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中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (03) : 155 -162. doi: 10.3877/cma.j.issn.2095-1221.2020.03.004

所属专题: 文献

论著

HOXA-AS2靶向miR-17对人脐静脉内皮细胞生物学功能以及炎症因子的影响
王磊1, 艾文2, 方叶青1, 陈之杰1, 邱小燕1, 李华英1, 谢培益1,()   
  1. 1. 518000 深圳,华中科技大学协和深圳医院心血管内科
    2. 518000 深圳,华中科技大学协和深圳医院科教科
  • 收稿日期:2019-11-28 出版日期:2020-06-01
  • 通信作者: 谢培益
  • 基金资助:
    深圳市科技创新委(201202174); 深圳市科技创新委(201202170)

Effect of HOXA-AS2 targeting miR-17 on cell biological function and inflammatory factors in EA.hy926

Lei Wang1, Wen Ai2, Yeqing Fang1, Zhijie Chen1, Xiaoyan Qiu1, Huaying Li1, Peiyi Xie1,()   

  1. 1. Internal Medicine-Cardiovascular Department, Shenzhen Union Hospital of Huazhong University of Science and Technology, Shenzhen 518000, China
    2. Science and Education Shenzhen Union Hospital of Huazhong University of Science and Technology, Shenzhen 518000, China
  • Received:2019-11-28 Published:2020-06-01
  • Corresponding author: Peiyi Xie
  • About author:
    Corresponding author: Xie Peiyi, Email:
引用本文:

王磊, 艾文, 方叶青, 陈之杰, 邱小燕, 李华英, 谢培益. HOXA-AS2靶向miR-17对人脐静脉内皮细胞生物学功能以及炎症因子的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2020, 10(03): 155-162.

Lei Wang, Wen Ai, Yeqing Fang, Zhijie Chen, Xiaoyan Qiu, Huaying Li, Peiyi Xie. Effect of HOXA-AS2 targeting miR-17 on cell biological function and inflammatory factors in EA.hy926[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(03): 155-162.

目的

探讨HOXA-AS2对动脉粥样硬化(AS)模型细胞生物学功能以及炎症因子的影响及分子机制。

方法

本实验共分为4个实验;实验1:用100 μg/mL的ox-LDL处理EA.hy926细胞作为ox-LDL组,正常培养的细胞作为对照组;实验2:将pcDNA3.1、pcDNA3.1-HOXA-AS2转染至EA.hy926细胞中再用100 μg/mL的ox-LDL处理,记为ox-LDL+pcDNA3.1组、ox-LDL+pcDNA3.1-HOXA-AS2组;实验3:将pcDNA3.1、pcDNA3.1-HOXA-AS2、si-NC、si-HOXA-AS2分别转染至EA.hy926细胞中,记为pcDNA3.1组、pcDNA3.1-HOXA-AS2组、si-NC组、si-HOXA-AS2组;实验4:将pcDNA3.1-HOXA-AS2与miR-NC、miR-17分别共转染至EA.hy926细胞中再用100 μg/mL的ox-LDL处理,记为ox-LDL+pcDNA3.1-HOXA-AS2+miR-NC组、ox-LDL+pcDNA3.1-HOXA-AS2+miR-17组。实时荧光定量PCR (RT-qPCR)检测HOXA-AS2和miR-17的表达水平;蛋白质印迹(Western blot)法检测细胞周期蛋白依赖性激酶抑制剂1A (P21)、cleaved caspase 3蛋白表达;MTT检测细胞增殖情况;流式细胞术检测细胞凋亡;ELISA法检测白细胞介素-1 (IL-1)、白细胞介素-6 (IL-6)水平;荧光素酶报告实验检测HOXA-AS2和miR-17的靶向关系。两组间比较采用独立样本t检验,多组间比较采用方差分析,组间两两比较采用LSD-t检验。

