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

doi:10.3877/cma.j.issn.2095-1221.2020.03.004

Abstract

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

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]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(03): 155-162.

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Figures/Tables 11

References 21

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基因	序列（5'-3'）	预期扩增长度（bp）
HOXA-AS2	上游CCCGTAGGAAGAACCGATGA	70 bp
?	下游TTTAGGCCTTCGCAGACAGC	?
GAPDH	上游GATACACGGAGCACCAGGTT	158 bp
?	下游TGTCAAAGTGCTTACAGTG	?
miR-17	上游GCAGGAAAAAAGAGAACATCACC	74 bp
?	下游GTCACCATAATGCTACAAG	?
U6	上游GCTTCGGCAGCACATATACTAAAAT	89 bp
?	下游CGCTTCACGAATTTGCGTGTCAT	?

基因	序列（5'-3'）	预期扩增长度（bp）
HOXA-AS2	上游CCCGTAGGAAGAACCGATGA	70 bp
?	下游TTTAGGCCTTCGCAGACAGC	?
GAPDH	上游GATACACGGAGCACCAGGTT	158 bp
?	下游TGTCAAAGTGCTTACAGTG	?
miR-17	上游GCAGGAAAAAAGAGAACATCACC	74 bp
?	下游GTCACCATAATGCTACAAG	?
U6	上游GCTTCGGCAGCACATATACTAAAAT	89 bp
?	下游CGCTTCACGAATTTGCGTGTCAT	?

分组	IL-1 （ng/L）	IL-6 （ng/L）
对照组	326.14±34.59	19.25±2.11
ox-LDL组	792.34±59.37^a	53.67±4.65^a
ox-LDL+pcDNA3.1组	802.21±60.18^a	55.21±5.10^a
ox-LDL+pcDNA3.1-HOXA-AS2组	446.25±46.84^bc	25.64±2.65^bc
F值	200.722	212.214
P值	< 0.001	<0.001

分组	IL-1 （ng/L）	IL-6 （ng/L）
对照组	326.14±34.59	19.25±2.11
ox-LDL组	792.34±59.37^a	53.67±4.65^a
ox-LDL+pcDNA3.1组	802.21±60.18^a	55.21±5.10^a
ox-LDL+pcDNA3.1-HOXA-AS2组	446.25±46.84^bc	25.64±2.65^bc
F值	200.722	212.214
P值	< 0.001	<0.001

分组	WT-HOXA-AS2	MUT-HOXA-AS2
miR-NC组	1.01±0.10	0.94±0.09
miR-17组	0.33±0.03^a	1.02±0.10
anti-miR-NC组	1.00±0.10	1.01±0.10
anti-miR-17组	2.29±0.21^b	0.99±0.10
F值	123.931	0.399
P值	< 0.001	0.758

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