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目前心血管疾病是世界上发病率和死亡率最高的疾病。急性心肌梗死(AMI)是心血管疾病的常见类型,已对人类健康构成巨大威胁。冠状动脉粥样硬化血管狭窄引起心肌缺血和缺氧,长期缺血甚至导致心肌细胞死亡,最终导致AMI [1-2]。然而,AMI发病机制并未完全阐明。非编码RNA(ncRNA)是一类缺乏蛋白质编码能力的RNA,最初被认为是“垃圾DNA”。近年研究[3]发现,人类基因组约98%为ncRNA,长链非编码RNA(lncRNA)和微小RNA(miRNA)是ncRNA的重要类型,参与细胞增殖、周期进展、迁移和凋亡等多种细胞过程,具有强大的基因调控功能。此外,lncRNA还可与miRNA相互作用防止目标mRNA被miRNA降解[4]。研究[5]显示,AMI病人血浆中lncRNA尿路上皮癌相关1(UCA1)表达显著降低,是急性心肌梗死潜在的诊断标志和治疗靶点。生物信息学数据库在线分析发现,UCA1与miR-503之间存在结合位点。miR-503高表达已被证实与糖尿病心肌梗死边缘区组织内血管新生能力的下降有关[6]。然而,lncRNA UCA1能否靶向miR-503在AMI中发挥作用尚未完全阐明。本研究在缺氧条件下培养H9c2心肌细胞以诱导细胞损伤,探讨UCA1在预防缺氧引起的心肌损伤中作用和可能机制。现作报道。
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相对于对照组,模型组AC16细胞中UCA1表达降低,miR-503表达升高,差异有统计学意义(P < 0.01)(见表 1)。
分组 n UCA1 miR-503 对照组 3 0.99±0.09 0.96±0.10 模型组 3 0.12±0.02 2.67±0.12 t — 16.34 18.96 P — < 0.01 < 0.01 表 1 UCA1、miR-503在缺氧处理的AC16细胞中的表达(x±s)
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相对于pcDNA组(0.12±0.01),pcDNA-UCA1组(0.63±0.04)AC16细胞中UCA1表达显著升高(t=21.42,P < 0.01)。相对于anti-miR-NC组(2.68±0.13),anti-miR-503组(1.16±0.06)AC16细胞中miR-503表达显著降低(t=18.39,P < 0.01)。
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相对于对照组,模型组AC16细胞存活率、Ki67蛋白表达、S期细胞比例降低,细胞凋亡率、Cleaved-caspase3蛋白表达、G0~G1期细胞比例升高,差异均有统计学意义(P < 0.05);相对于pcDNA组,pcDNA-UCA1组AC16细胞存活率、Ki67蛋白表达、S期细胞比例升高,细胞凋亡率、Cleaved-caspase3蛋白表达、G0~G1期细胞比例降低,差异均有统计学意义(P < 0.05)(见图 1、2和表 2)。
分组 n 存活率/% 凋亡率/% G0~G1 S G2~M Ki67 Cleaved-caspase3 对照组 3 95.88±5.11 6.73±0.39 33.59±1.16 33.70±1.20 32.71±1.11 0.89±0.05 0.13±0.01 模型组 3 44.94±1.47* 24.40±0.91* 49.10±1.10* 18.41±0.94* 32.49±0.91 0.23±0.01* 0.79±0.05* pcDNA 3 44.45±1.16 24.34±0.91 49.12±1.05 18.51±0.90 32.37±0.93 0.23±0.02 0.80±0.04 pcDNA-UCA1 3 83.25±2.15#△ 12.16±0.50#△ 37.29±0.94#△ 30.38±0.79#△ 32.33±0.70 0.71±0.04#△ 0.24±0.02#△ F — 244.52 461.55 170.61 202.18 0.10 296.61 328.87 P — < 0.01 < 0.01 < 0.01 < 0.01 >0.05 < 0.01 < 0.01 MS组内 — 8.560 0.515 1.135 0.939 0.854 0.001 0.001 q检验:与对照组比较*P < 0.05;与模型组比较#P < 0.05;与pcDNA组比较△P < 0.05 表 2 UCA1对缺氧处理的AC16细胞增殖凋亡的影响(x±s)
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靶基因预测数据库在线分析显示,UCA1与miR-503之间存在特异性结合位点(见图 3)。双荧光素酶报告实验显示,与转染miR-NC比较,转染miR-503 mimic可降低WT-UCA1的相对荧光素酶活性(P < 0.01),而对MUT-UCA1的相对荧光素酶活性影响无统计学意义(P>0.05)(见表 3)。RT-qPCR检测显示,与pcDNA组(0.96±0.10)比较,pcDNA-UCA1组(0.26±0.02)AC16细胞miR-503的表达水平降低(t=11.89,P < 0.01)。
