Cholesterol-Related lncRNAs as Response Predictors of Atorvastatin Treatment in Chilean Hypercholesterolemic Patients: A Pilot Study

Figures

• 

  Figure 1. Expression of genes (a) HMGCR; (b) SREBP2 and lncRNAs (c) RP1-13D10.2; (d) MANTIS; (e) LncHR1 in normal cholesterol group and hypercholesterolemic patients. The relative levels were quantified using real-time PCR applied to total RNA extracted from leukocyte cells of normal cholesterol individuals (NC) and hypercholesterolemic patients (HC). p-value obtained by unpaired Student’s t-test. Data were normalised against the RPL27 reference gene for genes and the U6 reference gene for lncRNAs. The results were obtained from technical duplicates. Circles represent normocholesterolemic individuals, and squares represent hypercholesterolemic patients.

• 

  Figure 2. Expression of the genes (a) HMGCR; (b) SREBP2 and the lncRNAs (c) RP1-13D10.2; (d) MANTIS; (e) LncHR1 in hypercholesterolemic patients treated with atorvastatin. The levels of expression were measured by real-time PCR based on total RNA extracted from leukocyte cells of hypercholesterolemic patients before (baseline) and after treatment with atorvastatin (ATV, 20 mg/day/4 weeks). p-value obtained by paired Student’s t-test. Data were normalised against the RPL27 reference gene for genes and the U6 reference gene for lncRNAs. The results were obtained from technical duplicates. Circles represent hypercholesterolemic patients before treatment with atorvastatin (Baseline) and squares represent the same individuals after treatment with atorvastatin (ATV).

• 

  Figure 3. Analysis by response quartiles of hypercholesterolemic patients treated with atorvastatin. (a) Percentage reduction of LDL-C in the hypercholesterolemic patients treated with atorvastatin 20 mg/day/4 weeks, grouped by extreme quartiles of response to the hypolipidemic drug. The quartiles with greatest and lowest response to the treatment are shown. Relative expression of the lncRNAs in extreme quartiles: (b) RP1-13D10.2; (c) MANTIS; (d) LncHR1. Data were normalised against the U6 reference gene. p-value obtained by unpaired Student’s t-test.

• 

  Figure 4. Dispersion diagrams to illustrate the correlation of expression of the genes (a) HMGCR and (b) SREBP2, associated with the pharmacodynamics of statins, and the downregulated lncRNA lncHR1 in hypercholesterolemic patients after treatment with atorvastatin 20 mg/day/4 weeks. Correlation results obtained by simple linear regression analysis. The line represents the trend line of the data obtained from the correlation of the evaluated genes. The points cor-respond to each of the gene pairs (x value and its respective y value). The asterisk (*) represents significant p value obtained from the analysis of variance.

• 

  Figure 5. THP-1 is differentiated to Mo macrophages by using 50 ng/mL of PMA for 48 h. Morphology of (a) Monocytic THP-1, (b) THP-1 differentiated to Mo macrophage. Cell size and granularity are shown in photographs taken through an Olympus CKX41 inverted microscope (objective 40X). Gene expression of (c) IL1-β, (d) MMP-9 and (e) TNF-α. RT-qPCR was normalised by RPL-27 reference gene. The bars represent the mean expression for each group ± standard deviation. p-value obtained by Student’s t-test. THP-1: monocytic cells; Mo: macrophage derived from THP-1. *** p < 0.0001.

• 

  Figure 6. Effect of atorvastatin on the viability of THP-1 differentiated to Mo macrophages (0; 2.5; 5; 10 and 20 μM atorvastatin). Cell viability tested by MTS assay. The data are expressed as mean ± standard deviation. They were analysed statistically by ANOVA, with Dunnett’s multiple comparison as a post test. CT: control, VH: methanol vehicle, ATV: atorvastatin.

• 

  Figure 7. Expression of the genes (a) HMGCR and (b) SREBP2 and the lncRNAs (c) RP1-13D10.2, (d) MANTIS and (e) lncHR1 in THP-1 cells differentiated to Mo macrophages (0; 2.5; 5; 10 and 20 μM atorvastatin). The data are expressed as mean ± standard deviation. Data were normalised against the RPL27 reference gene for genes and the U6 reference gene for lncRNAs. They were analysed statistically by ANOVA, with Dunnett’s multiple comparison as a post-test (* p ≤ 0.05, ** p ≤ 0.001 and *** p ≤ 0.0001). CT: control, VH: methanol vehicle, ATV: atorvastatin.