微RNA-140-5p靶向调节血管内皮生长因子表达对高糖诱导的人视网膜血管内皮细胞增殖、迁移和管腔形成的影响

Effect of microRNA-140-5p targeted regulating vascular endothelial growth factor expression on proliferation,migration and lumen formation of human retinal vascular endothelial cells induced by high glucose

目的 探讨微RNA(miR)-140-5p靶向调节血管内皮生长因子(VEGF)表达对高糖诱导的人视网膜血管内皮细胞(hRECs)增殖、迁移和管腔形成的影响。方法 将对数生长期hRECs细胞分为正常对照组、高糖组、miR-140-5p组、阴性对照(NC)组、高糖+miR-140-5p组。正常对照组和高糖组细胞不做任何转染,miR-140-5p组和高糖+miR-140-5p组细胞转染miR-140-5p模拟物(miR-140-5p mimics),NC组细胞转染阴性对照模拟物(mimics NC)。高糖组和高糖+miR-140-5p组细胞用含30 mmol·L^(-1)葡萄糖的培养基制备高糖模型。应用实时荧光定量聚合酶链式反应法检测各组细胞中miR-140-5p表达水平,噻唑蓝法检测各组细胞增殖能力,Transwell法检测各组细胞迁移能力,管腔形成实验检测各组细胞成管能力,Western blot法检测各组细胞中VEGF蛋白的表达。将40只Sprague Dawley大鼠随机分成正常对照组、糖尿病模型组、糖尿病+NC组、糖尿病+miR-140-5p组,每组10只。除正常对照组外,其余各组大鼠腹腔注射65 mg·kg^(-1)链脲佐菌素制备糖尿病模型,正常对照组和糖尿病模型组大鼠尾静脉注射200μL生理盐水,糖尿病+miR-140-5p组大鼠尾静脉注射200μL miR-140-5p,糖尿病+NC组大鼠尾静脉注射200μL mimics NC,每日1次,持续给药7 d;再继续饲养8周,大鼠麻醉后,取视网膜,苏木精-伊红染色观察miR-140-5p对糖尿病模型大鼠视网膜血管生成的影响。结果 高糖组、高糖+miR-140-5p组细胞中miR-140-5p相对表达量显著低于正常对照组,miR-140-5p组细胞中miR-140-5p相对表达量显著高于正常对照组(P<0.01);NC组与正常对照组细胞中miR-140-5p相对表达量比较差异无统计学意义(P>0.05)。miR-140-5p组、NC组、高糖+miR-140-5p组细胞中miR-140-5p相对表达量显著高于高糖组(P<0.01)。NC组、高糖+miR-140-5p组细胞中miR-140-5p相对表达量显著低于miR-140-5p组(P<0.01)。高糖+miR-140-5p组细胞中miR-140-5p相对表达量显著低于NC组(P<0.05)。培养24、48、72 h时,高糖组细胞增殖率均显著高于正常对照组(P<0.01),miR-140-5p组、NC组与正常对照组细胞增殖率比较差异均无统计学意义(P>0.05)。培养24 h时,高糖+miR-140-5p组与正常对照组细胞增殖率比较差异无统计学意义(P>0.05);培养48、72 h时,高糖+miR-140-5p组细胞增殖率显著高于正常对照组(P<0.01)。培养24、48、72 h时,miR-140-5p组、NC组、高糖+miR-140-5p组细胞增殖率均显著低于高糖组(P<0.05)。培养24、48、72 h时,NC组与miR-140-5p组细胞增殖率比较差异均无统计学意义(P>0.05)。培养24 h时,高糖+miR-140-5p组细胞增殖率与miR-140-5p组、NC组比较差异无统计学意义(P>0.05);培养48、72 h时,高糖+miR-140-5p组细胞增殖率显著高于miR-140-5p组和NC组(P<0.01)。高糖组迁移细胞数显著高于正常对照组,miR-140-5p组、NC组迁移细胞数显著低于正常对照组(P<0.01)。高糖+miR-140-5p组与正常对照组迁移细胞数比较差异无统计学意义(P>0.05)。miR-140-5p组、NC组、高糖+miR-140-5p组迁移细胞数显著低于高糖组(P<0.01)。NC组、高糖+miR-140-5p组迁移细胞数均显著高于miR-140-5p组(P<0.01)。高糖+miR-140-5p组迁移细胞数显著高于NC组(P<0.01)。高糖组管腔形成数量显著高于正常对照组,miR-140-5p组、NC组细胞管腔形成数量显著低于正常对照组(P<0.01);高糖+miR-140-5p组与正常对照组细胞管腔形成数量比较差异无统计学意义(P>0.05)。miR-140-5p组、NC组、高糖+miR-140-5p组细胞管腔形成数量均显著低于高糖组(P<0.01)。NC组、高糖+miR-140-5p组细胞管腔形成数量均显著高于miR-140-5p组(P<0.05)。高糖+miR-140-5p组细胞管腔形成数量显著高于NC组(P<0.05)。高糖组、高糖+miR-140-5p组细胞中VEGF蛋白相对表达量显著高于正常对照组,miR-140-5p组细胞中VEGF蛋白相对表达量显著低于正常对照组(P<0.05);NC组与正常对照组细胞中VEGF蛋白相对表达量比较差异无统计学意义(P>0.05)。miR-140-5p组、NC组、高糖+miR-140-5p组细胞中VEGF蛋白相对表达量显著低于高糖组(P<0.01)。NC组、高糖+miR-140-5p组细胞中VEGF蛋白相对表达量显著高于miR-140-5p组(P<0.01)。高糖+miR-140-5p组细胞中VEGF蛋白相对表达量显著高于NC组(P<0.05)。正常对照组大鼠靠近视乳头处的视网膜血管整体呈“树状结构”,内极部毛细血管分布较致密,外极部毛细血管分布较疏松,血管壁均匀。糖尿病模型组大鼠视网膜血管的“树状结构”被破坏,毛细血管分布不均匀,毛细血管瘤形成。糖尿病+NC组大鼠视网膜血管损伤与糖尿病模型组相当。糖尿病+miR-140-5p组大鼠视网膜血管的损伤情况有所好转,毛细血管瘤减少。结论 miR-140-5p可以抑制hRECs在高糖条件下的增殖、迁移及成管能力,缓解糖尿病大鼠视网膜血管病变,其作用可能是通过miR-140-5p靶向调控VEGF的表达而实现。

Objective To investigate the effect of microRNA(miR)-140-5p targeted regulating vascular endothelial growth factor expression on proliferation,migration and lumen formation of human retinal vascular endothelial cells(hRECs)induced by the high glucose.Methods The hRECs at logarithmic growth phase were divided into the normal control group,high glucose group,miR-140-5p group,negative control(NC)group and high glucose+miR-140-5p group.The cells in the normal control group and high glucose group did not recieve any transfection.The cells in the miR-140-5p group and high glucose+miR-140-5p group were transfected with miR-140-5p mimics.The cells in the NC group were transfected with negative control mimics(mimics NC).The cells in high glucose group and high glucose+miR-140-5p group were used to prepare high glucose models by using a culture medium containing 30 mmol·L^(-1) glucose.The expression level of miR-140-5p