Cholangiopathies are a group of diseases primarily or secondarily affecting bile duct cells, and result in cholangiocyte proliferation, regression, and/or transformation. Their etiopathogenesis may be associated with a broad variety of causes of different nature, which includes genetic, neoplastic, immune-associated, infectious, vascular, and drug-induced alterations, or being idiopathic. miRNAs, small non-coding endogenous RNAs that post-transcriptionally regulate gene expression, have been associated with pathophysiological processes in different organs and cell types, and are postulated as potential targets for diagnosis and therapy. In the current manuscript, knowledge regarding the role of miRNAs in the development and/or progression of cholangiopathies has been reviewed and the most relevant findings in this promising field of hepatology have been highlighted.

Cholangiocytes are the epithelial cells lining the bile ducts, and are key players in normal liver physiology[1]. They participate in fundamental secretory processes by modifying the composition and flow of primary bile generated at the canaliculi of hepatocytes upstream of the biliary tree. Thus, transport of water, ions or solutes between the blood and the bile-duct lumen finally result in fluidization and alkalinization of bile[1-4]. Although cholangiocytes only represent 3%-5% of the total liver cell population, they may account for up to 30% of total bile flow[1]. Cholangiocytes contain a single primary cilium extending from the apical membrane into the bile-duct lumen[5]. This antenna-like organelle possesses sensory properties that detect physical and chemical changes in bile that are transmitted into the cell to activate signaling pathways and finally modify cellular functions. The primary cilium of cholangiocytes functions as a mechano- (detecting changes in bile flow)[6], chemo- (interacting with different molecules[7] and vesicles[8]), and osmo- (identifying changes in osmolarity: hypo- and hyper-tonicity)[9] sensor.

In addition, cholangiocytes are primarily or secondarily affected in a group of diseases termed cholangiopathies, whose etiopathogenesis is of diverse nature (genetic, neoplastic, immune-associated, idiopathic, infectious, vascular or drug-induced)[10]. These biliary diseases may result in cholangiocyte proliferation, regression and/or transformation. Important advances are being achieved in understanding the molecular mechanisms involved in the development and progression of these disorders. In this regard, there is increasing evidence suggesting the role of miRNAs in the etiopathogenesis of cholangiopathies, which are postulated as potential targets for diagnosis and therapy.

miRNAs are small non-coding RNAs (approximately 22 nucleotides) that regulate the expression of multiple genes by binding to complementary sites of targeted mRNAs, causing translational repression (imperfect target duplexes) or degradation (perfect matches)[11,12]. They participate in the regulation of multiple cell types under physiological and pathological conditions, and are fundamental in different cellular processes such as development, proliferation, apoptosis, metabolism, morphogenesis, and in diseases[13,14]. In the current article, knowledge regarding the role of miRNAs in the development and/or progression of cholangiopathies has been highlighted.

Cholangiocarcinoma (CCA) is a malignant tumor affecting the biliary tree[15,16]. The incidence of CCA (1:50 000) is increasing worldwide and the therapeutic options are very limited because of high chemoresistance. CCA accounts for up to 10%-15% of primary hepatobiliary malignancies and 3% of all gastrointestinal tumors, affecting individuals of both sexes. Owing to slow growth and late metastasis, this cancer is usually diagnosed in patients older than 65 years when it is at an advanced stage, thus reducing the success of surgical procedures. Complications of CCA include bile-duct obstruction, liver failure, metastasis to other organs, infections, and uncontrolled vomiting. Although the pathogenesis of CCA is poorly understood, the presence of primary sclerosing cholangitis, chronic biliary irritation, or choledochal cysts appears to predispose to the development of this cancer.

CCAs can be anatomically classified into extrahepatic or intrahepatic[15,16]. Extrahepatic is more common than intrahepatic CCA, accounting for up to 80%-95% of all CCAs. In addition, depending on the tumor location in the extrahepatic biliary system, extrahepatic CCA can be divided into proximal or perihilar (also frequently referred to as Klatskin tumor), and distal. On the other hand, intrahepatic CCA originates within the liver and accounts for up to 5%-20% of all CCAs.

Increasing evidence suggests the importance of miRNAs in the pathogenesis of CCA. Thus, global changes in the miRNA profile of malignant cholangiocytes has been reported[17-21], which may alter different cholangiocyte features such as cell cycle, proliferation, migration and apoptosis (Table 1).

