Fig 2: Toll-like receptor (TLR)4 pathway induced changes in mRNA and miRNA expression of MEG-01 cells. Volcano plot depicting the number of differently expressed genes in untreated and lipopolysaccharide (LPS)-treated MEG-01 cells (n = 3/condition) at 4 h, observed by RNA-seq analysis, quantified by the DESeq method and plotted based on Log10 (Fold Change) and –log (p-value). There were 354 significantly upregulated, and 1060 downregulated transcripts in LPS-activated MEG-01 cells compared to control cells at a fold change (FC) of =1.5. Dotted lines indicate FC cut-off (A). Clustered heat map of the top 50 differentially expressed genes in LPS-treated MEG-01 cells at 4 h. Genes were displayed as Log (Fold Change) (B). We then validated SELP expression in MEG-01 cells after LPS treatment (n = 6–8/experiment), and TNF-a stimulation was used as a positive control (C). In parallel, miR-26b was significantly attenuated by LPS or TNF-a vs. untreated cells (D). These results were in accordance with the findings in ex vivo septic platelets. The functional relationship between miR-26b and SELP expression was analyzed using specific miRNA mimic transfection in LPS-stimulated MEG-01 cell cultures. The overexpression of miR-26b by mimic (E) resulted in lowered SELP mRNA levels compared to samples with control NEG-01 mimic (F). Mean ± SEM was depicted, * p < 0.05, ** p < 0.01 based on statistical analyses.

Fig 3: 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.