Variant rs8400 enhances ALKBH5 expression through disrupting miR-186 binding and promotes neuroblastoma progression

Abstract

Objective: AlkB homolog 5 (ALKBH5) has been proven to be closely related to tumors. However, the role and molecular mechanism of ALKBH5 in neuroblastomas have rarely been reported.

Methods: The potential functional single-nucleotide polymorphisms (SNPs) in ALKBH5 were identified by National Center for Biotechnology Information (NCBI) dbSNP screening and SNPinfo software. TaqMan probes were used for genotyping. A multiple logistic regression model was used to evaluate the effects of different SNP loci on the risk of neuroblastoma. The expression of ALKBH5 in neuroblastoma was evaluated by Western blotting and immunohistochemistry (IHC). Cell counting kit-8 (CCK-8), plate colony formation and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays were used to evaluate cell proliferation. Wound healing and Transwell assays were used to compare cell migration and invasion. Thermodynamic modelling was performed to predict the ability of miRNAs to bind to ALKBH5 with the rs8400 G/A polymorphism. RNA sequencing, N6-methyladenosine (m⁶A) sequencing, m⁶A methylated RNA immunoprecipitation (MeRIP) and a luciferase assay were used to identify the targeting effect of ALKBH5 on SPP1.

Results: ALKBH5 was highly expressed in neuroblastoma. Knocking down ALKBH5 inhibited the proliferation, migration and invasion of cancer cells. miR-186-3p negatively regulates the expression of ALKBH5, and this ability is affected by the rs8400 polymorphism. When the G nucleotide was mutated to A, the ability of miR-186-3p to bind to the 3'-UTR of ALKBH5 decreased, resulting in upregulation of ALKBH5. SPP1 is the downstream target gene of the ALKBH5 oncogene. Knocking down SPP1 partially restored the inhibitory effect of ALKBH5 downregulation on neuroblastoma. Downregulation of ALKBH5 can improve the therapeutic efficacy of carboplatin and etoposide in neuroblastoma.

Conclusions: We first found that the rs8400 G>A polymorphism in the m⁶A demethylase-encoding gene ALKBH5 increases neuroblastoma susceptibility and determines the related mechanisms. The aberrant regulation of ALKBH5 by miR-186-3p caused by this genetic variation in ALKBH5 promotes the occurrence and development of neuroblastoma through the ALKBH5-SPP1 axis.

Keywords: ALKBH5; Neuroblastoma; polymorphism; progression; risk.

Figure S1

Secondary structure energies of ALKBH5 3’UTR with different sequence lengths. To avoid sequence length-dependent effects, a nucleotide sequence around miR-186-3p seed binding site and SNP site was extended to a length of 40−52 nt toward the 5’ end. The final target sequence was determined to be 52 nt. SNP, single-nucleotide polymorphism; nt, nucleotide.

Figure 1

Downregulation of m⁶A demethylase gene ALKBH5 represents a characterization of neuroblastoma. (A) ALKBH5 is highly expressed in neuroblastoma tissues compared to normal tissues; (B) Five neuroblastoma cell lines [(SK-N-SH, SK-N-BE(2), SK-N-AS, SK-N-BE(2)-C and KELLY] ALKBH5 protein expression was higher compared to non-neuroblastoma cell lines (NT22 and HELS); (C) ALKBH5 expression increases at less than 18 months of age; (D) m⁶A expression levels are elevated in ALKBH5 knockdown neuroblastoma (si-ALKBH5) cells compared to controls; (E,F) mRNA and protein expression of ALKBH5 was significantly increased in late neuroblastoma (stages III and IV) tissues compared with early neuroblastoma (stages I and II) tissues; (G) Immunohistochemistry results show that ALKBH5 expression is higher in tissues of patients with advanced neuroblastoma (Magnification 10×). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure S2

Stable knockout cell line construction and number of differentially expressed genes prior to transcriptome analysis. (A) We constructed neuroblastoma cell lines with stable ALKBH5 knockdown using CRISPR-Cas9 technology prior to transcriptome analysis; (B) Neuroblastoma cells with knockout of ALKBH5 expression showed 34 differentially expressed genes (28 up-regulated genes and 6 down-regulated genes).

