Department of Otolaryngology, The First Affiliated Hospital of China Medical University, Shenyang 110001, China.
Int J Med Sci. 2018 Mar 8;15(5):447-455. doi: 10.7150/ijms.22410. eCollection 2018.
Keratinocytes are the predominant cell type in a cholesteatoma, and microRNA (miR)-203a has been shown to be essential for the growth and differentiation of keratinocytes. The regulatory mechanisms of miR-203a and Bmi1-the predicted target of miR-203a that is associated with cholesteatoma-have not been clarified. Real-time PCR and western blot were carried out for the detection of miRNAs, mRNAs, and proteins, including miR-203a, Bmi1, and phosphorylated (p-)Akt. Immunohistochemical staining was applied to observe the expression and distribution of Bmi1 and of p-Akt in cholesteatoma and in control retroauricular skin. The dual luciferase reporter assay was used to analyze the relationship between miR-203a and Bmi1. Ectopic miR-203a and Bmi1 were transfected into an immortalized line of human keratinocytes (HaCaT cells), and the roles of these molecules in cell proliferation, apoptosis, and migration were explored. Cholesteatoma tissues were characterized by downregulation of miR-203a and concomitant upregulation of Bmi1. Results of the dual-luciferase reporter assay indicated that Bmi1 was a direct target gene of miR-203a. Silencing of miR-203a increased Bmi1 expression; promoted proliferation, colony formation, and migration of HaCaT cells; and inhibited apoptosis. Moreover, p-Akt was significantly increased in cholesteatoma tissues and was positively correlated with Bmi1. Suppression of Bmi1 reduced p-Akt expression in HaCaT cells; subsequent inhibition of miR-203a reversed this phenomenon. Our results reveal that miR-203a may regulate cholesteatoma growth and proliferation by targeting Bmi1. These findings provide insight for the development of novel nonsurgical options for cholesteatoma.
角质形成细胞是胆脂瘤中的主要细胞类型，已经表明 microRNA（miR）-203a 对于角质形成细胞的生长和分化至关重要。miR-203a 和 Bmi1（miR-203a 的预测靶标，与胆脂瘤有关）的调节机制尚未阐明。进行了实时 PCR 和 Western blot 检测以检测 miRNA、mRNA 和蛋白质，包括 miR-203a、Bmi1 和磷酸化（p）-Akt。免疫组织化学染色用于观察胆脂瘤和对照耳后皮肤中 Bmi1 和 p-Akt 的表达和分布。双荧光素酶报告基因分析用于分析 miR-203a 和 Bmi1 之间的关系。将外源性 miR-203a 和 Bmi1 转染到永生化的人角质形成细胞系（HaCaT 细胞）中，探讨了这些分子在细胞增殖、凋亡和迁移中的作用。胆脂瘤组织表现为 miR-203a 下调和 Bmi1 上调。双荧光素酶报告基因分析结果表明，Bmi1 是 miR-203a 的直接靶基因。沉默 miR-203a 增加了 Bmi1 的表达；促进了 HaCaT 细胞的增殖、集落形成和迁移，并抑制了凋亡。此外，p-Akt 在胆脂瘤组织中显著增加，并且与 Bmi1 呈正相关。抑制 Bmi1 降低了 HaCaT 细胞中的 p-Akt 表达；随后抑制 miR-203a 逆转了这种现象。我们的结果表明，miR-203a 可能通过靶向 Bmi1 来调节胆脂瘤的生长和增殖。这些发现为胆脂瘤的新型非手术治疗方案的开发提供了思路。
