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

Qian Guan; Huiran Lin; Wenfeng Hua; Lei Lin; Jiabin Liu; Linqing Deng; Jiao Zhang; Jiwen Cheng; Zhonghua Yang; Yong Li; Jun Bian; Haixia Zhou; Suhong Li; Li Li; Lei Miao; Huimin Xia; Jing He; Zhenjian Zhuo

doi:10.21147/j.issn.1000-9604.2023.02.05

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

Chin J Cancer Res. 2023 Apr 30;35(2):140-162. doi: 10.21147/j.issn.1000-9604.2023.02.05.

Authors

Qian Guan^{1

2}, Huiran Lin³, Wenfeng Hua⁴, Lei Lin¹, Jiabin Liu¹, Linqing Deng¹, Jiao Zhang⁵, Jiwen Cheng⁶, Zhonghua Yang⁷, Yong Li⁸, Jun Bian⁹, Haixia Zhou¹⁰, Suhong Li¹¹, Li Li¹², Lei Miao¹, Huimin Xia¹, Jing He^{1

2}, Zhenjian Zhuo^{1

2

13}

Affiliations

¹ School of Medicine, South China University of Technology, Guangzhou 510006, China.
² Department of Pediatric Surgery, Guangzhou Institute of Pediatrics, Guangdong Provincial Key Laboratory of Research in Structural Birth Defect Disease, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China.
³ Faculty of Medicine, Macau University of Science and Technology, Macau 999078, China.
⁴ Research Institute for Maternal and Child Health, Guangdong Second Provincial General Hospital, Guangzhou 510317, China.
⁵ Department of Pediatric Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
⁶ Department of Pediatric Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China.
⁷ Department of Pediatric Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, China.
⁸ Department of Pediatric Surgery, Hunan Children's Hospital, Changsha 410004, China.
⁹ Department of General Surgery, Xi'an Children's Hospital, Xi'an Jiaotong University Affiliated Children's Hospital, Xi'an 710003, China.
¹⁰ Department of Hematology, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325027, China.
¹¹ Department of Pathology, Children Hospital and Women Health Center of Shanxi, Taiyuan 030013, China.
¹² Kunming Key Laboratory of Children Infection and Immunity, Yunnan Key Laboratory of Children's Major Disease Research, Yunnan Institute of Pediatrics Research, Yunnan Medical Center for Pediatric Diseases, Kunming Children's Hospital, Kunming 650228, China.
¹³ Laboratory Animal Center, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China.

PMID: 37180836
PMCID: PMC10167609
DOI: 10.21147/j.issn.1000-9604.2023.02.05

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.