lncRNA ZEB1-AS1 Mediates Oxidative Low-Density Lipoprotein-Mediated Endothelial Cells Injury by Post-transcriptional Stabilization of NOD2

Figures

• 

  FIGURE 1. Silence of lncRNA ZEB1-AS1 abrogates oxidized-low density lipoprotein (ox-LDL)-induced cell injury in HUVECs. (A) HUVECs were treated with 100 μg/ml ox-LDL for respective time durations. MTT assay showed that ox-LDL treatment decreased cell viability, ∗P < 0.05 (Mann–Whitney U-test) was used for analyzing statistical difference among multiple groups compared to previous group. (B) qRT-PCR showed that ZEB1-AS1 was silenced by transfection of specific ZEB1-AS1 siRNAs (si-ZEB1-AS1#1, si-ZEB1-AS1#2, and si-ZEB1-AS1#3), ∗P < 0.05, ∗∗P < 0.01 (Kruskal–Wallis test) compared to si-NC and blank control cell group (without transfection). (C,D) HUVECs (treated with ox-LDL or not) were transfected with si-NC or si-ZEB1-AS1#2. Cell viability (C) and LDH release (D) were detected were measured by MTT assay and ELISA, respectively, ∗P < 0.05 (Mann–Whitney U-test), ∗∗P < 0.01 (Kruskal–Wallis test). (E) HUVECs apoptosis was detected by using flow cytometry after treatment with ox-LDL and/or knockdown of ZEB1-AS1 for 24 h. The data showed that ZEB1-AS1 knockdown reversed the apoptosis induced by ox-LDL treatment. ∗P < 0.05, ∗∗P < 0.01 (Mann–Whitney U-test). (F) Nuclear apoptosis was evaluated by…

• 

  FIGURE 2. ox-LDL decreased p53 binding at ZEB1-AS1 promoter region, thereby activating the transcription of ZEB1-AS1. (A) The relative expression of ZEB1-AS1 was detected by qRT-PCR in HUVECs under normal control or ox-LDL condition, and ox-LDL treatment increased ZEB1-AS1 expression in a time-dependent manner. ∗P < 0.05 (Mann–Whitney U-test) compared to previous group. (B) Two binding sites of p53 (–1149, –534) at ZEB1-AS1 promoter were predicted by using online JASPAR software. (C) p53 was overexpressed in HUVECs by transfection of specific overexpression vector, ∗∗P < 0.01 (Mann–Whitney U-test). (D) Relative expression of ZEB1-AS1 was measured via qRT-PCR in cells overexpressed p53, ∗∗P < 0.01 (Mann–Whitney U-test). (E) ChIP assay was performed by using p53 antibody and two primers to detect the immunoprecipitations. (F) The enrichment of p53 at ZEB1-AS1 promoter region was evaluated via ChIP assay in cells treated with ox-LDL or not. The enrichment was significantly suppressed by ox-LDL treatment, ∗P < 0.05 (Mann–Whitney U-test).

• 

  FIGURE 3. NOD2 was verified as a functional target of ZEB1-AS1 in ox-LDL-induced ECs injury. (A) The expression of NOD2 protein in HUVECs under normal or ox-LDL condition was assessed by using immunofluorescence. ox-LDL significantly increased NOD2 expression, ∗P < 0.05 (Mann–Whitney U-test). (B) Western blot analysis showed that ox-LDL treatment decreased NOD2 protein level. (C) The expression of NOD2 mRNA (left panel) and protein (right panel) was downregulated by ZEB1-AS1 knockdown, ∗P < 0.05 (Mann–Whitney U-test). (D) ZEB1-AS1 was overexpressed in HUVECs by transfection of ZEB1-AS1 overexpression plasmid, ∗∗P < 0.01 (Mann–Whitney U-test). (E) NOD2 was silenced in HUVECs at both transcript (left panel) and protein (right panel) levels by transfection of specific siRNA, ∗∗∗P < 0.001 (Mann–Whitney U-test). (F,G) Cell viability (F) and apoptosis (G) were evaluated by MTT assay and flow cytometry assay, respectively. Knockdown of NOD2 attenuated the functional effect induced by ZEB1-AS1 overexpression, ∗P < 0.05 (Mann–Whitney U-test).

