Zhang Xiaoling, Chen Jian, Yao Min, Liu Xiaoyong, Zhou Qing
Department of Ophthalmology, First Affiliated Hospital, Jinan University, Guangzhou 510632, China.
Department of Ophthalmology, First Affiliated Hospital, Jinan University, Guangzhou 510632, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Oct;34(10):880-884.
Objective To investigate the effect of short hairpin RNA (shRNA) targeting hypoxia-inducible factor-1α (HIF-1α) on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in spontaneously immortalized human corneal epithelial cells (S-ihCECs) under hypoxic condition. Methods Three kinds of shRNAs targeting the sites of HIF-1α mRNA were designed and synthesized, and then transfected into S-ihCECs in vitro. After transfection, the cells were exposed to 1 mmol/L CoCl2 for 6 hours. HIF-1α, VEGF and PEDF mRNA expressions were detected by real-time quantitative PCR. HIF-1α and VEGF protein levels were analyzed by Western blotting. PEDF level was tested by ELISA. Results After the shRNA interference, the expressions of HIF-1α and VEGF mRNA decreased by (70.9±2.8)% and (51.9±15.7)%, respectively when compared with the hypoxia group. The expressions of HIF-1α and VEGF proteins were both down-regulated. The expression of PEDF mRNA increased (1.22±0.18) fold and the protein was up-regulated. Conclusion ShRNA targeting HIF-1α can effectively silence the expression of HIF-1α, reduce the expression of VEGF and up-regulate the expression of PEDF.
目的 探讨靶向缺氧诱导因子-1α（HIF-1α）的短发夹RNA（shRNA）对缺氧条件下永生化人角膜上皮细胞（S-ihCECs）中血管内皮生长因子（VEGF）和色素上皮衍生因子（PEDF）表达的影响。方法 设计并合成3种靶向HIF-1α mRNA位点的shRNA，体外转染S-ihCECs。转染后，细胞暴露于1 mmol/L CoCl2中6小时。采用实时定量PCR检测HIF-1α、VEGF和PEDF mRNA表达。通过蛋白质印迹法分析HIF-1α和VEGF蛋白水平。用酶联免疫吸附测定法检测PEDF水平。结果 shRNA干扰后，与缺氧组相比，HIF-1α和VEGF mRNA表达分别下降（70.9±2.8）%和（51.9±15.7）%。HIF-1α和VEGF蛋白表达均下调。PEDF mRNA表达增加（1.22±0.18）倍，蛋白表达上调。结论 靶向HIF-1α的shRNA可有效沉默HIF-1α表达，降低VEGF表达并上调PEDF表达。
