Chinese Journal Of Cell And Stem Cell >Archive >2020 >Volume 10 Number 3 June 2020 >Original Researches >contents

Journal content

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

Wang Lei, Ai Wen, Fang Yeqing, et al


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【Abstract】 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.
 

【Key words】 HOXA-AS2; MiR-17; Atherosclerosis; Proliferation; Apoptosis;  Inflammatory factors