The Pathogenic Role of Long Non-coding RNA H19 in Atherosclerosis via the miR-146a-5p/ANGPTL4 Pathway

Figure 1. LncRNA H19 was increasingly expressed in the atherosclerotic plaques of apoE−/− mice and macrophages after ox-LDL induction. (A,B) lncRNA H19 expression levels in the serum and aorta of apoE−/− and C57BL/6 mice were measured using qRT-PCR. (C) FISH analysis of the aorta roots in apoE−/− and C57BL/6 mice. Quantification of lncRNA H19 expression level in the aorta of apoE−/− and C57BL/6 mice. (D) The subcellular localization and expression level of lncRNA H19 in ox-LDL-stimulated THP-1-derived macrophages were detected using FISH (200×). Quantification of lncRNA H19 expression level. (E) THP-1 cells were treated with ox-LDL (50 and 100 μg/mL) or their controls for 24 h. The lncRNA H19 expression was detected utilizing RT-PCR. The experiment was repeated thrice independently. Data are represented as mean ± SD. **P < 0.01, and ***P < 0.001 vs. the control cohort.

Figure 2. lncRNA H19 facilitated lipid accumulation in ox-LDL-treated THP-1 cells. (A–C) THP-1 cells were transfected with sh-NC, sh-H19, OE-NC, or OE-H19 for 48 h. (A) lncRNA H19 expression was determined using RT-PCR (n = 3). (B) Oil red O-stained cells were treated with ox-LDL (100 μg/mL) and evaluated after lncRNA H19 knockdown or overexpression. Typical images of oil red O staining results (200×). Isopropanol extraction was performed for oil red O staining. Oil red O absorbance after isopropanol extraction was measured by quantifying lipids. Illustrative images of oil red O staining (200×). Scale bar = 50 μm. (C) Illustrative images demonstrating Dil-ox-LDL uptake after incubation for 1 h. The calculation of the fluorescence intensity was done utilizing Image J. Data are represented as mean ± SD of 3 separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control cohort.

Figure 3. LncRNA H19 aggravates atherosclerosis in apoE−/− mice. (A) Flow chart demonstrating the experimental protocol utilized in the in vivo experiments. (B,C) RT-PCR analysis of lncRNA H19 expression in the blood and aorta of atherosclerotic lesions of AAV-sh-NC-, AAV-sh-H19-, AAV-OE-NC-, and AAV-OE-H19-treated apoE−/− mice. (D) Plaques in the aortic arch of apoE−/− mice observed using a stereoscopic microscope. (E) Parts of the aortic root was stained with Mason, oil red O, or HE. The lesion area and proportion were computed utilizing the Image J software (n = 6). Scale bar = 100 μm. (F) The whole aorta was stained with oil red O, and the area of atherosclerotic lesion was computed by analyzing oil red O absorbance after isopropanol extraction (n = 6). Data are represented as mean ± SD of 3 separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control cohort.

Figure 4. Validation of lncRNA H19 as a ceRNA for miR-146a-5p. (A) Schematic illustration of the miR-146a-5p binding site in the lncRNA H19 sequence and matching mutant. (B) The luciferase reporter plasmids lncRNA H19-WT or lncRNA H19-Mut were co-transfected into HEK-293T cells with miR-146a-5p mimic or its negative control for 48 h, ensued by the identification of luciferase activity. (C) THP-1 cells were transfected with sh-NC, sh-H19, OE-NC, or OE-H19 for 48 h. RT-PCR was carried out to identify miR-146a-5p expression. (D,E) C57BL/6 and apoE−/− mice were supplied with a Western diet for 8 weeks (n = 6). miR-146a-5p expression in the blood and aorta were detected using RT-PCR. (F) THP-1 cells were treated with ox-LDL (100 μg/mL) or their controls for 24 h. The expression of miR-146a-5p was evaluated using RT-PCR. (G) THP-1 macrophages were transfected with NC inhibitor or miR-146a-5p inhibitor for 48 h, followed by oil red O staining. Data are represented as mean ± SD of 3 separate experiments. **P < 0.01 and ***P < 0.001.

Figure 5. Identification of ANGPTL4 as a target gene of miR-146a-5p. (A) Schematic illustration of the miR-146a-5p binding site in the 3′-UTR of ANGPTL4 and the matching mutant. The mutated nucleotides were marked purple. (B) The luciferase reporter plasmids (ANGPTL4-WT and ANGPTL4-Mut) were co-transfected into HEK-293T cells with miR-146a-5p mimic or its control. The luciferase activity was assessed after a duration of 48 h. (C–E) THP-1 cells were transfected with miR-146a-5p mimic/inhibitor or their negative controls for 48 h.; (C) RT-PCR analysis of miR-146a-5p expression. (D,E) The mRNA and protein levels of ANGPTL4 were detected utilizing RT-PCR and WB, in that order. Data are articulated as mean ± SD of 3 separate experiments. ***P < 0.001.

Figure 6. ANGPTL4 promoted lipid uptake in ox-LDL-treated THP-1 cells. (A) Heat map of 10 topmost downregulated and upregulated genes in BMDMs treated with oleic acid or control for 5 h. The data were obtained from a previously published dataset (GSE77104). (B) THP-1 cells were treated with ox-LDL (50 and 100 μg/mL) or their controls for 24 h. ANGPTL4 expression was evaluated using RT-PCR. (C,D) C57BL/6 and apoE−/− mice were supplied with a Western diet for 8 weeks (n = 6). ANGPTL4 expression in the blood and aorta was detected using RT-PCR. (E–G) THP-1 cells were transfected with si-ANGPTL4 or its negative control for 48 h. (E,F) The protein and mRNA levels of ANGPTL4 were detected using RT-PCR and WB, successively. (G) Oil red O assay. Data are articulated as mean ± SD of 3 separate experiments. **P < 0.01 and ***P < 0.001.

Figure 7. LncRNA H19 sponged miR-146a-5p to increase ANGPTL4 expression. (A,B) THP-1 cells were transfected with sh-NC, sh-H19, OE-NC, and OE-H19 for 48 h. The protein and mRNA levels of ANGPTL4 were detected utilizing RT-PCR and WB, successively. (C,D) THP-1 macrophages were transfected with miR-146a-5p inhibitor for 48 h and subsequently transduced with sh-H19 for 48 h (n = 3). The protein and mRNA levels of ANGPTL4 were measured utilizing RT-PCR and WB, successively. (E,F) THP-1 cells were transfected with miR-146a-5p inhibitor for 48 h and subsequently transduced with OE-H19 for 48 h (n = 3). The mRNA (E) and protein (F) levels of ANGPTL4 were determined utilizing RT-PCR and WB, in that order. Data were articulated as mean ± SD. **P < 0.01 and ***P < 0.001 vs, control cohort.