miR-221通过靶向CDKN1B调节脓毒症血管内皮细胞细胞凋亡和炎症反应-现代医学

miR-221通过靶向CDKN1B调节脓毒症血管内皮细胞细胞凋亡和炎症反应

单位：青海大学附属医院急诊ICU, 青海西宁 810000

分类号：R459.7

出版年·卷·期（页码）：2021·49·第九期（1063-1069）

摘要：

目的：探讨微小RNA-221（miR-221）对内毒素（LPS）诱导的脓毒症血管内皮细胞损伤的影响及机制。方法：使用100 ng·ml^-1的LPS诱导人脐静脉内皮细胞（HUVECs）构建脓毒症血管内皮细胞损伤模型，通过脂质体转染技术将miR-221 inhibitors转染至LPS诱导的HUVECs，实验分为对照（Con）组、模型（LPS）组、转染对照（LPS+anti-miR-NC）组和转染（LPS+anti-miR-221）组。实时荧光定量PCR （qRT-PCR）和蛋白免疫印迹法（Western blot）检测各组细胞中miR-221和CDKN1B的表达情况，酶联免疫吸附法（ELISA）检测白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）和肿瘤坏死因子α（TNF-α），流式细胞术检测细胞凋亡率，蛋白免疫印迹法（Western blot）分析凋亡相关蛋白B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关的X （Bax）和活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）的表达水平，生物信息学软件TargetScan预测miR-221的靶基因，双荧光素酶报告基因实验验证miR-221和CDKN1B之间的靶向作用关系。同时抑制miR-221和CDKN1B的表达，采用同样的方法检测细胞凋亡和炎症因子表达情况。结果：与Con组相比，LPS组miR-221、Bax和Cleaved Caspase-3的表达明显升高，CDKN1B和Bcl-2的表达明显降低，炎症因子IL-6、IL-1β和TNF-α的含量明显增多，凋亡率明显升高，差异均有统计学意义（P<0.05）；与LPS组和LPS+anti-miR-NC组相比，LPS+anti-miR-221组细胞凋亡率、Bax和Cleaved Caspase-3的表达水平均明显降低，Bcl-2的相对表达水平明显升高，而炎症因子IL-6、IL-1β和TNF-α的含量明显减少，差异均有统计学意义（P<0.05）。双荧光素酶报告基因实验证实了miR-221和CDKN1B存在靶向结合关系。结论：抑制miR-221能够逆转LPS对HUVECs损伤的影响；抑制CDKN1B可逆转抑制miR-221对LPS诱导的HUVECs损伤保护作用；miR-221可通过靶向CDKN1B调控脓毒症血管内皮细胞凋亡和的炎症反应。

Objective: To investigate the effect and mechanism of microRNA-221 (miR-221) on endotoxin (LPS)-induced vascular endothelial cell damage in sepsis. Methods: 100 ng·ml^-1 LPS was used to induce human umbilical vein endothelial cells (HUVECs) to construct a sepsis vascular endothelial cell injury model. The miR-221 inhibitors were transfected into LPS-induced HUVECs by liposome transfection technology. The experiment was divided into control (Con) group, model (LPS) group, transfection control (LPS+anti-miR-NC) group and transfection (LPS+anti-miR-221) group. Real-time fluorescent quantitative PCR (qRT-PCR) and Western blot were used to detect the expression of miR-221 and CDKN1B in each group of cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α). Flow cytometry to detect cell apoptosis rate. Western blot was used to detect the expression of apoptosis-related protein B-cell lymphoma (Bcl-2), Bcl-2 related X (Bax) and activated cysteine-containing aspartate proteolytic enzyme 3 (Cleaved Caspase-3). Bioinformatics software TargetScan was used to predict the target genes of miR-221. The dual-luciferase reporter gene experiment was used to verify the targeting relationship between miR-221 and CDKN1B. Simultaneously inhibit the expression of miR-221 and CDKN1B, and the same methods were used to detect cell apoptosis and inflammatory factor expression. Results: Compared with the Con group, the expressions of miR-221, Bax and Cleaved Caspase-3 in the LPS group were significantly increased, the expressions of CDKN1B and Bcl-2 were significantly reduced, and the inflammatory factors IL-6, IL-1β and TNF-α were significantly increased, the apoptosis rate was significantly increased, and the differences were statistically significant (P<0.05). Compared with the LPS group and the LPS+anti-miR-NC group, the apoptosis rate, the expression level of Bax and Cleaved Caspase-3 in the LPS+anti-miR-221 group were significantly reduced, while the expression level of Bcl-2 was significantly increased. However, the content of inflammatory factors IL-6, IL-1β and TNF-α were significantly reduced, the differences were statistically significant (P<0.05). The dual luciferase reporter gene experiment confirmed that miR-221 and CDKN1B have a targeted binding relationship. Conclusion: Inhibition of miR-221 can reverse the effect of LPS on the damage of HUVECs, inhibition of CDKN1B can reverse the protective effect of inhibiting miR-221 on LPS-induced HUVECs injury. miR-221 can regulate the apoptosis and inflammation of vascular endothelial cells in sepsis by targeting CDKN1B.

