Eugenic Genetics Laboratory, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430016, China.
Department of Otolaryngology, Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430016, China.
Biomed Res Int. 2022 Aug 29;2022:6496799. doi: 10.1155/2022/6496799. eCollection 2022.
Pendrin is encoded by SLC26A4, which is expressed in the apical membrane of inner ear epithelial cells and drives chloride reabsorption in the apical septum. In the inner ear, pendrin dysfunction and hypofunctional mutations lead to vestibular aqueduct (EVA) enlargement and sensory neural hearing loss. Mutations in SLC26A4 are a common reason of deafness. However, the underlying mechanisms of SLC26A4 mutants in hearing loss remain unknown.
In the present study, pEGFP-N1 carrying wild-type and mutant SLC26A4 (c.85G>A, c.2006A>T, and c.853G>A) were transfected into HEK-293T cells. GFP fluorescence and GFP levels were determined. SLC26A4 mRNA levels were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Then, the expression of chloride intracellular channel 1 (CLIC1) and CLIC2 was measured by Immunofluorescence assay. Intracellular chloride concentration and apoptotic rate were analyzed by flow cytometry. The levels of membrane/cytoplasmic pendrin, apoptosis-associated proteins, and the PI3K/Akt/mTOR pathway members were determined by Western blot.
Constructed SLC26A4 mutant 1 (c.85G>A), SLC26A4 mutant 2 (c.2006A>T), and SLC26A4 mutant 3 (c.853G>A). The wild-type and 3 mutations were stably expressed in HEK-293T. SLC26A4 mRNA expression was significantly increased after transfection with wild-type SLC26A4 and mutant SLC26A4 compared with the untransfected vector group ( < 0.01). Compared with the vector group, the expression levels of membrane pendrin, cytoplasmic pendrin, CLIC1, CLIC2, Bcl-2, p-PI3K, p-Akt, and p-mTOR were upregulated. Compared with the vector group, the chloride concentration, cell apoptotic rate, and the expression levels of caspase-3, caspase-9, and Bax were downregulated. Compared with the vector group, the above effects of SLC26A4 were reversed after the SLC26A4 mutant.
After SLC26A4 mutation, pendrin was transferred from the membrane, the chloride intracellular channel function was reduced, and the excessive accumulation of chloride in the cytoplasm induced cell apoptosis by inhibited PI3K/Akt/mTOR pathway phosphorylation.
Pendrin 由 SLC26A4 编码，在内耳上皮细胞的顶膜表达，并驱动顶隔中的氯离子重吸收。在内耳中，pendrin 功能障碍和功能低下突变导致前庭导水管（EVA）扩大和感觉神经性听力损失。SLC26A4 突变是耳聋的常见原因。然而，SLC26A4 突变体导致听力损失的潜在机制尚不清楚。
本研究将携带野生型和突变型 SLC26A4（c.85G>A、c.2006A>T 和 c.853G>A）的 pEGFP-N1 转染至 HEK-293T 细胞。测定 GFP 荧光和 GFP 水平。通过实时定量聚合酶链反应（qRT-PCR）检测 SLC26A4 mRNA 水平。然后，通过免疫荧光测定法测量氯离子细胞内通道 1（CLIC1）和 CLIC2 的表达。通过流式细胞术分析细胞内氯离子浓度和细胞凋亡率。通过 Western blot 测定膜/细胞质 pendrin、凋亡相关蛋白和 PI3K/Akt/mTOR 通路成员的水平。
构建了 SLC26A4 突变体 1（c.85G>A）、SLC26A4 突变体 2（c.2006A>T）和 SLC26A4 突变体 3（c.853G>A）。野生型和 3 种突变均稳定表达于 HEK-293T 细胞中。与未转染载体组相比，转染野生型 SLC26A4 和突变型 SLC26A4 后 SLC26A4 mRNA 表达明显增加（ < 0.01）。与载体组相比，膜 pendrin、细胞质 pendrin、CLIC1、CLIC2、Bcl-2、p-PI3K、p-Akt 和 p-mTOR 的表达水平上调。与载体组相比，氯离子浓度、细胞凋亡率以及 caspase-3、caspase-9 和 Bax 的表达水平下调。与载体组相比，SLC26A4 突变后 SLC26A4 的上述作用被逆转。
SLC26A4 突变后，pendrin 从膜转位，氯离子细胞内通道功能降低，细胞质中氯离子的过度积累通过抑制 PI3K/Akt/mTOR 通路磷酸化诱导细胞凋亡。
