Figure 8: Analysis of miRNA significantly differing between wt and CD44v6kd or Tspan8kd PaCa cells and TEX. (a) miRNA that is significantly higher in A818.4-wt than -CD44v6kd and/or -Tspan8kd cells and (c) miRNA that is significantly higher in A818.4-wt than -CD44v6kd and/or -Tspan8kd TEX; no significant differences: empty bar; (b, d) qRT-PCR examples of miRNA differing between wt and CD44v6kd and/or Tspan8kd cells or TEX (RQ values ± SD of 3 replicates; p values are indicated; the experiments were repeated 2 times). (e, f) IPA-based Reactome analysis was used for correlating miRNA with protein expression according to mRNA predictions by the miRNA, DIANA, and target scan databases. (e) miRNA higher in A818.4-wt than -CD44v6kd cells and their engagement in ECM interactions, proteoglycans in cancer, stem cell signaling, and cancer-related signaling are shown. (f) miRNA more abundant in wt than CD44v6kd TEX was analyzed as in (e); (e, f) crossed miRNA (4 miRNA higher in wt than CD44v6kd cells, 7 miRNA higher in wt than CD44v6kd TEX) are not engaged in the listed cancer-related activities. (g, h) Clustered heatmap analysis (p values under the selected threshold and 1) of the potential impact of miRNA that is reduced in CD44v6kd cells (g) and TEX (h) on predicted targets engaged in cancer relevant activities; pathway of interest is framed and underlined in blue; most frequently engaged miRNA are framed and underlined in green; in (g) miR-31-3p (framed and underlined in grey) was included as a negative control not affecting the pathways of interest. (i, j) Number of genes and significance of overrepresentation of predicted mRNA of selected CD44v6-dependent miRNA in cells (i) and TEX (j). Only CD44v6 affects miRNA processing and/or recruitment into ILV. The majority of miRNA distinctly processed with dependence on CD44v6 have predicted targets in cancer-related proteoglycans as well as in maintaining stemness and cancer-related signaling. Statistical evaluation of predicted genes overrepresentation confirmed a stronger impact on TEX than on cell miRNA.