Figure 2

Effects of ALKBH5 on transcriptome of neuroblastoma cells. (A) Volcano plots showing up-regulated genes (red) and down-regulated genes (blue) displayed by ALKBH5 knockout neuroblastoma cells; (B) Cluster analysis shows that SPP1, MIR6859-3, ANKRD1 and ADAMTS12 genes are up-regulated in neuroblastoma cell lines with down-regulated ALKBH5 expression; (C,D) GO enrichment analysis showed that genes related to RNA trafficking, localization, ribonucleic acid export, and DNA biosynthesis processes were down-regulated in ALKBH5 knockdown neuroblastoma cells (C), and genes related to the cellular response to tumor necrosis factor are up-regulated (D); (E) KEGG enrichment analysis of differential genes revealed that up-regulated genes are involved in pathways including transcriptional dysregulation in cancer, NOD-like receptor signaling and NF-kappa B signaling. GO, Gene Ontology; KEGG, KyotoEncyclopedia of Genes and Genomes.

Figure S3

Construction of ALKBH5 knockdown neuroblastoma cells. (A) Results of RT-qPCR analysis showed that ALKBH5 mRNA expression was significantly reduced in ALKBH5 knockdown neuroblastoma cell lines compared with the control group; (B) Western blotting data showed that ALKBH5 knockdown neuroblastoma cell lines had significantly reduced ALKBH5 protein expression; (C) EdU analysis also showed that proliferation of neuroblastoma cell lines with knockdown of ALKBH5 expression was inhibited. RT-qPCR, reverse transcription-quantitative real-time polymerase chain reaction. ***, P<0.001; ****, P<0.0001.

Figure 3

ALKBH5 promotes proliferation, migration and invasion of neuroblastoma cells. (A) CCK-8 assay showed that two neuroblastoma cell lines (si-ALKBH5) that interfered with ALKBH5 expression had significantly reduced rates of viable cell proliferation; (B) Colony formation assay (plate colony formation assay) was performed to evaluate the long-term proliferation of neuroblastoma cells with knockdown of ALKBH5 expression; (C) Flow cytometric analysis revealed an increased proportion of cells in G2/M phase after the downregulation of ALKBH5 expression; (D) Inhibition of ALKBH5 increased the expression of CDK2 and CDK4 and decreased the expression of p27; (E) Two cell lines that inhibited ALKBH expression [SK-N-SH, SK-N-BE(2)] had reduced wound closure ability compared to controls (Magnification 10×); (F) Downregulation of ALKBH5 inhibits neuroblastoma cell migration and invasion (Magnification 10×). CCK-8, cell counting kit-8. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure S4

Relationship between rs8400 G>A and ALKBH5 expression was analyzed by cis-eQTL. (A) Compared with rs8400 G genotype, individuals with rs8400 A allele had significantly higher ALKBH5 mRNA levels in the whole blood and adrenal gland, with consistent results in other tissues (cerebral cortex, tibial nerve, cervical vertebra C1 of the spinal cord and cultured fibroblasts); (B) RT-qPCR results showed that mRNA expression of ALKBH5 in cell lines overexpressing rs8400 A allele was higher than that in cell lines overexpressing rs8400 G allele or control vector; (C) Western blotting results showed that in cell lines overexpressing rs8400 A allele, the protein expression of ALKBH5 was higher than that in the cell lines overexpressing rs8400 G or the control vector. eQTL, expression quantitative trait loci. ****, P<0.0001.

Figure 4

rs8400 A allele increases ALKBH5 levels and promotes neuroblastoma cancer progression. (A) In adrenal tissues, individuals with rs8400 AA genotype had statistically higher levels of ALKBH5 mRNA than those with rs8400 GG or GA genotype; (B) Immunohistochemistry results show that tissue samples with the rs8400 A allele have higher ALKBH5 expression relative to rs8400 G allele (Magnification 10×); (C,D) Luciferase activity was significantly reduced in SK-N-SH (C), SK-N-BE(2) (D) cells with the rs8400 G allele construct compared with the construct with the rs8400 A allele; (E) Overexpression of rs8400 A significantly promoted neuroblastoma cell proliferation compared to overexpression of rs8400 G or the control vector; (F) Flow cytometry showed a higher proportion of cells in G2/M phase in neuroblastoma cells with rs8400 A allele relative to rs8400 G allele; (G) Colony formation experiments further demonstrated that rs8400 A-overexpressing cells had the most colonies; (H) Wound closure ability of stably overexpressed rs8400 A allele was increased compared with stably overexpressed rs8400 G allele or control vector or blank group without any treatment (Magnification 10×); (I) Stable overexpression of rs8400 A also significantly promoted the migration and invasion abilities of SK-N-AS cells compared with stably overexpressed rs8400G or control vector or blank group (Magnification 10×). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure 5

