摘要
目的探讨miR-30c-2-3p调控X-box结合蛋白-1(X box-binding protein-1,XBP1)对大鼠心肌细胞缺氧/复氧损伤的保护作用。方法制备新生大鼠心肌细胞,随机分为对照组、缺氧/复氧组和miR-30c-2-3p处理组。对照组心肌细胞正常培养,缺氧/复氧组和miR-30c-2-3p处理组心肌细胞进行缺氧3h/复氧6h处理;miR-30c-2-3p处理组心肌细胞于缺氧前48h采用miR-30c-2-3p抑制剂转染慢病毒,对照组和缺氧/复氧组感染慢病毒包装的无义寡核苷酸序列。复氧6h后,采用比色法检测3组心肌细胞乳酸脱氢酶(lactate dehydrogenase,LDH)漏出率,流式细胞术检测正常心肌细胞比率及凋亡水平,实时荧光定量PCR法检测miR-30c-2-3p和XBP1mRNA表达水平,Western blot法检测XBP1蛋白表达情况,双荧光素酶基因检测验证miR-30c-2-3p和XBP1的靶向关系。结果缺氧/复氧组和miR-30c-2-3p处理组心肌细胞LDH漏出率[(28.8±4.9)%、(22.2±3.0)%]、细胞凋亡率[(28.5±3.9)%、(19.2±2.6)%]、miR-30c-2-3p(2.87±0.57、1.57±0.21)、XBP1mRNA(1.68±0.68、3.96±0.69)和XBP1蛋白(1.96±0.40、2.73±0.61)表达均高于对照组[(4.6±1.1)%、(6.5±1.2)%、1、1、1],正常心肌细胞比率[(63.8±4.2)%、(71.6±4.0)%]低于对照组[(91.4±2.4)%](P<0.05);miR-30c-2-3p处理组心肌细胞LDH漏出率、细胞凋亡率及miR-30c-2-3p表达低于缺氧/复氧组,正常心肌细胞比率、XBP1mRNA和XBP1蛋白表达高于缺氧/复氧组(P<0.05);双荧光素酶基因检测结果证实,XBP1为miR-30c-2-3p的靶基因。结论在大鼠心肌细胞缺氧/复氧损伤模型,miR-30c-2-3p、XBP1及XBP1蛋白呈高表达,抑制miR-30c-2-3p可通过上调XBP1表达来减轻心肌细胞缺氧/复氧损伤。
Objective To explore the protective effect of miR-30c-2-3p modulating XBP1 on cardiomyocytes with anoxia/ reoxygenation （AR） injury in rats. Methods The cultured cadiomyocytes in newborn rats were randomly divided into control group, AR group and miR-30c-2-3p group. Control group was conventionally cultured. AR and miR-30c-2-3p groups were treated with anoxia for 3 h and reoxygenation for 6 h. MiR-30e-2-3p inhibitor was transfected with lentivirus in miR-30c-2-3p group 48 h before anoxia, and the other two groups were transfected with non-sense oligonucleotide. After reoxygenation for 6 h, lactate dehydrogenase （LDH） release rate was detected by colorimetric method and eardiomyocyte apoptosis was detected by the dead cell apoptosis kit with Annexin V-APC/7-ADD. The expressions of miR-30c-2-3p and XBP1 mRNA were detected by real-time PCR, and XBP1 protein was detected by Western blot. The dura luciferase report gene detection was done to measure a direct interaction between miR-30c-2-3p and XBP1. Results The LDH release rate, apoptosis rate, and the expression levels of miR-30c-2-3p, XBP1 mRNA and XBP1 protein were significantly higher in AR group （28. 8±4. 9） %, （28. 5±3. 9）%, 2. 87±0. 57, 1. 68±0. 68, 1. 96±0. 40） and miR-30c-2-3p group （（22.2±3.0）%, （19.2±2.6）%, 1.57±0. 21, 3.96±0.69, 2.73±0.61） than those in control group （（4.6±1.1）%, （6. 5±1. 2） % , 1, 1, 1） （P〈0.05）. The LDH release rate, apoptosis rate and miR-30e-2-3p level were significantly lower in miR-30c-2-3p group than those in AR group, and the levels of XBP1 mRNA and XBP1 protein were significantly higher in miR-30e-2-3p group than those in AR group （P（0.05）. The dual luciferase reporter gene system confirmed XBP1 was the target gene of miR-30c-2-3p. Conclusion In the cardiomyocyte AR injury rat models, miR-30c-2-3p, XBP1 mRNA and XBP1 protein are highly expressed, and inhibition of miR-30c-2-3p can alleviate the AR induced injury by up-regulating the expression of XBP1.
作者
李瑞萍
薛富善
杨桂珍
刘亚洋
李慧娴
廖旭
LI Rui-ping , XUE Fu-shan, YANG Gui-zhen, LIU Ya-yang, LI Hui-xian, LIAO Xu(Department of Anesthesiology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, Chin)
出处
《中华实用诊断与治疗杂志》
2018年第3期211-214,共4页
Journal of Chinese Practical Diagnosis and Therapy
基金
国家自然科学基金(81170128)
国家自然科学基金(81470019)
