Downregulation of LncRNA NORAD promotes Ox-LDL-induced vascular endothelial cell injury and atherosclerosis

Abstract

Long noncoding RNAs (lncRNAs) play important roles in the development of vascular diseases. However, the effect of lncRNA NORAD on atherosclerosis remains unknown. This study aimed to investigate the effect NORAD on endothelial cell injury and atherosclerosis. Ox-LDL-treated human umbilical vein endothelial cells (HUVECs) and high-fat-diet (HFD)-fed ApoE^-/- mice were used as in vitro and in vivo models. Results showed that NORAD-knockdown induced cell cycle arrest in G0/G1 phase, aggravated ox-LDL-induced cell viability reduction, cell apoptosis, and cell senescence along with the increased expression of Bax, P53, P21 and cleaved caspase-3 and the decreased expression of Bcl-2. The effect of NORAD on cell viability was further verified via NORAD-overexpression. NORAD- knockdown increased ox-LDL-induced reactive oxygen species, malondialdehyde, p-IKBα expression levels and NF-κB nuclear translocation. Proinflammatory molecules ICAM, VCAM, and IL-8 were also increased by NORAD- knockdown. Additionally, we identified the strong interaction of NORAD and IL-8 transcription repressor SFPQ in HUVECs. In ApoE^-/- mice, NORAD-knockdown increased the lipid disorder and atherosclerotic lesions. The results have suggested that lncRNA NORAD attenuates endothelial cell senescence, endothelial cell apoptosis, and atherosclerosis via NF-κB and p53-p21 signaling pathways and IL-8, in which NORAD-mediated effect on IL-8 might through the direct interaction with SFPQ.

Keywords: IL-8; NORAD; cell apoptosis; cell senescence; ox-LDL.

Figure 1

NORAD-knockdown aggravates ox-LDL-induced viability reduction and apoptosis of HUVECs. (A) Dose-dependent effect of ox-LDL on cell viability in HUVECs. Cell viability was measured after HUVECs were treated with 0-90 μg/mL of ox-LDL for 24 h by the CCK-8 assay. The data in each group were normalized with the group treated with 0 μg/mL ox-LDL. (n = 6, *P < 0.05 and ^##P < 0.01 vs. group treated with 0 μg/mL ox-LDL, respectively). (B) Dose-dependent upregulation of NORAD expression in HUVECs treated with 0-90 μg/mL of ox-LDL for 24 h (n = 3, *P < 0.05 vs. group treatment with 0 μg/mL ox-LDL). (C) HUVECs were transfected with siNORAD or scrambled siCTRL. NORAD levels were analyzed through qRT-PCR (n = 3, ^*P < 0.05 vs. siCTRL). (D) NORAD-knockdown suppressed the viability of ox-LDL-treated HUVECs. The effect of siNORAD on the cell viability was measured via a CCK-8 assay. Cells treated without both siRNA and ox-LDL were used as blank control. (n = 3, ^*P < 0.05 vs. siCTRL, ^#P < 0.05 vs. siCTRL+ox-LDL). The data of each group were normalized to the blank groups. (E) HUVECs were infected with Ad-NORAD or Ad-EGFP. NORAD levels were analyzed through qRT-PCR (n = 3, *P < 0.05 vs. Ad-EGFP). (F) The effect of NORAD overexpression on the cell viability was measured via a CCK-8 assay. Cells treated without both adenovirus and ox-LDL were used as blank control. (n = 6, **P < 0.01 vs. Ad-EGFP, ^##P < 0.01 vs. Ad-EGFP+ox-LDL). The data of each group were normalized to the blank groups. (G) NORAD- knockdown increased ox-LDL-induced cell apoptosis. The apoptosis rate was detected through flow cytometry by using annexin V-FITC/PI double staining. The apoptotic rate was analyzed in terms of the percentage of the lower and upper right quadrants (n = 3, ^*P < 0.05 vs. siCTRL, ^#P < 0.05 vs. siCTRL+ox-LDL). (H) Western blot was used to analyze the expression levels of Bcl-2, Bax and cleaved caspase-3. The results were analyzed with Image J. Values were shown as mean ± SD (n = 3). ^*P < 0.05 vs. siCTRL+ox-LDL.

Figure 2

NORAD-knockdown is results in the cell cycle arrest in G0/G1 phase and aggravates ox-LDL-induced senescence of HUVECs. HUVECs were transfected with siNORAD or scrambled siCTRL. After transfection for 24 h, the cells were treated with 60 μg/mL ox-LDL for 24 h. (A) HUVECs were stained to determine SA-β-gal. NORAD-knockdown increased the number of positive-stained cells after they were treated with ox-LDL. Results were representative of three separate experiments. (B) mRNA expression of p53 in ox-LDL-treated HUVECs through RT-qPCR (n = 3, ^*P < 0.05 vs. siCTRL, ^#P < 0.05 vs. siCTRL+ox-LDL). (C) mRNA expression of p21 in ox-LDL-treated HUVECs through RT-qPCR (n = 3, ^*P < 0.05 vs. siCTRL, ^##P < 0.01 vs. siCTRL+ox-LDL). (D) mRNA expression of p53 in young and senile HUVECs through RT-qPCR (n = 3, ^*P < 0.05 vs. Youth). (E) mRNA expression of p21 in young and senile HUVECs through RT-qPCR (n = 3, ^##P < 0.05 vs. Youth). (F) NORAD levels in young and senile HUVECs through qRT-PCR (n = 3, ^*P < 0.05 vs. Youth). (G) NORAD silencing induced ox-LDL-treated cell cycle arrest at G0/G1 phase. Flow cytometry was used to detect the cell cycle distribution of ox-LDL-treated HUVECs.

