Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A – E ) Male and female C57BL/6 mice were administered a single dose of AAV8-PCSK9 (w/o 3′-UTR) or AAV8 PCSK9-3′-UTR (with 3′-UTR) via tail vein injection ( n = 8 in each group) and fed an HFD for 12 weeks before killing. The gross appearance of mouse serum ( A ); serum levels of total cholesterol and triglycerides ( B ) ; FPLC detection of VLDL, LDL, and HDL ( C ); and representative Oil-red O staining of en face aortae ( D ) and aortic roots ( E ) are shown (original magnification, ×6; scale bars: 0.5 mm). In B , D , and E , data are mean ± SEM from 6–8 mice per group. Normally distributed data were analyzed by 2-tailed Student’s t test with Welch correction between 2 groups. * P < 0.05 vs. AAV8-PCSK9. AAV8, adeno-associated virus 8; PCSK9, proprotein convertase subtilisin/kexin type 9; AAV8-PCSK9-3′-UTR, AAV8-based recombinant virus encoding mouse Pcsk9 mRNA encompassing its native 3′-UTR; HFD, high-fat diet; FPLC, fast protein liquid chromatography; VLDL, very LDL-cholesterol.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Injection, Staining, Virus, Recombinant, Fast Protein Liquid Chromatography

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A and D ) The animals were used as described in . The hepatic Pcsk9 mRNA levels ( A ), ELISA detection of serum levels of PCSK9 ( A ), and hepatic levels of miR-191, -222, -224, and -483 ( D ) are shown. ( B ) Previously reported (blue lines) or newly predicted (green lines) miRs that bind to the h PCSK9 -3′-UTR are shown. ( C and E ) HepG2 cells were transfected with pre–miR-222 mimic (222), pre–miR-224 mimic (224), pre–miR-483 mimic (483), pre–miR-191 mimic (191), pre–miR-1912 mimic (1912), pre–miR-1295b mimic (1295b), or scramble miR control (Ctrl). In C , cells were cotransfected with Luc-PCSK9-3′-UTR reporter. Luciferase activity was measured with pRL-TK activity as a transfection control. In E , protein levels of PCSK9 and LDLR were determined by Western blot analysis; α-tubulin was a loading control. In A and D , data are mean ± SEM from 6–8 mice per group. Normally distributed data were analyzed by 2-tailed Student’s t test with Welch correction between 2 groups. In C and E , data are mean ± SEM from 3–4 independent experiments. Non-normally distributed data were analyzed using Mann-Whitney U test between indicated group and control. * P < 0.05 vs. AAV8-PCSK9 or Ctrl. miR, microRNA; PCSK9, proprotein convertase subtilisin/kexin type 9; HepG2, human hepatocellular carcinoma; LDLR, LDL receptor.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Control, Luciferase, Activity Assay, Western Blot, MANN-WHITNEY

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A ) Bioinformatics prediction of miR-483-5p binding sites in the 3′-UTR of human and mouse PCSK9 mRNA. In B – G , HepG2 cells were transfected with pre–miR-483 mimic (pre-483) or anti–miR-483 (anti-483) for 24 hours. ( B and C ) mRNA and protein levels of PCSK9 and LDLR. ( D ) HepG2 cells transfected with pre-483 or anti-483 were cotransfected with Luc-PCSK9-3′-UTR (WT) or Luc-mutated PCSK9-3′-UTR (MT). Luciferase activity was measured with pRL-TK activity as a transfection control. ( E ) Ago-1 or Ago-2 immunoprecipitation was performed, and miRISCs-associated miR-483, PCSK9 , and CTGF mRNA levels were quantified by qPCR. ( F and G ) mRNA and protein levels of PCSK9 and LDLR in WT HepG2 and mPCSK9 HepG2 cells were transfected with pre-483 or anti-483. # LDLR in the same samples were detected in parallel in a separate gel ( G ). Data are mean ± SEM from 3–4 independent experiments. In B – G , non-normally distributed data were analyzed using Mann-Whitney U test between indicated group and control. * P < 0.05 vs. control. miR, microRNA; PCSK9, proprotein convertase subtilisin/kexin type 9; HepG2, human hepatocellular carcinoma; LDLR, LDL receptor; Luc-PCSK9-3′-UTR (WT), WT PCSK9 3′-UTR; Luc-mutated PCSK9-3′-UTR (MT), mutant PCSK9 3′-UTR; miRISCs, miRNA-induced silencing complexes; CTGF , connective tissue growth factor.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Control, Immunoprecipitation, MANN-WHITNEY, Mutagenesis