Polycystic liver diseases (PCLDs) are genetic disorders characterized by bile-duct dilatation and/or cyst development, which become progressively more severe and require liver transplantation as the only therapeutic option[43]. The large volume of hepatic cysts causes symptoms such as abdominal distension, local pressure with back pain, gastroesophageal reflux, and dyspnea. The estimated prevalence of PCLDs is around 1:1000, and these patients often develop polycystic kidney disease. Different genetic mutations trigger the appearance and growth of cysts in PCLDs, and in some cases this phenomenon is also associated with hepatic fibrosis. Most of the proteins encoded by these genes are located in the primary cilium of cholangiocytes[44]. Many of these proteins interact with each other forming complexes that signal through common pathways. Mutations in genes encoding proteins located in the primary cilium of cholangiocytes result in physical and functional defects of this organelle, and outcome in the development of several forms of PCLD classified as ciliopathies. For this reason, and because the only cells in the liver that have cilia are cholangiocytes, the PCLDs affecting genes encoding proteins which are localized in the cilia are called cholangiociliopathies[44]. It has been recently demonstrated that hepatic cystogenesis in PCLDs is the result of hyperproliferation[45], hypersecretion[46], and alteration in the pattern of miRNAs in the bile duct cells[47], and that these alterations are intracellularly associated with an increase in the levels of cAMP and a decrease in calcium[45,48,49]. In this regard, global changes in the expression pattern of miRNAs were observed between cultured cholangiocytes from normal and PCK rats (animal model of hepatorenal polycystic disease, i.e., autosomal recessive polycystic kidney disease)[47,50]. In total, 121 and 148 miRNAs were detected in normal and PCK rat cholangiocytes, respectively. Twelve miRNAs were expressed in normal rat cholangiocytes and not in PCK rat cholangiocytes, and 39 were present only in PCK rat cholangiocytes. Moreover, there were changes in the expression of 109 common miRNAs between both cell lines. Interestingly, 97 of these common miRNAs (i.e., 87%) were found to be downregulated in PCK rat cholangiocytes compared to normal rat cholangiocytes, and 12 miRNAs (11%) were overexpressed in cystic cholangiocytes. Among those highly downregulated miRNAs in cultured PCK rat cholangiocytes, miRNA-15a is also downregulated in vivo in cholangiocytes from PCK rats and PCLD patients[47]. This miRNA-15a downregulation runs in parallel with the upregulation of its predicted target, the cell-cycle regulator cell division cycle 25A (Cdc25A). Experimental overexpression of miRNA-15a in PCK rat cholangiocytes results in decreased Cdc25A protein levels, inhibition of cell proliferation, and cyst growth reduction. Interestingly, experimental suppression of miRNA-15a in normal rat cholangiocytes accelerates cell proliferation, increases Cdc25A expression, and promotes cyst growth[47]. All these data suggest that the miRNA-15a/Cdc25A interplay could be a potential target to inhibit hepatic cystogenesis (Figure 1). Importantly, inhibition of Cdc25A with its inhibitor vitamin K3 suppresses hepatorenal cystogenesis in rodent models of polycystic kidney and liver disease, being a valuable potential pharmacological approach to test in clinical trials[51] (Figure 1). Moreover, it will be interesting to demonstrate in future studies the role of many other altered miRNAs in the pathogenesis of PCLDs.

