Central Research Laboratories, Research and Development Division, Senju Pharmaceutical Co., Ltd., 6-4-3, Minatojima-Minamimachi, Chuo-Ku, Kobe, Hyogo, 650-0047, Japan.
Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., 6-4-3, Minatojima-Minamimachi, Chuo-Ku, Kobe, Hyogo, 650-0047, Japan.
BMC Ophthalmol. 2021 Oct 25;21(1):377. doi: 10.1186/s12886-021-02141-9.
microRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. They are found within cells and in body fluids. Extracellular miRNAs have been shown to associate with the surrounding tissues. Therefore, we predicted that miRNAs in tears may contribute to regulate corneal epithelial cell function. However, information on the miRNA expression profile of tears is limited and the specific functions of tear miRNAs for corneal epithelial cells are still unknown. To study the role of tear miRNAs, we determined which miRNAs are highly expressed in tears and examined the involvement of miRNAs in corneal epithelial cell viability.
miRNAs extracted from monkey tears and sera were subjected to microarray analysis. miRNAs of which expression levels were higher in tears than in sera were selected, and their expression levels were quantified by quantitative polymerase chain reaction (qPCR). To examine miRNA function, mimics and inhibitors of miRNAs were transfected into human corneal epithelial (HCE-T) cells and incubated for 24 or 48 h. After transfection of miRNA mimics and inhibitors, the viability of HCE-T cells was measured using the water soluble tetrazolium salt (WST) assay, and microarray analysis and qPCR were performed using total RNA extracted from HCE-T cells. siRNAs of the candidate targets for miR-203 were transfected into HCE-T cells and the WST assay was performed. To determine a direct target gene for miR-203, a dual luciferase reporter assay was performed in HCE-T cells using a luciferase reporter plasmid containing 3'-UTR of human IGFBP5.
Microarray and qPCR analyses showed that miR-184 and miR-203 were expressed significantly more highly in tears than in sera (165,542.8- and 567.8-fold, respectively, p < 0.05). Of these two miRNAs, transfection of a miR-203 mimic significantly reduced the viability of HCE-T cells (p < 0.05), while a miR-203 inhibitor significantly increased this viability (p < 0.05). miR-203 mimic downregulated insulin-like growth factor-binding protein 5 (IGFBP5) and nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), while miR-203 inhibitor upregulated these two genes. Transfection of IGFBP5-siRNA decreased the viability of HCE-T cells. miR-203 mimic significantly diminished the luciferase reporter activity.
In this study, we identified miRNAs that are highly expressed in tears, and the inhibition of miR-203 increases the viability of corneal epithelial cells. Our results suggest that miR-203 contributes to regulating the homeostasis of corneal epithelial cells.
微小 RNA（miRNAs）是一种负调控基因表达的小型非编码 RNA。它们存在于细胞内和体液中。已经证明细胞外的 miRNAs 与周围组织有关联。因此，我们推测泪液中的 miRNAs 可能有助于调节角膜上皮细胞的功能。然而，有关泪液 miRNA 表达谱的信息有限，并且泪液 miRNA 对角膜上皮细胞的具体功能仍不清楚。为了研究泪液 miRNA 的作用，我们确定了在泪液中高度表达的 miRNA，并研究了 miRNA 对角膜上皮细胞活力的影响。
从猴泪和血清中提取 miRNA，进行微阵列分析。选择在泪液中表达水平高于血清的 miRNA，并通过定量聚合酶链反应（qPCR）定量其表达水平。为了研究 miRNA 的功能，将 miRNA 模拟物和抑制剂转染入人角膜上皮（HCE-T）细胞中，并孵育 24 或 48 小时。转染 miRNA 模拟物和抑制剂后，通过水溶性四唑盐（WST）测定法测量 HCE-T 细胞的活力，并使用从 HCE-T 细胞中提取的总 RNA 进行微阵列分析和 qPCR。将候选 miR-203 靶点的 siRNA 转染入 HCE-T 细胞中，并进行 WST 测定。为了确定 miR-203 的直接靶基因，在 HCE-T 细胞中使用包含人 IGFBP5 3'-UTR 的荧光素酶报告质粒进行双荧光素酶报告基因检测。
微阵列和 qPCR 分析显示，miR-184 和 miR-203 在泪液中的表达水平明显高于血清（分别为 165542.8 倍和 567.8 倍，p<0.05）。在这两种 miRNA 中，miR-203 模拟物的转染显著降低了 HCE-T 细胞的活力（p<0.05），而 miR-203 抑制剂则显著增加了这种活力（p<0.05）。miR-203 模拟物下调胰岛素样生长因子结合蛋白 5（IGFBP5）和核结合蛋白激酶和细胞周期蛋白依赖性激酶底物 1（NUCKS1），而 miR-203 抑制剂则上调这两个基因。IGFBP5-siRNA 的转染降低了 HCE-T 细胞的活力。miR-203 模拟物显著降低了荧光素酶报告基因的活性。
在这项研究中，我们鉴定了在泪液中高度表达的 miRNAs，并且抑制 miR-203 增加了角膜上皮细胞的活力。我们的结果表明，miR-203 有助于调节角膜上皮细胞的稳态。
