miR-15a对糖尿病性视网膜病变抗炎和抗新生血管生成的双重调节作用

Anti-inflammatory and Anti-angiogenic Effect of miR-15a on Diabetic Retinopathy

目的探讨miR-15a对糖尿病性视网膜病变抗炎和抗新生血管生成的调节作用。方法通过25mmol/L葡萄糖诱导人视网膜色素上皮细胞株HARPE-19建立高糖模型(HG),并以5mmol/L葡萄糖诱导的HARPE-19细胞作为对照(LG),将HG组和LG组分为4个亚组:miR-15amimic组、NC-mimic组、miR-15ainhibitor组和NC-inhibitor组,MTT增殖实验检测细胞增殖能力,细胞划痕实验检测细胞迁移能力,双荧光素酶报告基因实验验证miR-15a与酸性鞘磷脂酶(ASM)的关系,RT-PCR检测miR-15a、ASM、VEGF、IL-1β、IL-6、TNF-αmRNA表达。结果 NC-mimic细胞和NC-inhibitor细胞中,HG组miR-15a表达均显著低于LG组(均P<0.05)。miR-15amimic组ASM mRNA表达水平显著低于NC-mimic组(P<0.05),miR-15ainhibitor组ASM mRNA表达水平显著高于NC-inhibitor组(P<0.05)。miR-15amimic组细胞迁移距离显著大于NC-mimic组(P<0.05),miR-15ainhibitor组细胞迁移距离显著小于NC-inhibitor组(P<0.05)。转染24~120h,MTT法检测miR-15amimic组吸光度值显著高于NC-mimic组(P<0.05),miR-15ainhibitor组吸光度值显著低于NC-inhibitor组(P<0.05)。NC-mimic细胞和NC-inhibitor细胞中,HG组VEGF、IL-1β、IL-6、TNF-αmRNA表达水平均显著高于LG组(均P<0.05)。结论降低miR-15a表达能够促进视网膜细胞炎症反应和血管新生,miR-15a通过炎症反应和血管生成双重调节作用参与糖尿病性视网膜病变的发生。

Objective To explore the anti-inflammatory and anti-angiogenic effect of miR-15 ain diabetic retinopathy（DR）.Methods High glucose model（HG）was established by treatment with 25mmol/L glucose in human retinal pigment epithelial cell line HARPE-19,and HARPE-19 cells treated with 5mmol/L glucose served as control（LG）.HG group and LG group were divided into 4subgroups：miR-15 amimic group,NC-mimic group,miR-15 ainhibitor group and NC-inhibitor group.Cell proliferation ability was detected by MTT proliferation assay.Cell migration ability was detected by cell scratch assay.The relationship between miR-15 aand ASM was identified by 3′UTR luciferase reporter assay.miR-15 a,ASM,VEGF,IL-1β,IL-6,TNF-αmRNA expression was detected by RT-PCR method.Results In NC-mimic cells and NC-inhibitor cells,the expression of miR-15 a in HG group was significantly lower than that in LG group（all P0.05）.The expression level of ASM mRNA in miR-15 a mimic group was significantly lower than that in NC-mimic group（P0.05）,the expression level of ASM mRNA in miR-15 a inhibitor group was significantly higher than that in NC-inhibitor group（P0.05）.The cell scratch width in miR-15 amimic group was significantly higher than that in NC-mimic group（P0.05）.The cell scratch width in miR-15 ainhibitor group was significantly lower than that in NC-inhibitor group（P0.05）.The absorbance value in miR-15 amimic group was significantly higher than that in NC-mimic group（P0.05）,the absorbance value in miR-15 ainhibitor group was significantly lower than that in NC-inhibitor group（P0.05）.In NC-mimic cells and NC-inhibitor cells,the mRNA expression levels of VEGF,IL-1β,IL-6and TNF-αin HG group was significantly higher than those in LG group（all P0.05）.Conclusion miR-15 adownexpression induces inflammatory reaction and angiogenesis in retinal cells.miR-15 ais involved in the pathogenesis of DR by dual regulation of inflammation and angiogenesis.