MiR-2964a-5p binding site SNP regulates ATM expression contributing to age-related cataract risk

Abstract

This study was to explore the involvement of DNA repair genes in the pathogenesis of age-related cataract (ARC). We genotyped nine single nucleotide polymorphisms (SNPs) of genes responsible to DNA double strand breaks (DSBs) in 804 ARC cases and 804 controls in a cohort of eye diseases in Chinese population and found that the ataxia telangiectasia mutated (ATM) gene-rs4585:G>T was significantly associated with ARC risk. An in vitro functional test found that miR-2964a-5p specifically down-regulated luciferase reporter expression and ATM expression in the cell lines transfected with rs4585 T allele compared to rs4585 G allele. The molecular assay on human tissue samples discovered that ATM expression was down-regulated in majority of ARC tissues and correlated with ATM genotypes. In addition, the Comet assay of cellular DNA damage of peripheral lymphocytes indicated that individuals carrying the G allele (GG/GT) of ATM-rs4585 had lower DNA breaks compared to individuals with TT genotype. These findings suggested that the SNP rs4585 in ATM might affect ARC risk through modulating the regulatory affinity of miR-2964a-5p. The reduced DSBs repair might be involved in ARC pathogenesis.

Keywords: DNA damage; age-related cataract; ataxia telangiectasia mutated (ATM) gene; microRNA; single-nucleotide polymorphisms (SNPs).

Figure 1. The effect of miR-2964a-5p on miRSNP rs4585

(A) The schema showed predicted binding site of miR-2964a-5p in the 3’-UTR of the ATM. (B) MiR-2964a-5p mimics or miR-con was co-transfected with the reporter constructs containing G allele or T allele into HEK293T or HepG2 cells. *: P<0. 05, compared with G allele group; **: P<0. 01, compared with miR-con group. (C) MiR-2964a-5p mimics, inhibitors or miR inhibitor control was co-transfected with the reporter gene containing T allele into HepG2 or HEK293T cells. *: P<0. 01, compared with miR inhibitor control group.

Figure 2. The correlation of SNP rs4585 with ATM expression in vitro

(A) Analysis of ATM mRNA expression in HEK293T cells (TT) transfected with miR-2964a-5p mimics and inhibitors. (B) Analysis of ATM mRNA levels in HLEPIC-LECs (TT) transfected with the miR-2964a-5p mimics and inhibitors. (C) Western blot analysis and quantification (D) of ATM expression in HLEPIC-LECs (TT) transfected with the miR-2964a-5p mimics and inhibitors. *: P<0.01, compared with control group.

Figure 3. The correlation of SNP rs4585 with ATM expression in anterior capsules

(A) ATM mRNA expression was lower in ARC group than the non-ARC group regardless of genotypes. (B) After stratifying ARC by the subtypes, ATM mRNA levels were markedly decreased in the cortical, posterior subcapsular and mixed types of ARC regardless of genotypes. (C) Levels of miR-2964a-5p in anterior capsules of three genotypes were similar in ARC, P=0.2190. (D) ATM mRNA levels in ARC were lower in TT group than the GG or GT group. (E) Western blot analysis and quantification (F) of ATM expression in samples from GG, GT or TT group of ARC. *: P<0.01.

Figure 4. The correlation of rs4585-T with DNA breaks and ARC risk

(A) ARC had more DNA breaks than the control and TT carriers had more DNA damage than GG+GT carriers in ARC. (B) TT carriers had more DNA damage than GG+GT carriers in cortical, posterior subcapsular and mixed cataract patients, in addition to nuclear cataracts. *: P< 0.01, compared with control group of GG+GT genotype (n= 27), ^#: P< 0.01, compared with control group of TT genotype (n=5), **: P< 0.05, compared with corresponding ARC patients with GG+GT carriers.