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

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。

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

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