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Comparative Study
, 120 (5), 1046-54

Expression Profiling Identifies microRNA Signature in Pancreatic Cancer

Affiliations
Comparative Study

Expression Profiling Identifies microRNA Signature in Pancreatic Cancer

Eun Joo Lee et al. Int J Cancer.

Abstract

microRNAs are functional, 22 nt, noncoding RNAs that negatively regulate gene expression. Disturbance of microRNA expression may play a role in the initiation and progression of certain diseases. A microRNA expression signature has been identified that is associated with pancreatic cancer. This has been accomplished with the application of real-time PCR profiling of over 200 microRNA precursors on specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. Hierarchical clustering was able to distinguish tumor from normal pancreas, pancreatitis and cell lines. The PAM algorithm correctly classified 28 of 28 tumors, 6 of 6 normal pancreas and 11 of 15 adjacent benign tissues. One hundred microRNA precursors were aberrantly expressed in pancreatic cancer or desmoplasia (p < 0.01), including microRNAs previously reported as differentially expressed in other human cancers (miR-155, miR-21, miR-221 and miR-222) as well as those not previously reported in cancer (miR-376a and miR-301). Most of the top aberrantly expressed miRNAs displayed increased expression in the tumor. Expression of the active, mature microRNA was validated using a real-time PCR assay to quantify the mature microRNA and Northern blotting. Reverse transcription in situ PCR showed that three of the top differentially expressed miRNAs (miR-221, -376a and -301) were localized to tumor cells and not to stroma or normal acini or ducts. Aberrant microRNA expression may offer new clues to pancreatic tumorigenesis and may provide diagnostic biomarkers for pancreatic adenocarcinoma.

Figures

FIGURE 1
FIGURE 1
18S rRNA expression in pancreatic tissue. The expression of the 18S rRNA internal control is shown in pancreatic tumors, adjacent benign tissue, normal pancreas, chronic pancreatitis and pancreatic cancer cell lines. 18S rRNA expression, determined using real-time PCR as described in Material and Methods, is presented as 2−CT. Dashed line, mean value.
FIGURE 2
FIGURE 2
miRNA precursor expression in pancreatic samples. (a) The relative expression of each miRNA precursor was determined by real-time PCR; data are presented as ΔCT. Hierarchical clustering was performed on a subset of 112 genes that are differentially expressed (p < 0.001) among groups (tumor, chronic pancreatitis, cell lines and normal tissue) as determined by ANOVA multi-group comparison test. A median expression value equal to 1 was designated black; red, increased expression; green, reduced expression; grey, undetectable expression. (b) Dendrogram representing the results of hierarchical clustering analysis of the miRNA precursor expression pattern in 62 samples. Samples include primary pancreatic tumors (N = 28), normal pancreatic tissues (N = 6), adjacent benign pancreas (N = 15), chronic pancreatitis (N = 4) and pancreatic cancer cell lines (N = 9). (c) Three-dimensional expression terrain map was created from the filtered miRNA precursor expression data presented in (a). Each mountain represents an individual sample (tumor, adjacent benign, chronic pancreatitis, normal pancreas or pancreatic cancer cell line). The individual mountains sort into small groups based upon their similarities or differences to each other. Colored dots represent the same clusters as in (b). Black dots represent the 6 normal pancreases. The lines connecting pairs of samples indicate those samples which have very similar patterns of miRNA expression with average correlation above the threshold (>0.8).
FIGURE 3
FIGURE 3
Estimated probabilities for the training and test data. All training data including 6 normal pancreas samples and 18 of the samples known to be pancreatic tumors are correctly classified (a). Eleven out of 15 adjacent benign samples and 10 samples known to be pancreatic tumors are correctly classified in the testing group (b). Samples are partitioned by the true class (a) and the predicted class (b).
FIGURE 4
FIGURE 4
miRNA expression by Northern blotting. The expression of miR-100, miR-375 and miR-155 was determined in tissue specimens of pancreatic cancer (T), adjacent benign tissue (B) or normal pancreas (N). Blots were stripped and reprobed. tRNA, visualized by ethidium bromide staining, was used as a loading control.
FIGURE 5
FIGURE 5
Validation of precursor and mature miRNA levels. The expression of 8 miRNAs was validated in 6 normal pancreas specimens, 10 adjacent benign tissues and 16 pancreatic adenocarcinomas. The relative expression of the miRNA precursors (open bars) was determined using a real-time PCR assay to the miRNA precursors while the relative expression of the mature miRNA (closed bars) was determined using a real-time PCR assay to the mature miRNAs. The mean differences in miRNA expression between the normal pancreas black) and tumors (red) was significant p < 0.01 (student’s t-test). (a) let-7i, (b) miR-221, (c) miR-100, (d) miR-301, (e) miR-21, (f) miR-181a,c (precursor) and miR-181a (mature), (g) miR-125b-1 (precursor) and miR-125b (mature), (h) miR-212.
FIGURE 6
FIGURE 6
Histologic and molecular analyses of pancreatic cancer for microRNA expression. Panel (a) (×400) depicts the hematoxylin and eosin analysis of a pancreatic adenocarcinoma. The normal pancreatic glands (small arrow) are being invaded by the poorly formed glands of the carcinoma (large arrow). Serial section analysis of miR-221 after in situ amplification of the corresponding cDNA showed that many of the tumor cells contained the target sequence; note the cytoplasmic localization (arrows, panel (b) – ×400 and at higher magnification, panel (c) – ×1,000; the signal is blue due to NBT/BCIP with negative cells counterstained with fast red). The signal was lost with either omission of the primers or substitution with HPV-specific primers (panel (d), ×400). The adjacent serial section also showed many of the tumor cells expressed miR-376a after in situ amplification of the cDNA (e, f). Panel (e) (×400) shows the positive tumor cells (large arrow) and the negative stromal cells in the areas of desmoplasia (small arrow) while panel (f) (×400) depicts the positive tumor cells (large arrow) adjacent to the negative benign pancreatic gland acini (small arrow).

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