目的:SERCA2是基因ATP2A2编码的P型泵之一,是内质网(ER)唯一的钙回流通道,参与维持钙稳态。本研究旨在探索SERCA2在听觉毛细胞中的表达模式以及SERCA2对顺铂诱导的耳毒性的可能机制。
方法:通过Westernblot(WB)和免疫荧光染色检测耳蜗毛细胞和HEI-OC1细胞中SERCA2的表达模式。用TUNEL法和WB法检测细胞凋亡及其相关因子。内质网应激相关因子的表达水平,ATF6,PERK,IRE1α,和GRP78,通过WB测量。至于SERCA2过表达和敲低的判定,构建质粒和慢病毒载体,分别。
结果:我们发现SERCA2在耳蜗毛细胞和HEI-OC1细胞中高表达。值得注意的是,新生小鼠SERCA2表达水平明显高于成年小鼠。在30μM顺铂的暴露下,与对照组相比,SERCA2显著下调。此外,顺铂给药引发内质网应激和细胞凋亡的发生。这些事件通过过度表达SERCA2逆转。相反,SERCA2敲低可加重上述过程。
结论:本研究的发现表明,第一次,SERCA2在耳蜗毛细胞中大量表达,顺铂引起的SERCA2抑制可引发内质网稳态破坏,因此暗示SERCA2可能是预防顺铂诱导的毛细胞毒性的有希望的靶标。
OBJECTIVE: SERCA2, one of the P-type pumps encoded by gene ATP2A2, is the only calcium reflux channel of the endoplasmic reticulum (ER) and participates in maintaining calcium homeostasis. The present study was designed to explore SERCA2 expression pattern in auditory hair cells and the possible mechanism underlying the effects of
SERCA2 on cisplatin-induced ototoxicity.
METHODS: The
SERCA2 expression pattern in cochlea hair cells and HEI-OC1 cells was measured by Western blot (WB) and immunofluorescence staining. The apoptosis and its related factors were detected by TUNEL assay and WB. The expression levels of ER stress-related factors, ATF6, PERK, IRE1α, and GRP78, were measured via WB. As for the determination of SERCA2 overexpression and knockdown, plasmids and lentiviral vectors were constructed, respectively.
RESULTS: We found that SERCA2 was highly expressed in cochlea hair cells and HEI-OC1 cells. Of note, the level of SERCA2 expression in neonatal mice was remarkably higher than that in adult mice. Under the exposure of 30 μM cisplatin, SERCA2 was down-regulated significantly compared with the control group. In addition, cisplatin administration triggered the occurrence of ER stress and apoptosis. Those events were reversed by overexpressing
SERCA2. On the contrary,
SERCA2 knockdown could aggravate the above processes.
CONCLUSIONS: The findings from the present study disclose, for the first time, that SERCA2 is abundantly expressed in cochlea hair cells, and the suppression of SERCA2 caused by cisplatin could trigger ER homeostasis disruption, thereby implying that SERCA2 might be a promising target to prevent cisplatin-induced cytotoxicity of hair cells.