microRNA-203通过靶向SRC抑制人角膜上皮细胞增殖与迁移  

microRNA-203 Inhibits Proliferation and Migration of Human Corneal Epithelial Cells by Targeting SRC

作　　者：徐微微 罗广营 XU Wei-Wei;LUO Guang-Ying(State Key Laboratory of Optometry and Visual Science,Department of Optometry,Wenzhou Medical University,Wenzhou 325027,Zhejiang,China)

机构地区：[1]温州医科大学附属眼视光医院,省部共建眼视光学和视觉科学国家重点实验室,浙江温州325027

出　　处：《中国生物化学与分子生物学报》2021年第7期900-907,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：浙江省自然科学基金(No.LY21H120005);温州市科研项目(No.Y20190633)资助。

摘　　要：角膜上皮损伤修复对于维持角膜的完整性和透明度至关重要,其分子调控机制尚未明了。本室前期研究发现,miR-203在小鼠角膜上皮损伤修复中显著下调,提示miR-203在该进程中发挥重要作用。本研究首先通过小鼠在体实验,证实miR-203在角膜上皮损伤修复中的急剧下调。体外实验将miR-203转染入人角膜上皮细胞(human corneal epithelial cells, HCECs),使miR-203过表达。应用MTS法、EdU检测、流式细胞术、划痕实验和小孔迁移实验检测转染后细胞增殖、细胞周期及细胞迁移的变化情况。结果发现,过表达miR-203能抑制HCECs增殖(P<0.01),其细胞周期各时相比例中G1期上调了10.63%,并能诱导人角膜上皮细胞发生凋亡(P<0.001),划痕实验及小孔迁移检测均证实,过表达miR-203能抑制HCECs迁移。通过生物信息学预测及Western印迹验证SRC原癌基因(SRC proto-oncogene)是miR-203作用的靶基因。进一步在HCECs中敲减SRC,发现人角膜上皮细胞增殖速率放缓(P<0.001)。与阴性对照组相比,SRC敲减组细胞G1期上调了17.19%,且凋亡率增加(P<0.01)。划痕实验及小孔迁移实验也证实,降低SRC表达能抑制HCECs迁移。该研究表明,miR-203可能通过下调靶基因SRC的表达,进而影响HCECs的增殖及迁移。本研究将为miR-203在角膜损伤修复过程中的作用机制提供理论依据,并为临床治疗角膜损伤提供新靶点。Corneal epithelial wound healing(CEWH) is vital for maintaining tissue integrity and transparency, but its detailed molecular mechanism is unclear. Previous our study has shown that the expression of miR-203 is dramatically down-regulated during CEWH. Such changes suggest that miR-203 is an important effector of CEWH. In this study, we confirmed down-regulation of miR-203 during CEWH in mice. In vitro experiments, we transfected miR-203 into human corneal epithelial cells(HCECs) to overexpress miR-203. The MTS method, EdU detection, flow cytometry, scratch tests and transwell experiments were used to detect changes in cell proliferation, cell cycle, and cell migration after transfection. As a result, it was found that miR-203 can inhibit the proliferation of HCECs(P<0.01). The cell cycle at the G1 phase of HCECs was up-regulated by 10.63% compared to the negative control group, and cell apoptosis was increased(P<0.01). Scratch experiments and transwell experiments have confirmed that overexpression of miR-203 can inhibit the migration of HCECs. Bioinformatics prediction and Western blot identified that SRC(SRC proto-oncogene) was a target gene of miR-203. The proliferation rate of HCECs was slowed down when the expression of SRC was knocked down(P<0.001). Compared with the negative control group, the G1 phase of the SRC knockdown group was up-regulated by 17.19%, and the apoptosis rate was increased(P<0.01). Scratch experiments and transwell experiments also confirmed that reducing the expression of SRC can inhibit the migration of HCECs. This study shows that miR-203 may affect HCECs proliferation and migration by down-regulating the expression of SRC, which will provide novel insight regarding the role played by miR-203 in mediating CEWH and identify more selective drug targets for the treatment of patients with damaged corneas.

关 键 词：角膜损伤修复 miR-203 增殖 迁移 

分 类 号：R779.1[医药卫生—眼科]