MiR-16-5p靶向TXNIP调控脂多糖诱导的心肌细胞的氧化应激和凋亡

MiR-16-5p Targets TXNIP to Regulate LPS-Induced Oxidative Stress and Apoptosis in Cardiomyocytes

心肌细胞损伤与多种心血管疾病的发生发展有关,而氧化应激和细胞凋亡是造成心肌损伤的重要原因。硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)可调控细胞氧化还原状态,并诱导氧化应激的产生,最终可诱导炎症或细胞凋亡。miR-16-5p能抑制脂多糖(lipopolysaccharide,LPS)诱导的炎症反应和A549细胞损伤。但TXNIP是否受miR-16-5p的调控还鲜有报道。本实验旨在研究miR-16-5p与TXNIP的关系,以及miR-16-5p是否通过结合TXNIP影响心肌细胞氧化应激和凋亡。通过TargetScan数据库预测到TXNIP与miR-16-5p存在结合位点。双荧光素酶报告结果验证miR-16-5p靶向调控TXNIP。转染miR-16-5p过表达载体和TXNIP抑制表达载体后,用流式细胞术检测细胞凋亡率。结果显示,细胞凋亡率(10.44±1.13 vs 29.65±2.87,P<0.01)、(13.54±1.33 vs 29.14±2.76,P<0.01)均降低。采用丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)试剂盒分别检测丙二醛含量和SOD、GSH-Px活力。结果显示,miR-16-5p组丙二醛含量降低(13.58±1.55 vs 42.18±4.69,P<0.01),SOD活力(79.68±7.65 vs 34.87±3.49,P<0.01)、GSH-Px活力(687.99±35.42 vs 376.48±29.87,P<0.01)升高;si-TXNIP组丙二醛含量(18.69±1.84 vs 45.21±4.33,P<0.01)降低,SOD活力(71.65±7.32 vs 31.69±4.05,P<0.01)、GSH-Px活力(654.12±34.57 vs 367.43±33.54,P<0.01)升高。以上结果表明,过表达miR-16-5p或可抑制TXNIP表达,均可抑制脂多糖诱导的心肌细胞凋亡和氧化应激反应。综上所述,miR-16-5p可通过抑制TXNIP表达抑制脂多糖诱导的心肌细胞损伤。

Myocardial cell injury is related to the occurrence and development of various cardiovascular diseases, and oxidative stress and apoptosis are important causes of myocardial injury. Thioredoxin-interacting protein(TXNIP) regulates the cell redox state and induces oxidative stress, inflammation or apoptosis. miR-16-5 p inhibits lipopolysaccharide(LPS)-induced inflammatory response and cell damage in A549 cells. However, there are few reports on whether TXNIP is regulated by miR-16-5 p. The purpose of this study is to investigate the relationship between miR-16-5 p and TXNIP and whether miR-16-5 p affects oxidative stress and apoptosis in cardiac myocytes by binding to TXNIP. The TargetScan database predicted the existence of binding sites between TXNIP and miR-16-5 p, and double luciferase reporting experiments verified that miR-16-5 p targets and regulates TXNIP. After transfection of miR-16-5 p overexpression and TXNIP inhibitory expression plamids, the apoptotic rate was detected by flow cytometry. The results showed that the apoptotic rate(10.44 ± 1.13 vs. 29.65 ± 2.87, P<0.01),(13.54 ± 1.33 vs. 29.14 ± 2.76, P<0.01) were all reduced. Malondialdehyde(MDA), superoxide dismutase(SOD), and glutathione peroxidase(GSH-Px) kits was used to detect the MDA contents and SOD, GSH-Px activity, respectively. The results showed that the MDA content was decreased(13.58 ± 1.55 vs. 42.18 ± 4.69, P<0.01), SOD activity(79.68 ± 7.65 vs. 34.87 ± 3.49, P<0.01), GSH-Px activity(687.99 ± 35.42 vs. 376.48 ± 29.87, P<0.01) increased in the miR-16-5 p group;the MDA content(18.69 ± 1.84 vs. 45.21 ± 4.33, P<0.01) was decreased, SOD activity(71.65 ± 7.32 vs. 31.69 ± 4.05, P<0.01), and GSH-Px activity(654.12 ± 34.57 vs. 367.43 ± 33.54, P <0.01) was increased in the si-TXNIP group. The results above indicated that overexpression of miR-16-5 p or inhibition of TXNIP expression could inhibit the LPS-induced cardiomyocyte apoptosis and oxidative stress response. In summary, miR-16-5 p could inhibit LPS-induced cardiomyocyte injury by inhibiting TXNIP expression.