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. 2025 Oct;45(10):1969-1971.
doi: 10.1161/ATVBAHA.124.322353. Epub 2025 Jul 31.

Effective Transcriptional Induction of the CARMN/miR-143/145 Complex Locus in Smooth Muscle Cells Using CRISPR Activation

Francesca Vacante  1   2 , Sandra Sanchez-Esteban  1 , Themistoklis M Tsarouchas  3   2 , Julie Rodor  1 , Matthew Bennett  1 , Melissa S Carroll  4 , Abdelaziz Beqqali  1 , Andrew H Baker  1   5
Affiliations

Effective Transcriptional Induction of the CARMN/miR-143/145 Complex Locus in Smooth Muscle Cells Using CRISPR Activation

Francesca Vacante et al. Arterioscler Thromb Vasc Biol. 2025 Oct.
No abstract available

Keywords: Cis-activation; RNA, long noncoding; cardiovascular diseases; microRNAs; smooth muscle cell.

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Conflict of interest statement

No potential competing interest was reported by the author(s).

Figures

Figure.
Figure.
Effective activation of the human smooth muscle cell (SMC)–specific CARMN/miR-143/145 locus. A, (i) Schematic representation of CARMN/miR-143/145 locus, ChIP-Seq (H3K4me3) tracks, and CAGE-seq (Cap Analysis Gene Expression sequencing) peaks. The figure provides details of the location of the sgRNAs used to activate transcriptional start site (TSS) 1 and TSS2 and the SYBR green primers used in this study; (ii) Table displaying the sequences of sgRNA, SYBR green primers, and TaqMan probes used to amplify CARMN and miR-143/145 transcripts. B, (i) qRT-PCR expression of CARMN/miR-143/145 in HEK293T after transfection with plasmids expressing the VPR system and the sgRNAs activating promoters 1 and 2 (n=3). One-way ANOVA with Bonferroni test for multiple comparisons was used for statistical evaluation; (ii) Expression of pri-microRNAs (miRNAs) after activation of promoters 1 and 2 (n=3). One-way ANOVA with Bonferroni test for multiple comparisons was used for statistical evaluation. C, (i) Simplified representation of exons amplified with primers designed throughout the locus; (ii) qRT-PCR expression of different exon regions detected after activation (n=3). Student t test with Mann-Whitney U test was used for statistical evaluation. D, Expression of CARMN/miR-143/145 after delivery of an adenovirus expressing the VPR system and the sgRNA targeting promoter 1 or control virus after cholesterol loading assay with CMBCD (10 µg/mL, 72 hours) or control treatment (0.2% BSA; n=5). One-way ANOVA with Holm-Sidak test for multiple comparisons was used for statistical evaluation. E, (i) qRT-PCR of SMC markers ACTA2 (smooth muscle actin alpha 2) and MYH11 (myosin heavy chain 11) and (ii) proinflammatory markers CD68 and LGALS3 after treatment with CMBCD or control in sgCTR and sgRNA targeting promoter 1 (sgP1) conditions (n=5). One-way ANOVA with Holm-Sidak test for multiple comparisons was used for statistical evaluation. F, Immunostaining showing ACTA2 expression after treatment with CMBCD or control in sgCTR and sgP1 conditions. DAPI (4',6-diamidino-2-phenylindole) was used to stain cell nuclei. Scale bar 50 µm. UBC and RNU48 were used as housekeeping controls for qRT-PCR data. Fold changes were calculated by using the 2−ΔΔCt method. Data are presented as bar charts of the SEM with data points for individual replicates (GraphPad 10). Schematics were created using biorender.com.
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References

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