A Regulator SNP Affecting the Transcription Factor SP1 Binding Site in the PIK3R3 Gene Contributes to Noise-Induced...

Noise-induced hearing loss (NIHL) is a key occupational hazard, and its specific pathogenic mechanism is not fully elucidated. Here, we performed this study to explore the potential role of single nucleotide polymorphisms (SNPs) located in the PI3K/AKT pathway in human susceptibility to NIHL.

A case-control study including 688 NIHL patients and 667 normal subjects was conducted. Genotype analysis was tested by multiplex polymerase chain reaction (PCR) with next generation sequencing. Luciferase assays were conducted to explore the effect of SP1 on PIK3R3 promoter activity. siRNA and cell transfection were used to determine the regulatory effect of SP1/PIK3R3 on HEI-OC1 cells. qRT-PCR and Western blotting were used to determine the expression of genes and proteins related to PI3K/AKT, autophagy, and apoptosis. Cell counting kit 8 (CCK-8) assay was used to detect cell viability.

Subjects with PIK3R3 rs7536272 GG genotype were more sensitive to NIHL than those with the AA/AG genotypes and had a lower expression level of PIK3R3. Luciferase assay indicated that rs7536272 regulated PIK3R3 expression by binding to SP1. Knockdown of SP1 by siRNA and transfection into HEI-OC1 cells inhibited the PI3K/AKT pathway, indicated by the decreased phosphorylation levels of PI3K and AKT. Concurrently, SP1 deficiency promoted autophagy, as evidenced by an elevated LC3-II/LC3-I ratio and induced apoptosis, characterized by the reduction of Bcl2 and the increase of Bax and caspase-3. Overexpression of PIK3R3 partially restored the decreased activity of PI3K/AKT pathway and attenuated the enhanced autophagy and apoptosis caused by SP1 deficiency. CCK-8 assay revealed that SP1 knockdown led to a time-dependent decrease in cell viability, which was significantly rescued by the overexpression of PIK3R3.

All findings demonstrate that rs7536272 influences NIHL susceptibility by regulating PIK3R3 expression in an allele-specific manner by binding to SP1. SP1 inhibits autophagy and apoptosis by activating PIK3R3-mediated PI3K/AKT pathway.