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

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

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100μg/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100μg/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100μg/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 100μg/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 100μg/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 100μg/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.