结果

与对照组比较,ox-LDL组EA.hy926细胞中HOXA-AS2表达水平(0.23±0.02比1.02±0.10),细胞增殖率[(47.83±5.01)﹪比(100.06±10.20)﹪]均降低,细胞凋亡率[(26.81±2.47)﹪比(8.23±0.80)﹪]、P21 (0.82±0.08比0.20±0.02)、cleaved caspase 3 (0.67±0.06比0.14±0.01)、IL-1[(792.34±59.37)ng/L比(326.14±34.59) ng/ L]和IL-6表达水平[(53.67±4.65)ng/L比(19.25±2.11)ng/L]均升高,差异具有统计学意义(P均< 0.05)。与ox-LDL+pcDNA3.1组比较,ox-LDL+pcDNA3.1-HOXA-AS2组EA.hy926细胞中HOXA-AS2表达水平(0.87±0.09比0.22±0.02)、细胞增殖率[(89.94±8.34)﹪比(48.21±4.86)﹪]均升高,细胞凋亡率[(12.33±1.18)﹪比(26.83±2.48)﹪]、P21 (0.33±0.03比0.81±0.08)、cleaved caspase 3 (0.24±0.02比0.69±0.06)、IL-1[ (446.25±46.84)ng/L比(802.21±60.18)ng/L]和IL-6表达水平[(25.64±2.65)ng/L比(55.21±5.10)ng/L]均降低,差异具有统计学意义(P均< 0.001)。与ox-LDL+pcDNA3.1-HOXA-AS2+miR-NC组比较,ox-LDL+pcDNA3.1-HOXA-AS2+miR-17组EA.hy926细胞中miR-17表达水平(2.14±0.21比1.05±0.10)、细胞凋亡率[(23.31±2.33)﹪比(13.75±1.44)﹪]、IL-1水平[(684.26±62.38)ng/L比(451.21±43.58)ng/L]、IL-6水平[(41.29±4.37)ng/L比(26.11±2.39)ng/L]均升高,细胞增殖率[(53.67±5.46)﹪比(90.21±9.16)﹪]降低,差异具有统计学意义(P均< 0.001)。HOXA-AS2与miR-17存在结合位点,荧光素酶报告实验显示,与miR-NC组比较,miR-17组中转染WT-HOXA-AS2的EA.hy926细胞荧光素酶活性降低(0.33±0.03比1.01±0.10,P < 0.05),而转染MUT-HOXA-AS2的EA.hy926细胞荧光素酶活性差异无统计学意义(P > 0.05);与anti-miR-NC组比较,anti-miR-17组中转染WT-HOXA-AS2的EA.hy926细胞荧光素酶活性升高(2.29±0.21比1.00±0.10,P < 0.05),而转染MUT-HOXA-AS2的EA.hy926细胞荧光素酶活性差异无统计学意义(P > 0.05)。

结论

过表达HOXA-AS2促进细胞增殖,抑制ox-LDL引起的细胞凋亡和炎症因子的释放,其机制可能与miR-17有关。

Objective

To investigate the effects of HOXA-AS2 on the cell biological functions and inflammatory factors of the atherosclerosis model and its molecular mechanism.

Methods

This experiment was divided into 4 experiments. Experiment 1: EA.hy926 cells, treated with 100 μg/mL ox-LDL, were set as the ox-LDL group, and normally cultured cells as the control group; Experiment 2: pcDNA3.1, pcDNA3.1-HOXA-AS2, transfected into EA.hy926 and then treated with 100 μg/mL ox-LDL, were recorded as ox-LDL+pcDNA3.1 group and ox-LDL+pcDNA3.1-HOXA-AS2 group; Experiment 3: pcDNA3.1, pcDNA3.1-HOXA-AS2, si-NC and si-HOXA-AS2, transfected into EA.hy926, were recorded as pcDNA3.1 group, pcDNA3.1-HOXA-AS2 group, si-NC group and si-HOXA-AS2 group; Experiment 4: pcDNA3.1-HOXA-AS2 was co-transfected with miR-NC and miR-17 into EA.hy926 respectively, and then treated with 100 μg/ mL ox-LDL, which were recorded as ox-LDL+pcDNA3.1-HOXA-AS2+miR-NC group and ox-LDL+pcDNA3.1-HOXA-AS2+miR-17 group. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of HOXA-AS2 and miR-17, Western blot to detect cyclin-dependent kinase inhibitor 1A (P21) and cleaved cysteine-containing aspartate-specific proteases-3 (cleaved caspase 3) protein expression, MTT assay to detect cell proliferation, flow cytometry to detect apoptosis, enzyme-linked immunosorbent assay (ELISA) to detect interleukin-1 (IL-1) and interleukin-6 (IL-6) levels, and luciferase reporter assay to detect the targeting relationship between HOXA-AS2 and miR-17. The experimental data were analyzed by SPSS 20.0. The comparisons between two groups were conducted by t test, while the comparison among multiple groups was made by one-way ANOVA.