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A – H ) HepG2 and mPCSK9 HepG2 cells were transfected with pre-483 or anti-483 as indicated. Fluorescent-labeled LDL was incubated with HepG2 and mPCSK9 HepG2 cells. LDL uptake was detected by flow cytometry ( A and B ) or confocal microscopy ( C and D ) (original magnification, ×20; scale bars: 10 μm). ( E and F ) Levels of PCSK9 in conditioned media were measured by ELISA and Western blot analysis. ( G and H ) HepG2 and mPCSK9 HepG2 cells were incubated with 1 μM atorvastatin for 24 hours. mRNA and protein levels of PCSK9 and LDLR were determined by qPCR and Western blot analysis. In MT HepG2 cells, # LDLR in the same samples were detected in parallel in a separate gel ( H ). Data are mean ± SEM from at least 4 independent experiments. In A , B , and F , non-normally distributed data were analyzed using Mann-Whitney U test between 2 groups. In C – E , normally distributed data were analyzed by 2-tailed Student’s t test with Welch correction between 2 groups. In G and H , non-normally distributed data were analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. * P < 0.05 vs. Ctrl or between 2 indicated groups. miR, microRNA; HepG2, human hepatocellular carcinoma; LDL-C, LDL-cholesterol; PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Transfection, Labeling, Incubation, Flow Cytometry, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A ) Male and female C57BL/6 mice were fed an HFD or chow diet for 6 weeks. A single dose of AAV8-null (AAV-null) or AAV8-pri-miR-483 (AAV-483) was administered by tail vein injection at the end of week 2. ( B and C ) Hepatic miR-483-5p levels were determined by qPCR, protein levels of PCSK9, LDLR, and CTGF were detected by Western blot. # CTGF in the same samples were detected in parallel in a separate gel ( C ). ( D ) Ago-1 was immunoprecipitated from fixed liver tissue, and Ago1-associated miR-483-5p, Pcsk9 , and Ctgf mRNA levels were quantified by qPCR. ( E ) Total cholesterol levels measured by cholesterol assay. ( F ) Serum levels of VLDL, LDL, and HDL were determined by FPLC. ( G ) The correlations between hepatic miR-483-5p expression levels and serum levels of PCSK9 (left) or total cholesterol (right) are shown. ΔCT represents the difference between the cycle threshold of miR-483-5p and U6. The numbers of mice used are shown in . Data are mean ± SEM. In B , non-normally distributed data were analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. In C and D , non-normally distributed data were analyzed using Mann-Whitney U test. In E , normally distributed data were analyzed by 1-way ANOVA test with a Bonferroni’s post hoc test between 2 indicated groups. In G , the correlation analysis was assessed by the Pearson method. * P < 0.05. miR, microRNA; LDL-C, LDL-cholesterol; HFD, high-fat diet; AAV8, adeno-associated virus 8; PCSK9, proprotein convertase subtilisin/kexin type 9; LDLR, LDL receptor; CTGF, connective tissue growth factor; VLDL, very LDL; IDL, intermediate-density lipoprotein.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Injection, Western Blot, Immunoprecipitation, Cholesterol Assay, Expressing, MANN-WHITNEY, Virus

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A ) Male and female C57BL/6 mice were administered AAV8-PCSK9-3′-UTR WT (WT) or AAV8-PCSK9-3′-UTR with a deleted miR-483 binding site (ΔBS) together with AAV-miR-483 or AAV-null by tail vein injection ( n = 6–13 in each group). All mice were fed an HFD for 8–10 weeks. ( B ) Levels of Pcsk9 and Ldlr mRNA in mouse liver were determined by qPCR. ( C ) Protein levels of hepatic LDLR were detected by Western blot analysis. ( D – F ) Serum levels of total cholesterol, VLDL, LDL, and HDL are shown. ( G ) Serum levels of PCSK9 assessed by ELISA are shown. The numbers of mice used are shown in . Data are mean ± SEM. In B , normally distributed data was analyzed by 1-way ANOVA test with a Bonferroni’s post hoc test between 2 indicated groups. In C , D , and G , non-normally distributed data were analyzed using Mann-Whitney U test between 2 indicated groups. * P < 0.05 vs. WT. miR, microRNA; LDL-C, LDL-cholesterol; AAV8, adeno-associated virus 8; PCSK9, proprotein convertase subtilisin/kexin type 9; AAV8-PCSK9-3′-UTR, AAV8-based recombinant virus encoding mouse Pcsk9 mRNA encompassing its native 3′-UTR; HFD, high-fat diet; LDLR, LDL receptor; VLDL, very LDL.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Binding Assay, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Virus, Recombinant