In this study we have comprehensively profiled the expression of miRNAs in PanINs and normal pancreatic duct samples. Many of the differentially expressed miRNAs we identified in PanIN lesions have been also identified as differentially expressed in invasive pancreatic cancers (Supplemental Table 7
). These differentially expressed miRNAs include overexpression of let-7f/g, miR-101, miR-103, miR-106b, miR-146a, miR-15b, miR-155, miR-18a, miR-182, miR-190, miR-193b, miR-194, miR-196b, miR-200a/b, miR-203, miR-21, miR-222, miR-29a/b/c, miR-31, miR-338-3p, miR-429, miR-486-3p, miR-93 and miR95, and underexpression of miR-107, miR-139-3p/5p, miR-216a/b, miR-217, miR-218 and miR-483-5p (13
). Several of these miRNAs have been found to have oncogenic or tumor suppressive functions. These include miR-107, miR-146a, miR-155, miR-200a/b, miR-203, miR-21, miR-216a/b and miR-217(13
The overexpression of let-7a, miR-200 and miR-21 we observed in PanIN lesions confirm the results of Du Rieu et al who found overexpression of these miRNAs in mouse PanIN lesions, and found that the miR-21 upregulation preceded phenotypic changes in pancreatic duct cells (26
). In contrast, LaConti et al found overexpression of miR-10, miR-16, miR-21, miR-100 and miR-155 in mouse PanINs, whereas we identified overexpression of only miR-21 and miR-155 in human PanINs (27
We also identified numerous differentially expressed miRNAs that have not been identified as differentially expressed in pancreatic cancers (Supplemental Table 7
). For example, miR-183, a member of miR-182/183/96 family (44
), targets EGR1 and PTEN, and promotes tumor cell migration (45
). We also identified several differentially expressed miRNAs that had differential expression only in low-grade PanINs (PanIN-1 or PanIN-1/ PanIN-2 lesions). Levels of one such candidate, miR-200c has been reported to influence E-cadherin expression, inhibit invasion of pancreatic cancer cells and correlate with patient survival (40
). Such a pattern of expression is perhaps not surprising given the different phenotypic and molecular profiles of low-grade and high-grade PanINs. Several miRNAs described as elevated in pancreatic cancers including miR-143/145 and miR-34a did not display significantly different expression patterns to normal pancreatic duct samples (Supplemental Tables 2
). None of the miRNAs identified as overexpressed are located at chromosomal loci that are known targets of chromosomal amplification.
The identification of markers of high-grade dysplasia and invasive pancreatic ductal adenocarcinoma such as miR-196b could have implications for pancreatic screening. We and others have been screening individuals with extensive family histories of pancreatic cancer (4
). Pancreatic screening by endoscopic ultrasound typically identifies subtle non-specific changes in the pancreas and when such pancreata are resected PanIN lesions are often found in association with lobulocentric atrophy (49
). However, these imaging alterations are non-specific and are also detected in patients without an increased risk of pancreatic cancer as well as among older individuals and patients with chronic pancreatitis (50
). One important goal of pancreatic screening is to identify high-grade lesions so that patients with significant lesions can be offered pancreatic resection prior to the development of invasive cancer. Markers are needed that are specific for high-grade PanIN-3 lesions and invasive pancreatic ductal adenocarcinoma among patients undergoing pancreatic screening. Such markers could potentially be detected in pancreatic fluid samples obtained after secretin infusion at the time of upper endoscopy. Our results indicate that miR-196b has such specific expression patterns and that miR-196b deserves further investigation to determine if it can identify PanIN-3 among patients undergoing screening.
In summary, we have performed a quantitative real-time PCR analysis of over 700 miRNAs in PanIN lesions to identify aberrantly expressed miRNAs at each PanIN stage. These aberrant miRNA expression patterns may provide insights into pancreatic neoplastic development. The almost exclusive expression of miR-196b in PanIN-3 lesions and pancreatic cancers compared to lower-grade PanINs and normal pancreata suggest that its detection could be useful for identifying PanIN-3 lesions in patients undergoing pancreatic screening.
Statement of Translational Relevance
Pancreatic cancer is the fourth leading cause of cancer death in the United States and is characterized by advanced disease at the time of diagnosis and resistance to most therapeutic treatments. Investigating the precursor neoplasms of pancreatic cancer can help elucidate the molecular mechanisms responsible for the development of pancreatic cancer and can also identify markers that could potentially be used to help identify high-grade precursor neoplasms among patients undergoing pancreatic screening. In this study, we performed comprehensive quantitative analysis of over 700 microRNAs in pancreatic intraepithelial neoplasms (PanINs) and normal pancreatic duct samples to identify differentially expressed miRNAs at each PanIN grade. We found numerous differentially expressed miRNAs. The most specifically overexpressed miRNA in PanIN-3 lesions (carcinoma-in-situ) was miRNA-196. MiRNA-196 has potential utility as a marker of PanIN-3 lesions.