Results

Compared with the control group, the expression level of HOXA-AS2 (0.23±0.02 vs 1.02±0.10) and the cell proliferation rate [ (47.83±5.01) ﹪ vs (100.06±10.20) ﹪] in the ox-LDL group were reduced. Apoptosis rate [ (26.81±2.47) ﹪ vs (8.23±0.80) ﹪], P21 (0.82±0.08 vs 0.20±0.02) , cleaved caspase 3 (0.67±0.06 vs 0.14±0.01) , IL-1 [ (792.34± 59.37) ng/L vs (326.14±34.59) ng/L] and IL-6 expression levels [ (53.67±4.65) ng/L vs (19.25±2.11) ng/L] were increased, and the difference was statistically significant (P < 0.05) . Compared with the ox-LDL + pcDNA3.1 group, the expression level of HOXA-AS2 (0.87±0.09 vs 0.22±0.02) and cell proliferation rate [ (89.94 ±8.34) ﹪ vs (48.21±4.86) ﹪] were all increased in ox-LDL+ pcDNA3.1-HOXA-AS2 group, and the apoptosis rate [ (12.33±1.18) ﹪ vs (26.83±2.48) ﹪], P21 (0.33±0.03 vs 0.81±0.08) , cleaved caspase 3 (0.24±0.02 vs 0.69±0.06) , IL-1 [ (446.25± 46.84) ng/L vs (802.21±60.18) ng/L], and IL-6 expression levels [ (25.64±2.65) ng/L vs (55.21±5.10) ng / L] were decreased, and the difference was statistically significant (P < 0.001) . Compared with the ox-LDL+pcDNA3.1-HOXA-AS2+ miR-NC group, the expression level of miR-17 in EA.hy926 of the ox-LDL+ pcDNA3.1-HOXA-AS2 + miR-17 group (2.14±0.21 vs 1.05±0.10) , apoptosis rate [ (23.31±2.33) ﹪ vs (13.75±1.44) ﹪], IL-1 level [ (684.26±62.38) ng/L vs (451.21±43.58) ng/L], IL-6 levels [ (41.29±4.37) ng/ L vs (26.11±2.39) ng/L] were increased, cell proliferation rate [ (53.67 ±5.46) ﹪ vs (90.21±9.16) ﹪] was decreased, and the difference was statistically significant (P < 0.001) . HOXA-AS2 had a binding site with miR-17. The luciferase report experiment showed that compared with the miR-NC group, the luciferase activity of EA.hy926 cells transfected with WT-HOXA-AS2 in the miR-17 group was reduced (0.33±0.03 vs 1.01±0.10, P < 0.05) , but no statistically significant difference (P > 0.05) was found in the luciferase activity of EA.hy926 cells transfected with MUT-HOXA-AS2; and compared with anti-miR-NC group, luciferase activity of EA.hy926 cells transfected with WT-HOXA-AS2 in anti-miR-17 group was increased (2.29±0.21 vs 1.00±0.10, P < 0.05) , and no statistically significant difference (P > 0.05) was observed in the luciferase activity of EA.hy926 cells transfected with MUT-HOXA-AS2.

Conclusion

Overexpression of HOXA-AS2 promotes cell proliferation, inhibits ox-LDL-induced apoptosis and release of inflammatory factors, and its mechanism may be related to miR-17.

表1 引物序列信息
图1 对照组与ox-LDL组细胞相关指标的差异
表2 HOXA-AS2的表达及ox-LDL处理对EA.hy926细胞增殖率、凋亡率及P21、cleaved caspase 3蛋白表达的影响(±sn = 3)
图2 过表达HOXA-AS2对ox-LDL作用的EA.hy926细胞增殖、凋亡的影响
表3 过表达HOXA-AS2对ox-LDL处理的EA.hy926细胞增殖率、凋亡率及P21、cleaved caspase 3、caspase3蛋白表达的影响(±sn = 3)
表4 过表达HOXA-AS2对ox-LDL作用的EA.hy926细胞中IL-1和IL-6表达的影响(±sn = 3)
图3 HOXA-AS2靶向miR-17
表5 双荧光素酶报告实验对HOXA-AS2和miR-17靶向关系的验证(±sn = 3)
图4 过表达miR-17在HOXA-AS2对ox-LDL作用的EA.hy926细胞增殖、凋亡中的影响
表6 过表达miR-17能逆转HOXA-AS2对ox-LDL作用的EA.hy926细胞中IL-1和IL-6表达及细胞增殖率的影响(±sn = 3)
表7 过表达miR-17能逆转HOXA-AS2对ox-LDL作用的EA.hy926细胞中P21、cleaved caspase 3、caspase 3及凋亡率的影响(±sn = 3)
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