in cells in each group was detected by real time fluorescence quantitative polymerase chain reaction.The proliferation ability of cells in each group was detected by thiazole blue method.The migration ability of cells in each group was detected by Transwell method.The tubular ability of cells in each group was detected by lumen formation experiment.The expression of VEGF in the cells in each group was detected by Western blot method.Forty Sprague Dawley rats were randomly divided into the normal control group,diabetes model group,diabetes+NC group,diabetes+miR-140-5p group,with 10 rats in each group.Except for the normal control group,rats in the other groups were intraperitoneally injected with 65 mg·kg^(-1) streptozotocin to prepare the diabetes model,and rats in the normal control group and the diabetes model group were injected with 200μL normal saline through the tail vein,the rats in the diabetes+miR-140-5p group were injected with 200μL miR-140-5p through the tail vein,the rats in the diabetes+NC group were injected with 200μL mimics NC,once a day for 7 days,and then continued to be fed for 8 weeks.After anesthesia,the retina of rats was taken and stained with hematoxylin-eosin to observe the effect of miR-140-5p on retinal angiogenesis in diabetes model rats.Results The relative expression of miR-140-5p in the cells in the high glucose group and the high glucose+miR-140-5p group was significantly lower than that in the normal control group,while the relative expression of miR-140-5p in the cells in the miR-140-5p group was significantly higher than that in the normal control group(P<0.01);there was no significant difference in the relative expression of miR-140-5p between the NC group and the normal control group(P>0.05);the relative expression of miR-140-5p in the cells in the miR-140-5p group,NC group and high glucose+miR-140-5p group was significantly higher than that in the high glucose group(P<0.01);the relative expression of miR-140-5p in the cells in the NC group and high glucose+miR-140-5p group was significantly lower than that in the miR-140-5p group(P<0.01);the relative expression of miR-140-5p in the cells in the high glucose+miR-140-5p group was significantly lower than that in the NC group(P<0.05).At 24,48 and 72 hours of cultivation,the cell proliferation rate of cells in the high glucose group was significantly higher than that in the normal control group(P<0.01);there was no significant difference in cell proliferation rate between the miR-140-5p group,NC group and the normal control group(P>0.05);at 24 hours of cultivation,there was no significant difference in cell proliferation rate between the high glucose+miR-140-5p group and the normal control group(P>0.05);at 48 and 72 hours of cultivation,the cell proliferation rate in the high glucose+miR-140-5p group was significantly higher than that in the normal control group(P<0.01);at 24,48 and 72 hours of cultivation,the cell proliferation rates in the miR-140-5p group,NC group,and high glucose+miR-140-5p group were significantly lower than those in the high glucose group(P<0.05);at 24,48 and 72 hours of cultivation,there was no significant difference in cell proliferation rate between the NC group and the miR-140-5p group(P>0.05);at 24 hours of cultivation,there was no significant difference in cell proliferation rate between the high glucose+miR-140-5p group and the miR-140-5p group,NC group(P>0.05);at 48 and 72 hours of cultivation,the cell proliferation rate in the high glucose+miR-140-5p group was significantly lower than that in the miR-140-5p group and NC group(P<0.01).The number of migrated cells in