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. 2022 Jul 14;12:11984. doi: 10.1038/s41598-022-16017-5

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Selection of effective and specific anti-microRNA oligonucleotide (AMOs) against miR-33a and miR-33b. (a) Experimental scheme for reporter assay. In the absence of anti-microRNA (miR), intrinsic miR inhibits luciferase expression (left). Adding anti-miR inhibits intrinsic miR, and the expression of luciferase will increase. Luciferase intensity is proportional with inhibition efficiency of anti-miRs (right). (b) Reactivities of miR-33a perfect match (33a PM) and miR-33b perfect match (33b PM) reporter vectors in HepG2 cells (human hepatocellular carcinoma cell line), n = 3. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ***P < 0.001. (c) Evaluation of binding specificity of the reporter vectors to miR-33a-mimic (33a mimic) and miR-33b-mimic (33b mimic). (d) Screening for candidates of AMOs against miR-33a and miR-33b by efficacy and specificity. (e) Expression levels of miR-33a (left, n = 3) and miR-33b (right, n = 3) in HepG2 cells transfected with the selected AMOs against miR-33a and miR-33b at a concentration of 5 nM. One-way ANOVA with Dunnett’s multiple comparison test. *P < 0.01. (f) Absolute copy number of miR-33a and miR-33b in THP-1 macrophages with or without tumor necrosis factor α (TNF-α) treatment. Two-way ANOVA with Holm–Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001. (g) Expression levels of sterol regulatory element-binding transcription factor (SREBF1) and ATP binding cassette transporter A1 (ABCA1) in THP-1 macrophages with or without TNF-α treatment determined using quantitative real-time PCR. Two-way ANOVA with Holm–Sidak’s multiple comparisons test. **P < 0.01 and ***P < 0.001. (h) Absolute copy number of miR-33a and miR-33b in HASMCs and EA.hy926 with or without TNF-α treatment (right). Expression levels of SREBF1 and ABCA1 in HASMCs and EA.hy926 with or without TNF-α treatment (left) determined using quantitative real-time PCR. Two-way ANOVA with Holm–Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001. ns is defined as not significant. All data represent mean ± SEM. Incubation with TNF-α was as follows: 3 h at 25 ng/mL for mouse peritoneal macrophages (PMs), 4 h at 10 ng/mL for mouse vascular smooth muscle cells (VSMCs), and 1 h at 50 ng/mL for mouse aortic endothelial cells (ECs).

Selection of effective and specific anti-microRNA oligonucleotide (AMOs) against miR-33a and miR-33b. (a) Experimental scheme for reporter assay. In the absence of anti-microRNA (miR), intrinsic miR inhibits luciferase expression (left). Adding anti-miR inhibits intrinsic miR, and the expression of luciferase will increase. Luciferase intensity is proportional with inhibition efficiency of anti-miRs (right). (b) Reactivities of miR-33a perfect match (33a PM) and miR-33b perfect match (33b PM) reporter vectors in HepG2 cells (human hepatocellular carcinoma cell line), n = 3. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. ***P < 0.001. (c) Evaluation of binding specificity of the reporter vectors to miR-33a-mimic (33a mimic) and miR-33b-mimic (33b mimic). (d) Screening for candidates of AMOs against miR-33a and miR-33b by efficacy and specificity. (e) Expression levels of miR-33a (left, n = 3) and miR-33b (right, n = 3) in HepG2 cells transfected with the selected AMOs against miR-33a and miR-33b at a concentration of 5 nM. One-way ANOVA with Dunnett’s multiple comparison test. *P < 0.01. (f) Absolute copy number of miR-33a and miR-33b in THP-1 macrophages with or without tumor necrosis factor α (TNF-α) treatment. Two-way ANOVA with Holm–Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001. (g) Expression levels of sterol regulatory element-binding transcription factor (SREBF1) and ATP binding cassette transporter A1 (ABCA1) in THP-1 macrophages with or without TNF-α treatment determined using quantitative real-time PCR. Two-way ANOVA with Holm–Sidak’s multiple comparisons test. **P < 0.01 and ***P < 0.001. (h) Absolute copy number of miR-33a and miR-33b in HASMCs and EA.hy926 with or without TNF-α treatment (right). Expression levels of SREBF1 and ABCA1 in HASMCs and EA.hy926 with or without TNF-α treatment (left) determined using quantitative real-time PCR. Two-way ANOVA with Holm–Sidak’s multiple comparisons test. *P < 0.05, **P < 0.01 and ***P < 0.001. ns is defined as not significant. All data represent mean ± SEM. Incubation with TNF-α was as follows: 3 h at 25 ng/mL for mouse peritoneal macrophages (PMs), 4 h at 10 ng/mL for mouse vascular smooth muscle cells (VSMCs), and 1 h at 50 ng/mL for mouse aortic endothelial cells (ECs).