Deregulation of miR-146a expression in a mouse model of pancreatic cancer affecting EGFR signaling

Abstract

Aberrant expression of microRNAs (miRNAs) plays important roles in the development and progression of pancreatic cancer (PC). Expression analysis of miR-146a in human PC tissues showed decreased expression in about 80% of samples compared to corresponding non-cancerous tissue. Moreover, expression of miR-146a in eight PC cell lines, and in pancreatic tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC) and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), showed down-regulation of miR-146a expression in KCI mice which was in part led to over-expression of its target gene, epidermal growth factor receptor (EGFR). Treatment of PC cells with CDF, a novel synthetic compound, led to re-expression of miR-146a, resulting in the down-regulation of EGFR expression. Moreover, re-expression of miR-146a by stable transfection or treatment with CDF in vivo (xenograft animal model) resulted in decreased tumor growth which was consistent with reduced EGFR, ERK1, ERK2, and K-Ras expression. Further knock-down of miR-146a in AsPC-1 cells led to the up-regulation of EGFR expression and showed increased clonogenic growth. In addition, knock-down of EGFR by EGFR siRNA transfection of parental AsPC-1 cells and AsPC-1 cells stably transfected with pre-miR-146a resulted in decreased invasive capacity, which was further confirmed by reduced luciferase activity in cells transfected with pMIR-Luc reporter vector containing miR-146a binding site. Collectively, these results suggest that the loss of expression of miR-146a is a fundamental mechanism for over-expression of EGFR signaling and that re-expression of miR-146a by CDF treatment could be useful in designing personalized strategy for the treatment of human PC.

Keywords: CDF; EGFR; Pancreatic cancer; Xenograft mouse model; miR-146a.

Figure 1

Comparative expression analysis of miR-146a in 29 FNA cell blocks from PC patients individually compared to 15 normal controls using qRT-PCR. 1.0 represents average of normal subjects (n=15). There was a significant down-regulation of miR-146a in most of the 29 PC patients compared to normal subjects (A). Basal levels of miR-146a expression (B) and EGFR expression (C) in human PC cell lines AsPC-1, BxPC-3, COLO-357, L3.6pl, MIAPaCa-2 (MIA-2), MIAPaCa-2-GR (MIA-2-GR), PANC-1 and PANC-28. The miR-146a expression was significantly lower in PC cells and was normalized using SNORD48 miRNA, whereas EGFR expression was higher and was normalized using GAPDH mRNA as assessed by qRT-PCR.

Figure 2

Comparative expression of miR-146a (A) and EGFR (B) in the pancreata of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre; (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI) mouse. There was a significant down-regulation in the expression of miR-146a in KC and KCI pancreata (tumors) compared to either K or C pancreata (normal pancreas). In contrast, the expression of EGFR was significantly up-regulated in KC and KCI tumors. The miR-146a expression was normalized using RNU48 miRNA and EGFR expression was normalized using GAPDH mRNA. P values represent comparison between K/C and KC/ KCI and were found to be significant (*) and less than 0.05.

Figure 3

CDF treatment significantly up-regulated miR-146a expression (A), and decreased the expression of EGFR (B) as assessed by qRT-PCR of AsPC-1, BxPC-3, MIAPaCa-2 and MIAPaCa-2-GR cells. The miR-146a expression was normalized using RNU48 miRNA and EGFR expression was normalized using GAPDH mRNA. UNT: untreated. P values, relative to individual controls, are mentioned over the bars. The relative values of the controls for each cell lines were normalized to 1.0 (unit value).

Figure 4

Over-expression of miR-146a using stably transfected Pre-miR-146a followed by CDF treatment in AsPC-1 cells in vivo showed decrease in tumor growth rate (A). Tumor weight was significantly reduced in pre-146a and pre-146a+CDF, compared to control, in a xenograft model (B). This was consistent with re-expression of miR-146a in miR-146a and miR-146a+CDF group (C), which was associated with decreased EGFR, ERK1, ERK2, and K-Ras expression, compared to control (D). The relative expression of EGFR, ERK1, ERK2, and K-ras proteins were quantified against β-actin (E). P values relative to controls are mentioned over the bars

Figure 5

Inactivation of miR-146a expression by ASO, led to reduced expression of miR-146a as assessed by qRT-PCR (A), increased cell migration by scratch assay (B), increased EGFR expression by western blot analysis (C) and increased in colony formation by clonogenic assay (D). These changes were overcome by CDF treatment. RNU48 miRNA was used to normalize miR-146a expression. Beta-actin was used as loading control for the western blot.

Figure 6

Inactivation of EGFR expression by siRNA in AsPC-1 and AsPC-1 cells transfected with miR-146a. Decrease in EGFR expression was confirmed both at the mRNA and protein levels, respectively (A). Re-expression of miR-146a in AsPC-1+miR-146a was significantly more than AsPC-1 cells by qRT-PCR (B), inhibition of invasive cells in AsPC-1+miR-146a was more than AsPC-1 cells as assessed by invasion assay (C), and it was associated with decrease in luciferase activity observed in miR-146a luciferase vector compared to control vector as assessed by luciferase gene assay in AsPC-1 cells (D). This effect was further enhanced in cells transfected with premiR-146a and with CDF treatment. RNU48 miRNA was used to normalize miR-146a expression, and GAPDH mRNA for EGFR expression. The relative expression of EGFR and miR-146a by qRT-PCR in control siRNA treated cells were normalized to 1.0 (unit value) and were compared with their respective EGFR siRNA treated cells.