基因变异rs8400通过破坏miR-186的结合来增强ALKBH5的表达，并促进神经母细胞瘤的进展。

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

作者信息

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

机构信息

School of Medicine, South China University of Technology, Guangzhou 510006, China.

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.

出版信息

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

DOI:10.21147/j.issn.1000-9604.2023.02.05

PMID:37180836

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10167609/

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 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 with the rs8400 G/A polymorphism. RNA sequencing, N6-methyladenosine (mA) sequencing, mA 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 . is the downstream target gene of the 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 mA demethylase-encoding gene increases neuroblastoma susceptibility and determines the related mechanisms. The aberrant regulation of by miR-186-3p caused by this genetic variation in promotes the occurrence and development of neuroblastoma through the ALKBH5-SPP1 axis.

摘要

目的

已证实烷基化修复同源蛋白5（ALKBH5）与肿瘤密切相关。然而，ALKBH5在神经母细胞瘤中的作用及分子机制鲜有报道。

方法

通过美国国立生物技术信息中心（NCBI）的dbSNP筛选和SNPinfo软件鉴定[具体基因名称]中的潜在功能性单核苷酸多态性（SNP）。采用TaqMan探针进行基因分型。使用多元逻辑回归模型评估不同SNP位点对神经母细胞瘤风险的影响。通过蛋白质免疫印迹法和免疫组织化学（IHC）评估神经母细胞瘤中ALKBH5的表达。使用细胞计数试剂盒-8（CCK-8）、平板克隆形成实验和5-乙炔基-2'-脱氧尿苷（EdU）掺入实验评估细胞增殖。采用伤口愈合实验和Transwell实验比较细胞迁移和侵袭能力。进行热力学建模以预测微小RNA（miRNA）与具有rs8400 G/A多态性的[具体基因名称]结合的能力。采用RNA测序、N6-甲基腺苷（m6A）测序、m6A甲基化RNA免疫沉淀（MeRIP）和荧光素酶实验鉴定ALKBH5对分泌型磷蛋白1（SPP1）的靶向作用。

结果

ALKBH5在神经母细胞瘤中高表达。敲低ALKBH5可抑制癌细胞的增殖、迁移和侵袭。miR-186-3p负向调节ALKBH5的表达，且这种能力受rs8400多态性的影响。当G核苷酸突变为A时，miR-186-3p与ALKBH5的3'-非翻译区（3'-UTR）结合的能力降低，导致[具体基因名称]表达上调。SPP1是[具体癌基因名称]的下游靶基因。敲低SPP1可部分恢复ALKBH5下调对神经母细胞瘤的抑制作用。下调ALKBH5可提高卡铂和依托泊苷对神经母细胞瘤的治疗效果。