参考文献：

[1] HUANG M, CAI S, SU J.The pathogenesis of sepsis and potential therapeutic targets[J]. Int J Mol Sci, 2019, 20(21):5376.
[2] SUN R, HUANG J, SUN B.Mobilization of endothelial progenitor cells in sepsis[J]. Inflamm Res, 2020, 69(1):1-9.
[3] JOFFRE J, HELLMAN J, INCE C, et al. Endothelial responses in sepsis[J]. Am J Respir Crit Care Med, 2020, 202(3):361-370.
[4] HIRSCHBERGER S, HVBNER M, STRAUß G, et al. Identification of suitable controls for miRNA quantification in T-cells and whole blood cells in sepsis[J]. Sci Rep, 2019, 9(1):15735-15747.
[5] MIRNA M, PAAR V, REZAR R, et al. MicroRNAs in inflammatory heart diseases and sepsis-induced cardiac dysfunction:a potential scope for the future[J]. Cells, 2019, 8(11):1352-1363.
[6] SHU Z, TAN J, MIAO Y, et al. The role of microvesicles containing microRNAs in vascular endothelial dysfunction[J]. J Cell Mol Med, 2019, 23(12):7933-7945.
[7] CHEN D, WANG X, HUANG J, et al. CDKN1B mediates apoptosis of neuronal cells and inflammation induced by oxyhemoglobin via miR-502-5p after subarachnoid hemorrhage[J]. J Mol Neurosci, 2020, 70(7):1073-1080.
[8] CHUROV A, SUMMERHILL V, GRECHKO A, et al. MicroRNAs as potential biomarkers in atherosclerosis[J]. Int J Mol Sci, 2019, 20(22):5547-5567.
[9] MENG Y, CAI X H, WANG L.Potential genes and pathways of neonatal sepsis based on functional gene set enrichment analyses[J]. Comput Math Methods Med, 2018, 2018:6708520-6708529.
[10] XU Y, XUE Y, LIU X, et al. Ferumoxytol attenuates the function of MDSCs to ameliorate LPS-induced immunosuppression in sepsis[J]. Nanoscale Res Lett, 2019, 14(1):379-389.
[11] WANG C, ZHU Z.MiR-499a suppresses LPS-induced human vascular endothelial cell inflammatory response and apoptosis by regulating STAT1[J]. Int J Clin Exp Pathol, 2019, 12(11):4232-4241.
[12] RUBIO I, OSUCHOWSKI M F, SHANKAR-HARI M, et al. Current gaps in sepsis immunology:new opportunities for translational research[J]. Lancet Infect Dis, 2019, 19(12):e422-e436.
[13] GUO H, TANG L, XU J, et al. MicroRNA-495 serves as a diagnostic biomarker in patients with sepsis and regulates sepsis-induced inflammation and cardiac dysfunction[J]. Eur J Med Res, 2019, 24(1):37-45.
[14] BINAS S, KNYRIM M, HUPFELD J, et al. miR-221 and-222 target CACNA1C and KCNJ5 leading to altered cardiac ion channel expression and current density[J]. Cell Mol Life Sci, 2020, 77(5):903-918.
[15] SONG Q, AN Q, NIU B, et al. Role of miR-221/222 in tumor development and the underlying mechanism[J]. J Oncol, 2019, 2019:7252013-7252027.
[16] TALEPOOR A G, KALANI M, DAHAGHANI A S, et al. Hydrogen peroxide and lipopolysaccharide differentially affect the expression of MicroRNAs 10a, 33a, 21, 221 in endothelial cells before and after coculture with monocytes[J]. Int J Toxicol, 2017, 36(2):133-141.
[17] WANG T, JIANG L, WEI X, et al. Inhibition of miR-221 alleviates LPS-induced acute lung injury via inactivation of SOCS1/NF-κB signaling pathway[J]. Cell Cycle, 2019, 18(16):1893-1907.
[18] ZHU L, GONG X, GONG J, et al. Notoginsenoside R1 upregulates miR-221-3p expression to alleviate ox-LDL-induced apoptosis, inflammation, and oxidative stress by inhibiting the TLR4/NF-κB pathway in HUVECs[J]. Braz J Med Biol Res, 2020, 53(6):e9346-e9353.
[19] STAVAST C J, ERKELAND S J.The non-canonical aspects of MicroRNAs:many roads to gene regulation[J]. Cells, 2019, 8(11):1465-1484.
[20] HU L, YE H, LIAO J.LncRNA TUG1 reverses LPS-induced cell apoptosis and inflammation of macrophage via targeting MiR-221-3p/SPRED2 axis[J]. Biosci Biotechnol Biochem, 2020, 84(12):2458-2465.
[21] CHEN D, WANG X, HUANG J, et al. CDKN1B mediates apoptosis of neuronal cells and inflammation induced by oxyhemoglobin via miR-502-5p after subarachnoid hemorrhage[J]. J Mol Neurosci, 2020, 70(7):1073-1080.

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