Figure 4 | Scientific Reports

Validation of CARHSP1 as a functional target of miR-155.

(A) The predicted binding site of miR-155 in the 3′-UTR of CARHSP1 is indicated. A CARHSP1 3′-UTR mutant with a mutation in the miR-155 binding site is also shown. (B,C) After co-transfection with the pRL-TK plasmid carrying a wild type or mutant 3′-UTR sequence and a miR-155 mimic (10 pmol) or a miR-155 inhibitor (10 pmol) into HEK-293T cells for 48 h, the luciferase activity was measured. (D,E) The protein and mRNA level of CARHSP1 was measured by western blot and qRT-PCR after transfection with the miR-155 mimic (100 nM), the miR-155 inhibitor (100 nM), the mimic control, or the inhibitor control into THP-1 cells at 48 h. (F,G) Lipid uptake by foam cells was detected by Oil Red O staining after transfection of the pCMV6-AC-CARHSP1 plasmid (1 μg), CARHSP1 siRNA (siCARHSP1, 50 pmol/ml), plasmid control, or siRNA control (siCtrl) for 24 h in macrophages, which were treated with oxLDL for 24 h (magnification, 400 × ). (H,I) Western blot analysis and Oil Red O staining were employed to examine the protein level of CARHSP1 and TNF-α after transfection with miR-155 mimic control, miR-155 mimic, pCMV6-AC-GFP (defined as Vector), or pCMV6-AC-CARHSP1 or co-transfection with miR-155 mimic and pCMV6-AC-CARHSP1, with β-actin as western blot control. ***P < 0.001. Results are presented as mean ± SD of 3 independent experiments as determined by performing a Student’s t test (B,C,E). Full-length blots are presented in Supplementary Figures S4 and S5.