Fig. 4

Regulation of CRHR1 by miR-34a. a Relative expression of miR-34a in the blood of CRPS patients (Responders n = 8, Poor responders n = 7) b Reporter assay confirming binding of miR-34a to the 3′UTR of CRHR1. Luciferase reporter assay was performed 24 h after cotransfection of HEK293 cells with CRHR1 3′UTR reporter plasmid and miR-34 or control miRNA. c Overexpression of miR-34a in Jurkat cells decreased endogenous CRHR1 mRNA. Jurkat cells were transfected with miR-34a or control miRNA. The relative expression of CRHR1 was determined using qPCR 24 h after transfection and 18S was used as the normalizer. d miR-34a decreased CRHR1 protein expression in Jurkat cells. Western blot analysis of CRHR1 expression in Jurkat cells 48 h after miR-34a or control miRNA transfection. Protein expression was normalized to GAPDH (n = 4, representative blot shown). e miR-34a can repress the CRH-induced increase in POMC expression. Jurkat cells transfected with miR-34a or control miRNA were incubated with 100 nM of CRH for 2 h. POMC expression was determined using qPCR before and after CRH stimulation. 18S was used as the normalizer. All in vitro data represent an average of three or more independent experiments. *p < 0.05. Unpaired t-test was used for statistical analysis. For all panels, data represent mean ± SEM