Figure 5.

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-α stimulation was used as a positive control (C). In parallel, miR-26b was significantly attenuated by LPS or TNF-α 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.