Fig. 2: CircRNA-RBCK1 binds to miR-133a in human endocardium endothelial cells (EEC). | Nature Communications

Fig. 2: CircRNA-RBCK1 binds to miR-133a in human endocardium endothelial cells (EEC).

From: Statins improve cardiac endothelial function to prevent heart failure with preserved ejection fraction through upregulating circRNA-RBCK1

Fig. 2

A and B Human EECs were transfected with a plasmid expressing circRNA-RBCK1 for 24 h under ox-LDL (100 μg/ml). Total cell lysates were subjected to perform biotinylated-circRNA-RBCK1 pull-down assay followed by quantitative PCR analyses of circRNA-RBCK1 in (A) and top five candidate miRNAs predicted by CircNet database in (B). C The biotinylated wildtype (WT) or mutant (MT) miR-133a was, respectively, transfected into HEK293 cells with circRNA-RBCK1 overexpression. The levels of circRNA-RBCK1 were tested by quantitative PCR after streptavidin capture. D Plasmid of luciferase reporter construction containing circRNA-RBCK1 sequences with WT or mutated miR-133a binding sites (MT1, MT2, MT3, MT1/2/3) was co-transfected with miRNA negative control (miR-NC) or miR-133a in HEK293 cells. The luciferase activities in total cell lysates were assayed. E Human EECs were pretreated lovastatin (10 μM) for 2 h followed by ox-LDL (100 μg/ml) for 24h incubation. FISH was conducted to determine the co-location between circRNA-RBCK1 and miR-133a in human EECs. Scale bar, 5 µM. Red, circRNA-RBCK1; Green, miR-133a; Blue, nucleus. N = 5 per group in (A–D). Representative microscopy image was obtained from five independent experiments in (E). A one-way ANOVA followed by Tukey post-hoc tests was used to determine P value in A and D. A two-sided unpaired Student’s t test was used to determine P value in (B) and (C). Data are presented as mean ± SD. Source data are provided as a Source Data file.

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