Figure 6.

Manipulation of miRNA-23b leads to altered cellular invasion, migration, and PTEN and MTSS1 expression. (A) Schematic of miR-23b binding site in PTEN mRNA 3′ UTR sequence at nucleotide position 1608 to 1615. (B) DIANA LAB miTG prediction score of the probability that miR-23b will target PTEN. The closer to 1.0 the miTG score, the higher the probability of targeting. (C) qPCR analysis of relative miR-23b expression levels in PDAC epithelial cells or PDAC CAFs. (D) PANC-1 cells were transiently transfected with miR-23b27b cluster plasmid. These cells showed robust loss of PTEN expression upon Western blot examination. (E) qPCR confirmation of PANC-1 cells transiently transfected with anti–miR-23b inhibitor. (F) PANC-1 cells treated with anti–miR-23b were significantly less able to travel across the wound in a scratch assay setting as compared to (−) control. (G) PANC-1 cells treated with anti–miR-23b were significantly less able to travel through a Matrigel-coated membrane in a Transwell invasion assay setting as compared to (−) control. (H) Treatment of PANC-1 cells with anti–miR-23b resulted in an increase in expression levels of both PTEN and MTSS1 as confirmed via Western blot analysis. (I) Schematic for the hypothesized mechanism of action for the downregulation of MTSS1 via PTEN loss. The dense tumor microenvironment, majorly composed of CAFs, contains higher amounts of miRNA-23b. miRNA-23b is then secreted to the PDAC tumor epithelium, where it targets PTEN, downregulating it. This downregulation leaves MTSS1 unstabilized from proteasomal degradation and thus results in loss of MTSS1 and thereby increased metastatic potential. *P value < .05, **P value < .001, ***P value < .0001.