• 

  FIGURE 4. ZEB1-AS1 directly interacts with LRPPRC in ECs. (A). Nuclear fraction experiment and qRT-PCR detected the abundance of ZEB1-AS1 in the nucleus and cytoplasm. GAPDH was set as the positive control for cytoplasm, and U1 served as the positive controls for the nucleus. (B) The subcellular distribution of ZEB1-AS1 was visualized by RNA Fluorescent in situ hybridization (FISH) in HUVECs. ZEB1-AS1 was mainly distributed in cytoplasm of HUVECs. (C). Prediction of 421–630 nt ZEB1-AS1 structure was based on minimum free energy (MFE) and partition function (http://rna.tbi.univie.ac.at/). (D) RNA pulldown assay was performed using ZEB1-AS1 incubated with cytoplasm extracts of HUVECs, followed by silver staining. An arrow indicates LRPPRC. (E) Verification of LRPPRC protein by using mass spectrometry. (F) The subcellular distribution of LRPPRC (cytoplasm) was visualized by immunofluorescence in HUVECs. (G) The interaction between ZEB1-AS1 and LRPPRC was confirmed by RNA pulldown and western blotting. GAPDH served as negative controls. (H) RIP was performed using anti-LRPPRC and control IgG antibodies, followed by qRT-PCR to examine the enrichment of ZEB1-AS1 and U6. U6 served as negative…

• 

  FIGURE 5. ZEB1-AS1 regulates mRNA stability of NOD2 via recruiting LRPPRC. (A) LRPPRC was silenced by specific siRNAs at both transcript (down panel) and protein (upper panel) levels, ∗∗P < 0.01 (Mann–Whitney U-test). (B) NOD2 expression was detected in HUVECs overexpressed with ZEB1-AS1 and/or silenced with LRPPRC, ∗P < 0.05 (Mann–Whitney U-test). (C) HUVECs lysates were incubated with in vitro synthesized, biotin-labeled control LacZ DNA probes or DNA probes against ZEB1-AS1 for the biotinylated oligonucleotide pulldown assay. The precipitates from the pulldown were analyzed by qRT-PCR to detect the interacting mRNAs. ∗∗P < 0.01 (Mann–Whitney U-test) compared to respective LacZ probes. (D) RIP was performed using anti-LRPPRC and control IgG antibodies, followed by qRT-PCR to examine the enrichment of NOD2 and U6. U6 served as negative controls. ∗∗P < 0.01 (Mann–Whitney U-test) compared to respective IgG controls. (E) HUVECs expressing si-ZEB1-AS1#2 or si-LRPPRC were treated with actinomycin D (5 μg/mL) for the indicated periods of time. ∗P < 0.05 (Mann–Whitney U-test) compared to si-NC group (F). HUVECs expressing si-NC, or p-ZEB1-AS1, or p-ZEB1-AS1 + si-LRPPRC were treated with…

• 

  FIGURE 6. ZEB1-AS1 modulates ox-LDL-induced ECs injury by stabilization of NOD2 mRNA. (A,B) HUVECs (treated with ox-LDL or not) were transfected with si-NC or si-LRPPRC. Cell viability (A) and LDH release (B) were detected were measured by MTT assay and flow cytometry, respectively, ∗P < 0.05 (Mann–Whitney U-test). (C,D) Cell viability (C) and apoptosis (D) were evaluated by MTT assay and flow cytometry assay, respectively, ∗P < 0.05 (Mann–Whitney U-test). (E) A scheme of the proposed mechanisms. ox-LDL treatment increases ZEB1-AS1 expression by sequestering p53 from binding to the promoter region of ZEB1-AS1 gene, which activates the transcription and upregulates ZEB1-AS1 level in HUVECs. Overexpression of ZEB1-AS1 upregulates NOD2 level by guiding LRPPRC to stabilize its mRNA, resulting in decreased viability and increased apoptosis of ECs.