Inhibition of long noncoding RNA HIF1A-AS2 confers protection against atherosclerosis via ATF2 downregulation

Fig. 1. High expression of lncRNA HIF1A-AS2 and ATF2 are identified in atherosclerotic mice. (A) Body weight and composition of ApoE−/− mice fed with ND or HFD. (B) Measurement of the food intake of ApoE−/− mice fed with ND or HFD. (C) Lipid deposition in the aorta observed after Oil red O staining. (D) The thoracic aortic injury area detected by HE staining (100×). (E) Lipid deposition in the thoracic aorta observed after Oil red staining (100×). (F) ELISA detection of TNF-α, IL-1β, and IL-6 levels in the serum of the ApoE−/− mice fed with ND or HFD. (G) Immunohistochemical detection of VCAM-1, ICAM-1, and MCP-1 expression in arterial tissues of the ApoE−/− mice fed with ND or HFD (400×). (H) The expressions of lncRNA HIF1A-AS2 and ATF2 in arterial tissues of the ApoE−/− mice fed with ND or HFD determined by RT-qPCR. (I) Correlation analysis of lncRNA HIF1A-AS2 expression and ATF2 expression. (J) The expression of ATF2 in arterial tissues of the ApoE−/− mice fed with ND or HFD detected by immunohistochemistry (400×). *p < 0.05 vs. the ApoE−/− mice fed with ND. Data (mean ± standard deviation) between two groups were analyzed using unpaired t test. n = 20 for mice fed with ND or HFD.

Fig. 2. Inhibition of lncRNA HIF1A-AS2 curbs ox-LDL-induced inflammation in ECs, SMCs and HCAECs. (A) RT-qPCR determination of the expression of lncRNA HIF1A-AS2 in cells transfected with si-HIF1A-AS2-1 or si-HIF1A-AS2-2. (B) The expression of lncRNA HIF1A-AS2 in ECs, SMCs and HCAECs transfected with si-HIF1A-AS2 measured by RT-qPCR. (C) ELISA detection of the expression of TNF-α, IL-1β and IL-6 in cell culture supernatants after lncRNA HIF1A-AS2 downregulation in ECs, SMCs and HCAECs. (D), The protein expression of VCAM-1, ICAM-1, and MCP-1 in ECs, SMCs and HCAECs after inhibiting lncRNA HIF1A-AS2 measured by Western blot analysis. (E) Lipid level in ox-LDL-treated cells following HIF1A-AS2 knockdown analyzed by Oil red O staining (400×). *p < 0.05 vs. cells transfected with si-NC or cells without treatment. #p < 0.05 vs. ox-LDL-treated cells transfected with si-NC. Data (mean ± standard deviation) were obtained from three independent experiments and data between two groups were analyzed using unpaired t test.

Fig. 3. Knockdown of lncRNA HIF1A-AS2 alleviates ox-LDL-induced inflammation in ECs, SMCs and HCAECs by inhibiting ATF2. (A) The protein expression of ATF2 determined by Western blot analysis after lncRNA HIF1A-AS2 was silenced in ox-LDL-exposed ECs, SMCs and HCAECs. (B) Measurement of ATF2 protein expression by Western blot analysis in response to downregulation of lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs. (C) ELISA detection of the expression of TNF-α, IL-1β and IL-6 in EC and SMC culture supernatants exposed to ox-LDL. (D) The protein expression of VCAM-1, ICAM-1, and MCP-1 in ox-LDL-exposed ECs, SMCs and HCAECs measured by Western blot analysis. *p < 0.05 vs. the untreated cells or ox-LDL-exposed cells co-transfected with si-NC and oe-NC. #p < 0.05 vs. the cells ox-LDL-exposed transfected with si-NC or ox-LDL-exposed cells co-transfected with si-HIF1A-AS2 and oe-NC. & p < 0.05 vs. the ox-LDL-exposed cells co-transfected with si-NC and oe-ATF2. Data (mean ± standard deviation) were obtained from three independent cell experiments and comparison among multiple groups was analyzed with one-way analysis of variance.

Fig. 4. Silencing of lncRNA HIF1A-AS2 enhances cell viability, but reduces apoptosis by inhibiting ATF2 expression. (A) Viability of ECs after ox-LDL-induced inflammation determined using CCK-8 assay. (B) Viability of SMCs after ox-LDL-induced inflammation determined using CCK-8 assay. (C) Viability of HCAECs after ox-LDL-induced inflammation determined using CCK-8 assay. (D) Cell apoptosis of ECs after ox-LDL-induced inflammation determined using flow cytometry. (E) Cell apoptosis of SMCs after ox-LDL-induced inflammation determined using flow cytometry. (F) Cell apoptosis of HCAECs after ox-LDL-induced inflammation determined using flow cytometry. *p < 0.05 vs. ox-LDL-exposed cells co-transfected with si-NC + oe-NC. #p < 0.05 vs. ox-LDL-exposed cells co-transfected with si-HIF1A-AS2 + oe-NC. &p < 0.05 vs. the ox-LDL-exposed cells co-transfected with si-NC + oe-ATF2. Data (mean ± standard deviation) were obtained from three independent cell experiments and comparison among multiple groups was analyzed with one-way analysis of variance.

