Reported here are the results of the first detailed miRNA expression profiling study in pancreatic ductal adenocarcinoma. Expression profiling identified a large number of miRNAs that are aberrantly expressed in pancreatic ductal adenocarcinoma. A bead-based flow cytometric assay was used previously to profile the expression of 217 miRNAs in nine samples of pancreatic adenocarcinoma. 38
A micro chip assay was recently used to profile the expression of miRNAs in 39 samples of endocrine pancreas cancer.39
Our results in pancreatic ductal adenocarcinoma more closely approximate those of Volinia, et al.,39
in endocrine pancreas tumors and show that the majority of miRNAs are increased in the tumor compared to normal pancreas. Many of the miRNAs that are increased in both pancreas adenocarcinoma and endocrine pancreas cancer are similar, including miR-221, -100, -125b and -21. On the other hand, Lu, et al.,38
report an almost universal decrease in miRNA expression in the pancreatic adenocarcinomas compared to normal pancreas.
miRNAs are believed to function primarily as negative regulators of gene expression following binding to conserved sequences within the 3′ untranslated region of target mRNAs. While the biological roles of miRNA are under intense investigation, they are believed to define and maintain cellular fate in a manner similar to transcription factors40
by regulating developmental timing and differentiation. 41
Since alterations in developmental pathways play a critical role in pancreatic cancer development,42,43
alterations in miRNA expression may be an important contributor to the development of pancreatic adenocarcinoma.
Our study is unique in that a sensitive, real-time PCR assay was used to profile a relatively small number of nonconding RNAs. Real-time PCR is the gold standard of RNA quantification and has much less technical noise and greater reproducibility than traditional cDNA micro-arrays. miRNA expression profiling correctly identified 28 of 28 tissues as tumor (). All 6 normal pancreases were correctly predicted and 11 of 15 adjacent benign tissues were classified as normal tissue (). The data presented here reinforces the argument that miRNA expression profiling may generate a unique molecular signature for a given cancer. This concept is supported by several recent studies. Profiling of various cancers revealed that the pattern of miRNA expression varies markedly across different tumors and that a small number of miRNAs define the cancer better than expression data from 16,000 mRNAs.38
A unique expression signature of only 13 miRNAs differentiated cases of the more aggressive form of chronic lymphocytic leukemia from the more indolent form and was associated with the presence or absence of disease progression.44
The 3 factors that are likely driving the differences in gene expression are the normal acini, stroma and tumor cells. We cannot conclude that each of the aberrantly expressed miRNAs () reflect a difference in expression between normal ductal epithelium and tumor and may reflect differences in expression among the different cell types (stoma, acini and tumor). However, we confirmed by RT in situ PCR that 3 of the top differentially expressed miRNAs miRNAs that were identified in the screen are localized to the tumor cells (). We cannot explain why some of the benign tissues failed to cluster with the normal pancreas (). While 2 levels of quality control were used to reduce the possibility of contaminating tumor cells, it is possible that some tumor cells were present in the benign tissue since the RNA was isolated from whole tissue and not microdissected tissue. Another possibility is that premalignant changes have already occurred in some of the benign tissues as those samples are obtained from tissue adjacent to tumor. It is interesting that most of the benign samples (and chronic pancreatitis as well) lie in between the normal pancreas and tumor on the expression terrain map (). This observation may indeed describe the premalignant alterations that have occurred in these benign tissues. Future studies using RT in situ PCR and perhaps laser microdissection will be able to address these issues in more detail.
Some of the differentially expressed miRNAs in pancreatic cancer were aberrantly expressed in other cancers. These include miR-155, which was increased in the present study and in diffuse large B-cell lymphoma18
; miR-21 was increased here and in glioblastomas, 20,21
and papillary thyroid cancer22
; miR-221 was increased in pancreatic cancer, in glioblastoma21
and in thyroid cancer.22
miR-221 is located ~700 bp from miR-222 on the X chromosome; both miR-221 and miR-222 are predicted to bind to and regulate kit.22,45
miR-222 precursor was not among the top 20 differentially expressed miRNAs (); however, subsequent analysis of mature miR-222 by PCR showed that miR-222 was increased in pancreas cancer at levels that were similar to miR-221 (data not shown). Thus, deregulation of the miRNAs mentioned earlier may be unique to cancer in general. miRNAs differentially expressed in other cancers were not deregulated to the same degree in pancreatic cancer. The let-7 family, decreased in lung cancer, 16,17
was increased here. Expression of the miR-17-92 polycistron (encoding miR-17, -18, -19a, -19b-1 and -92-1) was increased in lymphoma and colorectal cancer24
but was not significantly altered in pancreatic cancer. We report deregulation of a number of miRNAs in pancreatic cancer such as miR-376a and miR-301 that have not been reported in any other cancers to our knowledge. Also of interest is the fact that most of the deregulated miRNAs reported here show increased expression in the tumors compared to the normal pancreas. A few miRNAs had reduced expression in pancreatic cancer including miR-375 (). miR-375 was cloned from pancreas and is believed to be islet cell specific.12
We report significant changes in miRNA expression between pancreatic adenocarcinoma and normal pancreas. Since each miRNA may regulate scores of mRNAs,46
the impact on gene expression in pancreatic cancer may be profound. The miRNA field is currently hampered by a lack of methods to sort through the many hundreds of predicted miRNA target genes. As more sophisticated approaches become available to identify and validate miRNA targets, the role of aberrant miRNA expression in pancreas cancer will become better understood.