Wang Qiaoling, Li Qian, Wei Ning
Department of Ophthalmology, The Second People's Hospital of Jinan, No.148, Jingyi Road, Jinan, 250000, Shandong Province, China.
Mol Biol Rep. 2025 Jul 16;52(1):714. doi: 10.1007/s11033-025-10810-x.
The repair of corneal epithelial injury is essential to maintain the cornea integrity and transparency, and the molecular regulation mechanism is still unclear. CALR promotes wound healing through a variety of biological effects. Therefore, this study explored effect and mechanism of CALR on corneal epithelial wound healing.
The model of repairing corneal epithelium injury in mice was established, and corneal epithelial tissues were collected from the model group and the control group. oe-CALR or sh-Wnt7a was transfected into HCE-2[50.B1] cells by Lipofectamine 2000 to over-express CALR or knock down Wnt7a in vitro. CALR mRNA expression was detected by RT-qPCR. CCK-8, clone formation assay, cell senescence, flow cytometry, wound healing and Transwell migration assays were used to detect the changes in proliferation, cell senescence, cell cycle and cell migration after transfection. CALR, Wnt7a and β-catenin proteins expression were detected by Western blot. Interaction between CALR and Wnt7a was detected by Co-immunoprecipitation.
CALR expression was increased in mice corneal epithelial injury repair, suggesting that CALR might play vital role in this process. CALR overexpression promoted HCE-2[50.B1] proliferation and migration, inhibited cell senescence of HCE-2[50.B1], and relieved S phase block and increased the number of HCE-2[50] cells in G0/G1 phase. Wnt7a and CALR proteins expression were respectively detected in the protein complexes co-precipitated by anti-CALR antibody and anti-Flag antibody. The interaction between CALR and Wnt7a could activate the downstream β-catenin signaling pathway. Wnt7a knockdown attenuated the effect of CALR overexpression on HCE-2[50.B1] cells proliferation, senescence and migration.
CALR promotes proliferation and migration, inhibited senescence of HCE-2[50.B1] cells by Wnt7a, thus promoting corneal epithelial wound healing. This study will provide a theoretical basis for mechanism of CALR in corneal injury repair, and provide a new target for corneal injury clinical treatment.
角膜上皮损伤的修复对于维持角膜的完整性和透明度至关重要，但其分子调控机制仍不清楚。钙网蛋白（CALR）通过多种生物学效应促进伤口愈合。因此，本研究探讨了CALR对角膜上皮伤口愈合的作用及机制。
建立小鼠角膜上皮损伤修复模型，从模型组和对照组收集角膜上皮组织。通过脂质体2000将过表达CALR的载体（oe-CALR）或敲低Wnt7a的载体（sh-Wnt7a）转染至HCE-2[50.B1]细胞，以在体外过表达CALR或敲低Wnt7a。通过逆转录定量聚合酶链反应（RT-qPCR）检测CALR信使核糖核酸（mRNA）表达。采用细胞计数试剂盒-8（CCK-8）、克隆形成实验、细胞衰老检测、流式细胞术、伤口愈合实验和Transwell迁移实验检测转染后细胞增殖、细胞衰老、细胞周期和细胞迁移的变化。通过蛋白质免疫印迹法检测CALR、Wnt7a和β-连环蛋白的蛋白表达。通过免疫共沉淀检测CALR与Wnt7a之间的相互作用。
在小鼠角膜上皮损伤修复过程中CALR表达增加，提示CALR可能在此过程中发挥重要作用。CALR过表达促进了HCE-2[50.B1]细胞的增殖和迁移，抑制了HCE-2[50.B1]细胞的衰老，并缓解了S期阻滞，增加了处于G0/G1期的HCE-2[50]细胞数量。在抗CALR抗体和抗Flag抗体共沉淀的蛋白复合物中分别检测到Wnt7a和CALR蛋白表达。CALR与Wnt7a之间的相互作用可激活下游β-连环蛋白信号通路。敲低Wnt7a减弱了CALR过表达对HCE-2[50.B1]细胞增殖、衰老和迁移的影响。
CALR通过Wnt7a促进HCE-2[50.B1]细胞的增殖和迁移，抑制其衰老，从而促进角膜上皮伤口愈合。本研究将为CALR在角膜损伤修复中的机制提供理论依据，并为角膜损伤的临床治疗提供新的靶点。
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