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-KRASG12D (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.
Highlights
MicroRNAs display altered patterns of expression in clinical specimens of pancreatic ductal adenocarcinoma (PDAC), with some miRNAs increased and others decreased in the tumors[10,11,12]
The processed, mature miR-216a, miR-216b and miR-217 are pancreas enriched[13,14] with predominate, abundant expression in pancreatic acini15. miR-216a, miR-216b and miR-217 are expressed in brain[16] and to a lesser degree in kidneys, lung and small intestine17. miR-216a, miR-216b and miR-217 expression is reduced in mice and humans with pancreatitis[18,19,20], during PanIN progression[21,22] and in PDAC in both mice and humans[12,19,23,24]
The NIH Genotype-Tissue Expression (GTEx) project data set on 51 normal human tissues was mined to confirm these findings (SFig. 2)
Summary
MicroRNAs (miRNAs) display altered patterns of expression in clinical specimens of pancreatic ductal adenocarcinoma (PDAC), with some miRNAs increased and others decreased in the tumors[10,11,12]. Further evidence supporting a potential tumor suppressive role for these miRNAs in PDAC comes from an evaluation of their reported target genes that include KRAS28,29, ANLN30, E2F331, JAK232 and TPT133. In order to establish a functional role for the three mature miRNAs, we deleted a 27.9 kbp region of miR217HG in mice, the deletion was embryonic lethal at around E9.523. While the specific cause(s) of the lethality are unknown, possibilities include simultaneous deletion of all three miRNAs or deletion of a functional miR217HG lncRNA. To evaluate these possibilities in greater detail, we generated individual germ line KO mice for miR-216a, miR-216b and miR-217 using CRISPR/Cas[9] gene editing. We conclude that miR-216a, miR-216b and miR-217 are not tumor suppressive in pancreas and our work emphasizes the need to evaluate miRNA function beyond in vitro systems
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