory effect of miR-17-5p on JAK1 expression levels in AML cells was detected by qRT-PCR. B The negative regulatory effect of miR-17-5p on JAK1 expression levels in AML cells was detected by WB. C, D SUCLG2-AS1 can affect the proliferation of AML cells through miR-17-5p by CCK-8 assay and EdU assay. E miR-17-5p can affect the migration and invasion of AML cells through JAK1 by Transwell assay. F miR-17-5p can regulate the apoptosis of AML cells through JAK1 by flow cytometry

Fig. 9

The m6A modification was enriched in SUCLG2-AS1 and improved its transcripts stability. A The m6A methylation level of SUCLG2-AS1 in AML cells and control cells were determined by MeRIP-qPCR. B The knockdown effect of sh-WTAP was verified by Western blot (WB) analysis in THP-1 cells. C m6A methylation level in THP-1 cells after WTAP was knocked down. D Changes in m6A modified SUCLG2-AS1 levels upon WTAP was knockdown in THP-1 cells. E Transcript levels of WTAP and SUCLG2-AS1 in negative control and sh-WTAP THP-1 cells. F Reduction of SUCLG2-AS1 RNA stability in WTAP knockdown THP-1 cells as compared to control. Cells were treated with actinomycin D and RNA was isolated at 0, 2, and 4 h. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. The experiments were independently repeated at least three times

Fig. 10

Overview of m6A gene locus and gene information. A The location of mutations in m6A regulators. B Waterfall plot of m6A regulators mutation genes and mutation types. C Comparison of gene expression levels of 23 regulators between the normal and tumor tissue cohorts. D Landscape and inner crosslink between 23 m6A regulators. E The Kaplan–Meier survival analysis of WTAP. **P < 0.01, ***P < 0.001. m6A, N6-methyladenosine

Fig. 11

WTAP expression and m6A modification in AML cells. A The expression of WTAP in AML cells and control cells were determined by qRT-PCR. B The m6A level of WTAP in AML cells. C, D WTAP expression in AML Cells were stained for WTAP (red), and nuclei were stained with DAPI (blue). Scale bar: 100 μm. *P < 0.05, ***P < 0.001. ns, no significance