the high glucose group was significantly higher than that in the normal control group,while the number of migrated cells in the miR-140-5p group and NC group was significantly lower than that in the normal control group(P<0.01);there was no significant difference in the number of migrated cells between the high glucose+miR-140-5p group and the normal control group(P>0.05);the number of migrated cells in the miR-140-5p group,NC group and high glucose+miR-140-5p group was significantly lower than that in the high glucose group(P<0.01);the number of migrated cells in the NC group and the high glucose+miR-140-5p group was significantly higher than that in the miR-140-5p group(P<0.01);the number of migrated cells in the high glucose+miR-140-5p group was significantly higher than that in the NC group(P<0.01).The number of lumen formation in the high glucose group was significantly higher than that in the normal control group,while the number of lumen formation in the miR-140-5p group and NC group was significantly lower than that in the normal control group(P<0.01);there was no significant difference in the number of cell lumen formation between the high glucose+miR-140-5p group and the normal control group(P>0.05);the number of cell lumen formation in the miR-140-5p group,NC group,and high glucose+miR-140-5p group was significantly lower than that in the high glucose group(P<0.01);the number of cell lumen formation in the NC group and the high glucose+miR-140-5p group was significantly higher than that in the miR-140-5p group(P<0.05);the number of cell lumen formation in the high glucose+miR-140-5p group was significantly higher than that in the NC group(P<0.05).The relative expression of VEGF protein in the cells in the high glucose group and the high glucose+miR-140-5p group was significantly higher than that in the normal control group,while the relative expression of VEGF protein in the cells in the miR-140-5p group was significantly lower than that in the normal control group(P<0.05);there was no significant difference in the relative expression of VEGF protein between the NC group and the normal control group(P>0.05);the relative expression of VEGF protein in the cells in the miR-140-5p group,NC group and high glucose+miR-140-5p group was significantly lower than that in the high glucose group(P<0.01);the relative expression of VEGF protein in the cells in the NC group and high glucose+miR-140-5p group was significantly higher than that in the miR-140-5p group(P<0.01);the relative expression of VEGF protein in the cells in the high glucose+miR-140-5p group was significantly higher than that in the NC group(P<0.05).The retinal blood vessels of rats in the normal control group near the myopic nipple were generally in a"tree-like structure";the distribution of capillaries in the inner pole was relatively dense,while the distribution of capillaries in the outer pole was relatively loose,and the blood vessel wall was uniform.The"dendriform structure"of retinal blood vessels of rats in the diabetes model group was destroyed,the distribution of capillaries was uneven,and capillary hemangiomas were formed.The retinal vascular injury of rats in the diabetes+NC group was equivalent to that in the diabetes model group.In the diabetes+miR-140-5p group,the damage of retinal blood vessels of rats was improved,and the capillary hemangioma was reduced.Conclusion The miR-140-5p can inhibit the proliferation,migration and tube formation of hRECs under high glucose conditions,and alleviate retinal angiopathy in diabetes rats.Its mechanism may be through miR-140-5p targeting the regulation of VEGF expression.