分组 n WT-UCA1 MUT-UCA1 miR-NC 3 0.95±0.09 0.90±0.06 miR-503 3 0.38±0.02 0.93±0.06 t — 10.71 0.61 P — < 0.01 >0.05 表 3 双荧光素酶报告实验验证UCA1和miR-503的靶向关系(x±s)
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相对于Model组、anti-miR-NC组,anti-miR-503组AC16细胞存活率、Ki67蛋白表达、S期细胞比例显著升高,细胞凋亡率、Cleaved-caspase3蛋白表达、G0~G1期细胞比例显著降低,差异均有统计学意义(P < 0.05)(见图 4、5和表 4)。
分组 n 存活率/% 凋亡率/% G0~G1 S G2~M Ki67 Cleaved-caspase3 模型组 3 44.58±3.25 24.42±0.66 49.61±1.17 18.41±1.05 31.98±1.18 0.25±0.02 0.82±0.05 模型组+anti-miR-NC 3 44.79±0.10 24.38±0.77 49.24±0.98 18.38±0.89 32.38±0.94 0.23±0.02 0.78±0.05 Model+anti-miR-503 3 72.97±1.59△ 15.55±0.52△ 40.44±0.79△ 28.12±0.64△ 32.44±0.77 0.56±0.02△ 0.34±0.03△ F — 18.22 180.90 82.18 123.14 0.20 256.75 108.20 P — < 0.01 < 0.01 < 0.01 < 0.01 >0.05 < 0.01 < 0.01 MS组内 — 4.367 0.433 0.985 0.768 0.956 0.000 0.002 q检验:与anti-miR-NC组比较△P < 0.05 表 4 干扰miR-503对缺氧处理的AC16增殖凋亡的影响(x±s)
lncRNA UCA1抑制miR-503减轻缺氧诱导的心肌细胞损伤机制研究
lncRNA UCA1 alleviates hypoxia-induced cardiomyocyte injury by targeting miR-503
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摘要:
目的探讨长链非编码RNA(lncRNA)尿路上皮癌相关1(UCA1)靶向miR-503对缺氧条件下心肌细胞增殖、凋亡的影响。 方法构建体外心肌细胞AC16缺氧模型。实时荧光定量PCR(RT-qPCR)检测UCA1和miR-503表达。细胞计数试剂盒(CCK-8)检测细胞活力;流式细胞术检测细胞凋亡和周期分布。将UCA1过表达载体、miR-503抑制物分别转染AC16,检测上调UCA1或下调miR-503对缺氧条件下AC16细胞增殖和凋亡的影响。双荧光素酶报告基因实验和RT-qPCR确定UCA1对miR-503的靶向作用。 结果缺氧处理后AC16细胞存活率、S期细胞比例降低,凋亡率、G0~G1期细胞比例升高,差异均有统计学意义(P < 0.05)。上调UCA1或下调miR-503表达后,缺氧处理的AC16细胞存活率、S期细胞比例升高,凋亡率、G0~G1期细胞比例降低,差异均有统计学意义(P < 0.05)。miR-503是UCA1的靶基因,UCA1负性调控miR-503表达。 结论lncRNA UCA1能够显著提高缺氧条件下心肌细胞存活率,抑制缺氧诱导的心肌细胞凋亡,其机制可能与靶向下调miR-503表达有关。 Abstract:ObjectiveTo investigate the effect of long-chain non-coding RNA(lncRNA) urothelial carcinoma is associated 1(UCA1) targeting miR-503 on proliferation and apoptosis of cardiomyocyte with hypoxia treatment. MethodsThe cardiomyocyte AC16 hypoxia model was established in vitro.The expression of UCA1 and miR-503 were detected by real-time quantitative PCR (RT-qPCR).The cell viability was measured by CCK-8.Apoptosis and cycle distribution were detected by flow cytometry.The UCA1 overexpression vector and miR-503 inhibitor were transfected into AC16 cells, respectively.And the effects of up-regulating UCA1 or down-regulating miR-503 on the proliferation and apoptosis of AC16 cells treated with hypoxic were detected.Dual luciferase reporter gene assay and RT-qPCR confirmed the targeting effect of UCA1 on miR-503. ResultsAfter