转录组学和免疫组织化学分析进展性圆锥角膜显示上皮和Bowman 层中 WNT10A 的改变。

Transcriptomic and Immunohistochemical Analysis of Progressive Keratoconus Reveal Altered WNT10A in Epithelium and Bowman's Layer.

机构信息

The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.

Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany.

出版信息

Invest Ophthalmol Vis Sci. 2021 May 3;62(6):16. doi: 10.1167/iovs.62.6.16.

PURPOSE

To identify global gene expression changes in the corneal epithelium of keratoconus (KC) patients compared to non-KC myopic controls.

METHODS

RNA-sequencing was performed on corneal epithelium samples of five progressive KC and five myopic control patients. Selected results were validated using TaqMan quantitative PCR (qPCR) on 31 additional independent samples, and protein level validation was conducted using western blot analysis on a subset. Immunohistochemistry was performed on tissue microarrays containing cores from over 100 KC and control cases. WNT10A transcript levels in corneal epithelium were correlated with tomographic indicators of KC disease severity in 15 eyes. Additionally, WNT10A was overexpressed in vitro in immortalized corneal epithelial cells.

RESULTS

WNT10A was found to be underexpressed in KC epithelium at the transcript (ratio KC/control = 0.59, P = 0.02 per RNA-sequencing study; ratio = 0.66, P = 0.03 per qPCR) and protein (ratio = 0.07, P = 0.06) levels. Immunohistochemical analysis also indicated WNT10A protein was decreased in Bowman's layer of KC patients. In contrast, WNT10A transcript level positively correlated with increased keratometry (Kmax ρ = 0.57, P = 0.02). Finally, WNT10A positively regulated COL1A1 expression in corneal epithelial cells.

CONCLUSIONS

A specific Wnt ligand, WNT10A, is reduced at the mRNA and protein level in KC epithelium and Bowman's layer. This ligand positively regulates collagen type I expression in corneal epithelial cells. The results suggest that WNT10A expression in the corneal epithelium may play a role in progressive KC.

目的

与非 KC 近视对照相比，鉴定圆锥角膜 (KC) 患者角膜上皮的全局基因表达变化。

方法

对 5 例进行性 KC 和 5 例近视对照患者的角膜上皮样本进行 RNA 测序。使用 TaqMan 定量 PCR (qPCR) 在 31 个额外的独立样本上验证了选定的结果，并在亚组上使用 Western blot 分析验证了蛋白质水平。在包含 100 多个 KC 和对照病例核心的组织微阵列上进行了免疫组织化学分析。在 15 只眼中，角膜上皮中的 WNT10A 转录水平与 KC 疾病严重程度的层析指标相关。此外，在永生化角膜上皮细胞中体外过表达 WNT10A。

结果

发现 WNT10A 在 KC 上皮中的转录水平（比值 KC/对照=0.59，P=0.02；比值=0.66，P=0.03）和蛋白水平（比值=0.07，P=0.06）均表达不足。免疫组织化学分析还表明，KC 患者的 Bowman 层中的 WNT10A 蛋白减少。相比之下，WNT10A 转录水平与角膜曲率（Kmax ρ=0.57，P=0.02）的增加呈正相关。最后，WNT10A 可正向调节角膜上皮细胞中 COL1A1 的表达。