SNP rs8400 A/G causes different binding abilities of miR-186-3p to ALKBH5 3’UTR. (A) We found the binding sites for ALKBH5 3’-UTR and miR-186-3p by prediction software (Bartel, 2009); (B) When a miRNA binds to a target, the target first transitions to an intermediate state by unraveling the seed target nucleotides, and then forms a complex upon binding to the miRNA; (C,D) Using RNAfold and RNAcofold WebSever, we found that the rs8400 A allele (D) binds miR-186-3p to a reduced capacity compared with the rs8400 G allele (C); (E) Western blotting results showed that inhibiting miR-186-3p could promote expression of ALKBH5, and increasing miR-186-3p could inhibit the expression of ALKBH5; (F) Luciferase analysis showed that miR-186-3p could bind to the 3’-UTR of ALKBH5 gene, and rs8400 A allele reduced the binding ability of miR-186 to the 3’-UTR. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 6

SPP1 is identified as a downstream target of ALKBH5. (A) m⁶A consensus motif of GGAC is highly concentrated in SK-N-AS cells; (B) The study of m⁶A peaks showed that there were 12,508 and 10,945 in control cells and ALKBH5 knockdown, respectively, of which 1,500 were common; (C) There were 7,092 and 7,617 genes associated with the peak, with 1,931 and 2,465 uniquely associated genes, respectively; (D) Among the shared genes of differential peak-related genes of m⁶A-seq and differential genes of RNA-seq, there are 5 genes related to the ALKBH5 effect, namely SPP1, COL15A1, ADAMTS12, SYT14 and RP11-440G9.1; (E-I) When ALKBH5 was knocked down, m⁶A modification peak of the target gene was significantly increased; (J,K) RT-qPCR (J) and Western blotting (K) analysis demonstrated that SPP1 is inversely regulated by ALKBH5 in three cell types with significant differences. RT-qPCR, reverse transcription-quantitative real-time PCR. *, P <0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Figure S5

Distribution and counts of m⁶A peaks in control and knockout ALKBH groups. (A,B) m⁶A peak counts in control (A) and knockout ALKBH5 (B) groups; (C,D) Distribution of m⁶A peaks in control (C) and knockout ALKBH5 (D) groups. m⁶A, N6-methyladenosine.

Figure 7

ALKBH5 decreases SPP1 mRNA levels in an m⁶A manner. (A) MeRIP-qPCR detected m⁶A modification of SPP1. Compared with the control group, the overexpression of ALKBH5 inhibited m⁶A abundance of SPP1 mRNA, and increased m⁶A abundance of SPP1 mRNA after ALKBH5 was knocked out; (B) Detected the luciferase activity of SPP1 wild type and mutant type in overexpressed or knockdown ALKBH5 cells, respectively. MeRIP-qPCR, methylated RNA immunoprecipitation quantitative polymerase chain reaction; **, P<0.01.

Figure 8

Oncogenic effects of ALKBH5 were reversed by overexpression of SPP1. (A) CCK-8 showed that SPP1 expression inhibition promoted neuroblastoma cell progression and partially reversed ALKBH5 knockdown-induced reduction in neuroblastoma proliferation; (B) Co-precipitation results in Co-IP experiments indicated that ALKBH5 interacts with SPP1; (C,D) EdU (C) and colony (D) assays showed that inhibition of SPP1 expression promoted neuroblastoma cell proliferation and could partially reverse the decreased proliferation of neuroblastoma cells caused by ALKBH5 knockdown; (E) Knockdown of SPP1 also significantly abolished the reduced invasiveness caused by ALKBH5 deletion (Magnification 20×). CCK-8, cellcounting kit-8; Co-IP, coimmunoprecipitation. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 9

ALKBH5 inhibition promotes therapeutic effects of chemotherapeutic drugs on neuroblastoma cells. (A,B) SK-N-BE(2) (A) and SK-N-SH (B) neuroblastoma cell lines with ALKBH5 knockdown were significantly more sensitive to low doses of carboplatin compared to controls; (C,D) SK-N-BE(2) (C) and SK-N-SH (D) cell lines with ALKBH5 knockdown were significantly more sensitive to low doses of etoposide compared to control. *, P<0.05; **, P<0.01; ****, P<0.0001.

Figure S6

Workflow of current study. GTEx, Genotype-Tissue Expression; CCK-8, cell counting kit-8; ALKBH5, AlkB homolog 5; GO, Gene Ontology; KEGG, the Kyoto Encyclopedia of Genes and Genomes; SNP, single-nucleotide polymorphism; RT-qPCR, reverse transcription-quantitative real-time polymerase chain reaction; MeRIP, methylated RNA immunoprecipitation.

Figure 10

rs8400 G>A of ALKBH5 gene affects binding of miR-186-3p to the ALKBH5 binding site, making miR-186-3p unable to inhibit expression of ALKBH5, resulting in upregulation of its mRNA and protein levels. Upregulation of ALKBH5 further inhibited downregulation of tumor suppressor gene SPP1 and promoted progression of neuroblastoma.