Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer

Abstract

Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC.

Circulating microRNAs: New biomarkers in diagnosis, prognosis and treatment of cancer (Review)

“…Moreover, they verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC (155).…”

A review of the clinical diagnosis and therapy of cholangiocarcinoma

“…17 Shigehara et al demonstrated that some miRNAs (miR-9, miR-302c, miR199a-3p and miR-222) in human bile were more highly expressed in biliary tract cancer than in benign conditions, so miR-9 might be helpful in the diagnosis and clinical management of biliary tract cancer. 18 Markers for the precise prediction of the prognosis of CCA have been difficult to identify. Recently, it has been shown that single nucleotide polymorphisms (SNPs) were able to predict the outcome of CCA and the B-cell-lymphoma-2 (Bcl-2) À938C> A polymorphism was associated with a favourable clinical outcome.…”

Barking up the wrong tree: MicroRNAs in bile as markers for biliary complications

“…14 Moreover, extracellular miRNAs are more resistant against ribonuclease activity than longer messenger RNAs at room temperature or after repeated freezethaw cycles. 12,14,15 Abbreviations: DCD, donation after circulatory death; ITBL, ischemic-type biliary lesions; LT, liver transplantation; miR, micro-RNA; miRNA, microRNA.…”