treated with hypoxia, the survival rate of AC16 cells and the proportion of S-phase cells were significantly decreased, and the apoptosis rate and the proportion of G0-G1 phase cells were significantly increased (P < 0.05).After up-regulating UCA1 expression or down-regulating miR-503 expression, the survival rate and the proportion of S-phase in AC16 cells treated with hypoxia were significantly increased, while the apoptosis rate and the proportion of G0-G1 phase cells were significantly decreased (P < 0.05).miR-503 was the target gene of UCA1, and UCA1 negatively regulated the expression of miR-503. ConclusionslncRNA UCA1 can significantly improve the survival rate and inhibit the apoptosis in hypoxia-induced cardiomyocyte, which may be attributed to the targeted down-regulation of miR-503 expression. -
表 1 UCA1、miR-503在缺氧处理的AC16细胞中的表达(x±s)
分组 n UCA1 miR-503 对照组 3 0.99±0.09 0.96±0.10 模型组 3 0.12±0.02 2.67±0.12 t — 16.34 18.96 P — < 0.01 < 0.01 表 2 UCA1对缺氧处理的AC16细胞增殖凋亡的影响(x±s)
分组 n 存活率/% 凋亡率/% G0~G1 S G2~M Ki67 Cleaved-caspase3 对照组 3 95.88±5.11 6.73±0.39 33.59±1.16 33.70±1.20 32.71±1.11 0.89±0.05 0.13±0.01 模型组 3 44.94±1.47* 24.40±0.91* 49.10±1.10* 18.41±0.94* 32.49±0.91 0.23±0.01* 0.79±0.05* pcDNA 3 44.45±1.16 24.34±0.91 49.12±1.05 18.51±0.90 32.37±0.93 0.23±0.02 0.80±0.04 pcDNA-UCA1 3 83.25±2.15#△ 12.16±0.50#△ 37.29±0.94#△ 30.38±0.79#△ 32.33±0.70 0.71±0.04#△ 0.24±0.02#△ F — 244.52 461.55 170.61 202.18 0.10 296.61 328.87 P — < 0.01 < 0.01 < 0.01 < 0.01 >0.05 < 0.01 < 0.01 MS组内 — 8.560 0.515 1.135 0.939 0.854 0.001 0.001 q检验:与对照组比较*P < 0.05;与模型组比较#P < 0.05;与pcDNA组比较△P < 0.05 表 3 双荧光素酶报告实验验证UCA1和miR-503的靶向关系(x±s)
分组 n WT-UCA1 MUT-UCA1 miR-NC 3 0.95±0.09 0.90±0.06 miR-503 3 0.38±0.02 0.93±0.06 t — 10.71 0.61 P — < 0.01 >0.05 表 4 干扰miR-503对缺氧处理的AC16增殖凋亡的影响(x±s)
分组 n 存活率/% 凋亡率/% G0~G1 S G2~M Ki67 Cleaved-caspase3 模型组 3 44.58±3.25 24.42±0.66 49.61±1.17 18.41±1.05 31.98±1.18 0.25±0.02 0.82±0.05 模型组+anti-miR-NC 3 44.79±0.10 24.38±0.77 49.24±0.98 18.38±0.89 32.38±0.94 0.23±0.02 0.78±0.05 Model+anti-miR-503 3 72.97±1.59△ 15.55±0.52△ 40.44±0.79△ 28.12±0.64△ 32.44±0.77 0.56±0.02△ 0.34±0.03△ F — 18.22 180.90 82.18 123.14 0.20 256.75 108.20 P — < 0.01 < 0.01 < 0.01 < 0.01 >0.05 < 0.01 < 0.01 MS组内 — 4.367 0.433 0.985 0.768 0.956 0.000 0.002 q检验:与anti-miR-NC组比较△P < 0.05 -
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