Knockout of Acinar Enriched microRNAs in Mice Promote Duct Formation But Not Pancreatic Cancer

Abstract

The pancreatic acinar-enriched miR-216a, miR-216b and miR-217 are encoded within the miR217HG. These miRNAs have been purported to play a tumor suppressive role as their expression is reduced in both human and mouse pancreatic ductal adenocarcinoma (PDAC). To examine this possibility, we generated individual, germline knockout (KO) mice of miR-216a, miR-216b or miR-217. Unlike our previous study showing germline deletion of the miR217HG was embryonic lethal, CRISPR-Cas9 deleted portions of the 5' seed region of the miRNAs produced live births. To investigate possible phenotypes during pancreatic acinar ductal metaplasia (ADM), pancreatic acini from wild type and KO mice were plated on collagen and allowed to transdifferentiate over 4 days. Acini from each of the three miRNA KO mice produced greater numbers of ducts compared to controls. Evaluation of the gene expression during in vitro ADM demonstrated an increase in Krt19 and a reduction in acinar genes (Carboxypeptidase A1, Amylase2a) on day 4 of the transdifferentiation. Recovery was delayed for the miR-216a and miR-216b KOs following caerulein-induced acute pancreatitis. Also predominate in the caerulein treated miR-216a and miR-216b KO mice was the presence of pancreatic duct glands (PDGs). To further establish a phenotype, miRNA KO mice were crossed with EL-KRAS^G12D (EK) mice and followed up to 13 months of age. While all mice developed severe dysplasia and cystic papillary neoplasms, there existed no apparent phenotypic difference in the miRNA KO/EK mice compared to EK mice. Our data does not support a tumor suppressor role for miR-216a, miR-216b or miR-217 in PDAC and emphasizes the need for phenotypic evaluation of miRNAs in complex in vivo models beyond that performed using cell culture.

Figure 1

miRNA expression in the pancreata of individual knockout mice. TaqMan qRT-PCR was performed on miRNAs isolated from the pancreata of 13 month old A. miR-216a, B. miR-216b and C. miR-217 knockout mice. The expression of each individual miRNA, relative to 18S rRNA, is depicted on the y-axis of each graph. ND, not detectable.

Figure 2

miRNA knockout accelerates in vitro pancreatic acinar ductal metaplasia. Mouse pancreatic acinar cells were plated onto collagen and the transdifferentiation occurred over 4 days. Bright field images were captured at Day 1 (A–D) and Day 4 (E–H) of culture for the wild type (A,E), miR-217 (B,F), miR-216a (C,G) and miR-216b (D,H) KOs. (I) Number of ducts after 4 days of culture in the wild type and individual knockout mice. RNA was isolated from the cultures at days 1 to 4 of the transdifferentiation and the expression of J. cytokeratin 19, K. amylase 2A and L. carboxypeptidase A2 was determined by qRT-PCR. Data are presented relative to 18S rRNA (mean ± SEM) and were normalized to the day 0 controls. *P < 0.05 (Student’s t-test).

Figure 3

Recovery from acute pancreatitis. A. Wild type or miR-217, miR-216b or miR-216a knockout mice (2 month old) were dosed daily with eight injections of caerulein for two days. B. Mice were sacrificed at 2, 4 and 7 days following the last caerulein dose and the pancreata were processed for histology. 20X magnification.

Figure 4

Presence of pancreatic ductal glands during acute pancreatitis. The presence of pancreatic ductal glands (PDGs, yellow arrows) was observed in the miR-216b and miR-216a knockout mice 2, 4 and 7 days, following caerulein-induced acute pancreatitis. Pancreata were stained with A,C hematoxylin and eosin or B,D periodic acid Schiff (PAS). Green asterisks represent enlarged ducts on the PAS stained sections. 20X magnification.

Figure 5

Histopathology of miRNA knockout and Kras^G12D mouse crosses. The miR-216a, miR-216b and miR-217 KO mice were crossed with the EL-KRAS^G12D mice. The histopathology from the pancreata of 3 month old mice are shown at 4× (upper panels) and 20× (lower panels) magnification.