[1]赵鑫,杨静,辛向阳.基于miR-224-5P调控的IL6ST/JAK/STAT信号通路在糖尿病视网膜病变发生发展中的作用[J].眼科新进展,2023,43(5):357-362.[doi:10.13389/j.cnki.rao.2023.0072]
　ZHAO Xin,YANG Jing,XIN Xiangyang.Effect of micro-ribonucleic acid-224-5P in the occurrence and development of diabetes retinopathy via regulating interleukin-6 signal transducer/Janus-activated kinase/signal transducer and activator of transcription signaling pathway[J].Recent Advances in Ophthalmology,2023,43(5):357-362.[doi:10.13389/j.cnki.rao.2023.0072]
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基于miR-224-5P调控的IL6ST/JAK/STAT信号通路在糖尿病视网膜病变发生发展中的作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
43卷
期数:
2023年5期
页码:
357-362
栏目:
实验研究
出版日期:
2023-05-05

文章信息/Info

Title:
Effect of micro-ribonucleic acid-224-5P in the occurrence and development of diabetes retinopathy via regulating interleukin-6 signal transducer/Janus-activated kinase/signal transducer and activator of transcription signaling pathway
作者:
赵鑫; 杨静; 辛向阳
014040　内蒙古自治区包头市，内蒙古科技大学包头医学院基础医学与法医学院（赵鑫，杨静）；014010　内蒙古自治区包头市，内蒙古包钢医院眼科（赵鑫，辛向阳）
Author(s):
ZHAO Xin1; 2; YANG Jing1; XIN Xiangyang2
1.School of Basic Medicine and Forensic Medicine，Inner Mongolia University of Science and Technology Baotou Medical College，Baotou 014040，Inner Mongolia，China
2.Department of Ophthalmology，Inner Mongolia Baogang Hospital，Baotou 014010，Inner Mongolia，China
关键词:
糖尿病视网膜病变; miR-224-5P; IL6ST/JAK/STAT通路
Keywords:
diabetic retinopathy;  micro-ribonucleic acid-224-5P;  interleukin-6 signal transducer/Janus-activated kinase/signal transducer and activator of transcription signaling pathway
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2023.0072
文献标志码:
A
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。
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.

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备注/Memo

备注/Memo:
内蒙古自治区卫生健康科技计划项目（编号：202201522）
更新日期/Last Update: 2023-05-05