Fig. 5. LncRNA HIF1A-AS2 modulates the expression of ATF2 via binding to USF1. (A) Prediction of the lncRNA-TF-mRNA interaction based on the LncMAP database. (B) The protein expression of USF1 and ATF2 in ECs, SMCs and HCAECs exposed to ox-LDL measured by Western blot analysis. (C) The binding of lncRNA HIF1A-AS2 to USF1 in ECs, SMCs and HCAECs detected by RNA pull-down. (D) The binding of lncRNA HIF1A-AS2 to USF1 in ECs detected by RIP assay. (E) The binding site of USF1 on the ATF2 promoter predicted from JASPAR website. (F) The binding of USF1 to the ATF2 promoter region detected by the dual-luciferase reporter assay. (G) The binding of USF1 to the ATF2 promoter in ECs with or without silencing lncRNA HIF1A-AS2 detected by ChIP assay. *p < 0.05 vs. the untreated cells or ox-LDL-exposed cells treated with oe-NC or IgG. Data (mean ± standard deviation) were obtained from three independent experiments and those between groups were analyzed by unpaired t test.

Fig. 6. Knockdown of lncRNA HIF1A-AS2 downregulates ATF2 expression via USF1 to inhibit inflammation in ox-LDL-exposed ECs, SMCs and HCAECs. (A) The expression of lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs determined by RT-qPCR. (B) Measurement of USF1 and ATF2 protein expression in ox-LDL-exposed ECs, SMCs and HCAECs by Western blot analysis. (C) The expression of TNF-α, IL-1β and IL-6 in the supernatant of ox-LDL-exposed ECs, SMCs and HCAECs detected by ELISA. (D) The protein expression of VCAM-1, ICAM-1, and MCP-1 in ox-LDL-exposed ECs, SMCs and HCAECs measured by Western blot analysis. *p < 0.05 vs. the ox-LDL-exposed cells co-transfected with si-NC and oe-NC. #p < 0.05 vs. the ox-LDL-exposed cells co-transfected with si-HIF1A-AS2 and oe-NC. & p < 0.05 vs. the ox-LDL-exposed cells co-transfected with si-NC and oe-USF1. Data (mean ± standard deviation) were obtained from three independent experiments and those among multiple groups analyzed by one-way analysis of variance.

Fig. 7. Knockdown of lncRNA HIF1A-AS2 or ATF2 inhibits inflammation in atherosclerotic ApoE−/− mice in vivo. (A) Determination of lncRNA HIF1A-AS2 and ATF2 expression in mouse arterial tissues by RT-qPCR. (B) Lipid deposition in injured aorta areas observed after Oil red O staining. (C) HE staining for detection of thoracic aortic injury area (100×). (D) Lipid deposition in the thoracic aorta observed after Oil red O staining (100×). (E) ELISA detection of TNF-α, IL-1β, IL-6 expression in the serum of ApoE−/− mice fed with HFD. (F) VCAM-1, ICAM-1, and MCP-1 protein expression in mouse arterial tissues measured by Western blot analysis. *p < 0.05 vs. the ApoE−/− mice fed with HFD and injected with lentivirus vector expressing sh-NC; #p < 0.05 vs. the ApoE−/− mice fed with HFD and injected with lentivirus vector expressing sh-HIF1A-AS2. Data (mean ± standard deviation) among multiple groups were analyzed by one-way analysis of variance. n = 20 for mice in each group.

Fig. 8. The map illustrating mechanisms associated with lncRNA HIF1A-AS2-mediated inflammation in the atherosclerosis. The upper panel shows that lncRNA HIF1A-AS2 forms a complex with USF1, which is then recruited into the ATF2 promoter to elevate ATF2 expression, thus promoting the development of atherosclerotic inflammation. The lower panel shows that downregulation of lncRNA HIF1A-AS2 reduces the expression of ATF2 by reducing the binding of USF1 to the ATF2 promoter regions, thereby inhibiting atherosclerotic inflammation, corresponding to decreased inflammatory factors TNF-α, IL-1β and IL-6 in serum and protein levels of adhesion molecules VCAM-1, ICAM-1, and MCP-1, as well as increased cell viability and reduced apoptosis in ox-LDL-induced inflammation in ECs, SMCs, and HCAECs.

Supplementary Fig. 1. Flow chart of animal experiments.