【摘要】 目的 探讨miR-224-5P在糖尿病视网膜病变（DR）患者中的表达变化及其在高糖诱导的人视网膜色素上皮细胞（ARPE-19）中的作用和机制。方法 抽取DR患者及健康人群各6名的外周血，RT-qPCR检测miR-224-5P相对表达量。将正常培养的ARPE-19细胞随机分成高糖组(给予30 mmol·L^-1高糖)、高糖+miR-224-5P mimics组(转染100 nmol·L^-1miR-224-5P mimics后给予30 mmol·L^-1高糖)、高糖+miR-224-5P inhibitor组(转染100 nmol·L^-1miR-224-5P inhibitor后给予30 mmol·L^-1高糖)、高糖+OE-IL6ST组(转染100 nmol·L^-1OE-IL6ST后给予30 mmol·L^-1高糖)和高糖+miR-224-5P mimics+OE-IL6ST组(转染100 nmol·L^-1miR-224-5P mimics和100 nmol·L^-1 OE-IL6ST后给予30 mmol·L^-1高糖),CCK-8检测细胞活力，Transwell检测细胞迁移率，双荧光素酶实验验证miR-224-5P与IL6ST的靶向关系，Western blot检测IL6ST、P-JAK及P-STAT蛋白表达，ELISA检测细胞中IL-1β、 IL-6及TNF-α蛋白表达水平。通过过表达IL6ST进一步验证miR-224-5P是否通过IL6ST/JAK/STAT通路发挥作用。结果 DR患者外周血中miR-224-5P mRNA相对表达量显著低于健康人群（P<0.001）。与高糖组相比，高糖+miR-224-5P mimics组细胞活力、细胞迁移率增高，高糖+miR-224-5P inhibitor组细胞活力、细胞迁移率降低（均为P<0.001）。双荧光素酶实验检测结果证实IL6ST是miR-224-5P调控的靶基因。与高糖组相比，高糖+miR-224-5P mimics组细胞的IL6ST蛋白表达水平降低，P-JAK和P-STAT蛋白表达水平升高（均为P<0.05）;高糖+miR-224-5P inhibitor组细胞的IL6ST蛋白表达水平升高（P<0.05）,P-JAK和P-STAT蛋白表达水平降低（均为P<0.05）。与高糖组相比，高糖+miR-224-5P mimics组细胞的IL-1β、IL-6及TNF-α蛋白表达水平均下降（均为P<0.01）,高糖+miR-224-5P inhibitor组细胞的IL-1β、 IL-6及TNF-α蛋白表达水平均显著上升（均为P<0.01）。高糖+miR-224-5P mimics+OE-IL6ST组细胞活力和细胞迁移率均较高糖+miR-224-5P mimics组显著降低（均为P<0.001）。与高糖+OE-IL6ST组相比，高糖+miR-224-5P mimics+OE-IL6ST组细胞的P-JAK和P-STAT蛋白表达水平均升高（均为P<0.001）;与高糖+miR-224-5P mimics组相比，高糖+miR-224-5P mimics+OE-IL6ST组细胞的P-JAK和P-STAT蛋白表达水平均降低（均为P<0.001）。结论 miR-224-5P在DR患者血液中表达降低，过表达miR-224-5P可降低高糖引起的炎症因子表达，并可通过IL6ST/JAK/STAT信号通路增加高糖诱导的ARPE-19细胞活力及细胞迁移率，从而调控DR的发生和发展。更多还原

【Abstract】 Objective To investigate the expression changes of micro-ribonucleic acid-224-5P（miR-224-5P） in diabetes retinopathy（DR） patients and its effect and mechanism in high glucose-induced adult retinal pigment epithelial cell-19（ARPE-19 cells）. Methods The peripheral blood was extracted from 6 DR patients and healthy people, respectively. Reverse transcription-quantitative polymerase chain reaction was used to detect the relative expression of miR-224-5P. The normally cultured ARPE-19 cells were randomly divided into the high glucose group(administered with 30 mmol·L^-1 high glucose), high glucose+miR-224-5P mimics group(administered with 30 mmol·L^-1 high glucose after transfection with 100 nmol·L^-1 miR-224-5P mimics), high glucose+miR-224-5P inhibitor group(administered with 30 mmol·L^-1 high glucose after transfection with 100 nmol·L^-1 miR-224-5P inhibitor), high glucose+OE-IL6ST group(administered with 30 mmol·L^-1 high glucose after transfection with 100 nmol·L^-1 OE-IL6ST), and high glucose+miR-224-5P mimics+OE-IL6ST group(administered with 30 mmol·L^-1 high glucose after transfection with 100 nmol·L^-1 miR-224-5P mimics and 100 nmol·L^-1 OE-IL6ST). Cell viability was analyzed by Cell Counting Kit-8; cell migration rate was detected by Transwell; targeting relationship between miR-224-5P and interleukin-6 signal transducer（IL6ST） was verified by dual-luciferase assay; Western blot was used to determine the protein expressions of IL6ST, phosphorylated Janus-activated kinase（P-JAK） and phosphorylated signal transducer and activator of transcription（P-STAT）; the protein expressions of interleukin（IL）-1β, IL-6 and tumor necrosis factor-α（TNF-α） were detected by enzyme-linked immunosorbent assay. Additionally, the IL6ST was overexpressed to confirm whether miR-224-5P functioned through IL6ST/JAK/STAT signaling pathway. Results The relative messenger ribonucleic acid（mRNA） expression of miR-224-5P in the peripheral blood of DR patients was lower than the healthy people（P<0.001）. Compared with the high glucose group, the viability and migration rate of cells increased in the high glucose+miR-224-5P mimics group, and decreased in the high glucose+miR-224-5P inhibitor group（all P<0.001）. The results of the dual-luciferase assay confirmed that IL6ST was the target gene regulated by miR-224-5P. Compared with the high glucose group, the protein expression of IL6ST decreased and protein expressions of P-JAK, P-STAT increased（all P<0.05） in the high glucose+miR-224-5P mimics group; the protein expression of IL6ST increased and protein expressions of P-JAK, P-STAT decreased（all P<0.05） in the high glucose+miR-224-5P inhibitor group. Compared with the high glucose group, the protein expressions of IL-1β, IL-6 and TNF-α decreased in the high glucose+miR-224-5P mimics group（all P<0.01）, while significantly increased in the high glucose+miR-224-5P inhibitor group（all P<0.01）. Compared with the high glucose+miR-224-5P mimics group, the viability and cell migration rate of cells decreased in the high glucose+miR-224-5P mimics+OE-IL6ST group（both P<0.001）. Compared with the high glucose+OE-IL6ST group, the protein expressions of P-JAK and P-STAT increased in the high glucose+miR-224-5P mimics+OE-IL6ST group（both P<0.001）. Compared with the high glucose+miR-224-5P mimics group, the protein expressions of P-JAK and P-STAT decreased in the high glucose+miR-224-5P mimics+OE-IL6ST group（both P<0.001）. Conclusion The expression of miR-224-5P is lower in DR patients, and the expression of high glucose-induced inflammatory factors can be reduced by overexpression of miR-224-5P. The viability and migration rate of ARPE-19 cells induced by high glucose can be increased by regulating IL6ST/JAK/STAT signaling pathway so as to affect the occurrence and development of DR.更多还原