Figure 7.

Investigation of the functional role of Dicer enzyme in attenuated miRNA levels in sepsis. First, to mimic sepsis-induced alteration of the Dicer1 level, DICER1 expression was downregulated via siRNA transfection in MEG-01 cells for the analysis of miR-26b and its target SELP mRNA. Dicer1 specific siRNA was successfully transfected into MK cell cultures based on the highly elevated level of Dicer1 siRNA measured by RT-qPCR (A). This resulted in decreased DICER1 mRNA expression compared to control samples with NEG-01 siRNA (B). In these cells, reduced miR-26b (C) with elevated SELP mRNA levels were quantified (E). In addition, Dicer1-independent miR-451 (D) was analyzed to double-check the specificity of siRNA transfection that showed no change due to manipulation. Second, calpain inhibition was applied to alter miRNA levels in MEG-01 cells among inflammatory conditions in vitro. Calpeptin significantly restored miR-26b (F) levels when was used during LPS treatment, suggesting the role of abnormal Dicer function in miRNA expression of sepsis. Mean ± SEM was depicted (n = 4–6/experiment). * p < 0.05, ** p < 0.01 vs. NEG-01 or untreated samples based on statistical analyses.