Figure 3

NORAD-knockdown aggravates ox-LDL-induced oxidative stress and p-IKBα expression level in HUVECs. HUVECs were transfected with siNORAD or scrambled siCTRL. After transfection for 24 h, the cells were treated with 60 μg/mL ox-LDL for 24 h. (A) NORAD knockdown aggravated ox-LDL-induced ROS production. HUVECs were treated with 60 μg/mL ox-LDL for 3 h and incubated with DCF-DA for 25 min. The ROS levels were observed under an inverted fluorescence microscope. Images were representative of three separate experiments (n = 3). (B) Flow cytometry was used to detect the intracellular ROS levels. Data were from three separate experiments and described as mean ± SD. ^*P < 0.05 vs. the siCTRL group. (C) MDA content in HUVECs treated with 60 μg/mL ox-LDL for 24 h. Data were shown as mean ± SD of three separate experiments. ^**P < 0.01 vs. siCTRL, ^#P < 0.05 vs. siCTRL+ox-LDL. (D) NORAD-knockdown increased the p-IKBα expression observed through western blot. The ratio of p-IKBα to IKBα was analyzed with ImageJ. Values were shown as mean ± SD (n = 3). ^**P < 0.01 vs. siCTRL. (E) NORAD-knockdown increased NF-κB nuclear translocation by immunofluorescence. p65 was stained red with Cy3. Nuclei were stained blue with DAPI. Co-localization of p65 with nucleus is shown in purple.

Figure 4

Effects of NORAD-knockdown on ox-LDL-induced proinflammatory molecules in HUVECs. (A) mRNA expression of ICAM-1 in ox-LDL-treated HUVECs observed through qRT-PCR (n = 3, ^*P < 0.05 vs. siCTRL, ^##P < 0.01 vs. siCTRL+ox-LDL). (B) mRNA expression of VCAM in ox-LDL-treated HUVECs observed through qRT-PCR (n = 3, ^**P < 0.01 vs. siCTRL, ^#P < 0.05 vs. siCTRL+ox-LDL). (C) mRNA expression of IL-8 mRNA in ox-LDL-treated HUVECs observed through qRT-PCR (n = 3, ^**P < 0.01 vs. siCTRL, ^#P < 0.05 vs. siCTRL+ox-LDL). (D) RIP assay confirmed the interaction of NORAD and SFPQ. HUVEC lysates were incubated with the anti-SFPQ or anti-IgG antibody, and the precipitated complexes were analyzed with qRT-PCR to investigate the expression of NORAD (n = 3, ^*P < 0.05 vs. IgG).

Figure 5

NORAD-knockdown aggravates the abnormal serum lipid condition and increased the proinflammatory molecule expression induced by HFD in ApoE^−/− mice. ApoE^−/− mice were given Ad-NORAD or control Ad-EGFP injection after 8 weeks of HFD, and HFD was maintained for another 8 weeks. C57BL/6 J mice fed with the normal diet were set as the WT control group. (A) NORAD levels in the aortic sinus were analyzed through qRT-PCR (n = 3, ^*P < 0.05 vs. Ad-EGFP). Serum levels of lipids, TC (B), TG (C), and LDL-C (D) were detected. Values were shown as mean ± SD (n = 8). ^*P < 0.05, ^**P < 0.01 vs. WT group, and ^##P < 0.01 vs. Ad-EGFP group. (E) Western blot was used to detect the ICAM, VCAM, and IL-8 expression levels (n = 8, ^*P < 0.05 vs. WT group, and ^#P < 0.05 vs. Ad-EGFP group). The mRNA levels of ICAM (F), VCAM (G), and IL-8 (H) in thoracic aortas were detected through qRT-PCR (n = 8, ^*P < 0.05 vs. WT group, and ^#P < 0.05 vs. Ad-EGFP group).

Figure 6

NORAD-knockdown promotes atherosclerosis development in ApoE^−/− mice. ApoE^−/− mice were given Ad-NORAD or control Ad-EGFP injection after 8 weeks of HFD, and the HFD was maintained for another 8 weeks. C57BL/6 J mice fed with the normal diet were used as the blank WT control group. (A) Representative micrographs and quantification of lesion area in the aortic sinus observed through H&E staining. (B) Representative micrographs and quantification of the collagen fibers area in the aortic sinus observed through Masson staining. (C) Representative en face images of the entire aorta with Oil Red O staining. The atherosclerotic lesion areas were stained red. Oil Red O-positive areas were analyzed with ImageJ. Data were shown as mean ± SD, n = 8. ^**P < 0.01 vs. WT group, ^*P < 0.05 vs. WT group, and ^#P < 0.05 vs. Ad-EGFP group.