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Figure 1 miRNA-15a/Cdc25a interplay participates in hepatic cystogenesis of polycystic liver diseases. miRNA-15a directly targets the cell cycle regulator Cdc25a. In polycystic rat and human cholangiocytes, miR15a is downregulated, thus resulting in Cdc25a upregulation, cell proliferation and cystogenesis. The Cdc25a inhibitor vitamin K3 was demonstrated to target Cdc25a, blocking the hepatorenal cystogenesis of several rodent models of polycystic kidney and liver disease. This suggests the therapeutic potential of vitamin K3 for polycystic kidney and liver diseases.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease that mainly affects middle-aged women[52]. It is a rare disease, with an estimated prevalence of 1:8000 in countries such as the United Kingdom. Four stages of this disorder have been characterized by progressive bile-duct loss that runs in parallel with an increase in cholestasis and fibrosis[52]. The etiology of PBC remains unknown, and the pathogenesis is poorly understood. The serological hallmark of PBC is the development of high titers of antimitochondrial autoantibodies (AMAs) in serum associated with an immunological attack on the small intrahepatic bile ducts[52]. Interestingly, PBC patients usually have a poor response to immunosuppressants and the only therapeutic option that improves the outcome of the disease in two thirds of PBC patients is daily administration of ursodeoxycholic acid (UDCA), a hypercholeretic bile acid[52]. In addition, and importantly, PBC patients show alterations in the biliary secretion of bicarbonate. Thus, PBC patients show a failure in the secretin-stimulated biliary secretion of bicarbonate[53], as well as a reduction in the hepatobiliary expression of anion exchanger 2 (AE2/SLC4A2)[54,55]. AE2 is a Cl^-/HCO₃^- exchanger located in the canalicular membrane of hepatocytes and in the apical membrane of cholangiocytes that controls the intracellular pH (pH_i) and promotes the alkalinization and fluidization of bile[1,2,56]. An interesting hypothesis suggests that, in PBC patients, long-term maintained alteration of pH_i by AE2 downregulation might modify cholangiocyte and lymphocyte function[57]. To test the etiopathogenic role of AE2 downregulation in PBC, an Ae2_a,b1,b2^-/-mouse knockout was generated. Interestingly, these animals spontaneously reproduce over time many PBC features[57], such as: (1) portal infiltration of T lymphocytes and bile duct damage; (2) increased oxidative stress in cholangiocytes; (3) elevated production of interferon-γ and IL-12; (4) periductular hepatic fibrosis; (5) increased levels of IgM, IgG, and hepatic alkaline phosphatase; and (6) the presence of PBC-specific AMAs. Different agents such as hormones, metabolic influences, infectious agents, xenobiotics, cytokines/inflammatory mediators, and/or miRNAs could be involved in the AE2 downregulation present in PBC patients. Regarding miRNAs, a differentially expressed miRNA profile in livers from PBC patients compared with normal controls has been recently reported[58]. The change consists of 35 differentially expressed miRNAs (11 upregulated and 24 downregulated), which were predicted to target genetic transcripts involved in cell proliferation, apoptosis, inflammation, oxidative stress and metabolism[58]. Regarding those miRNAs that were overexpressed in the miRNA array, we observed by using bioinformatics approaches that miRNA-506 could be a potential direct AE2 regulator. Therefore, we tested if AE2 downregulation in PBC cholangiocytes is dependent on miRNAs, and specifically on miRNA-506. Our data showed that miR-506 is upregulated in cholangiocytes from PBC patients, binds to the 3’-UTR of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions[59]. These data suggest the ethiopathogenic role of miR-506 in the AE2 downregulation characteristic of PBC cholangiocytes (Figure 2). On the other hand, the reported AE2 downregulation in PBC lymphocytes was not associated with changes in the expression of miRNA-506[59]. This disease preferentially affects women, therefore, it is interesting to remark that the gene encoding miRNA-506 is located in the X chromosome. Further investigations are needed to characterize fully the role of miRNA-506 in the regulation of other targets and cellular features, as well as the analysis of its expression in other tissues. Moreover, the role of UDCA and estrogens on miRNA-506 expression will be valuable information to expand the ethiopathogenic role of this miRNA in PBC.

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Figure 2 Role of miRNA-506 in the etiopathogenesis of primary biliary cirrhosis. Different risk factors could induce genetic alterations leading to anion exchange 2 (AE2) downregulation in primary biliary cirrhosis (PBC) cholangiocytes. miRNA-506 is overexpressed in PBC cholangiocytes, thus resulting in the inhibition of both AE2 protein translation and subsequent Cl^-/HCO₃^- activity, and impairing the biliary secretory functions.

In summary, the available information clearly indicates that miRNAs represent a new research area in the field of biliary pathophysiology. As stated in this review, they may modify different features in cholangiocytes, such as secretion, apoptosis, proliferation, and migration, and can be regulated by different mechanisms and conditions, such as epigenetics, hypoxia, or circadian rhythms. There is still limited information about the role of miRNAs in biliary diseases, but future investigations will provide more evidence regarding their role in the development and progression of cholangiopathies, as well as in their potential use for diagnosis and therapy. Finally, it is important to remark upon the increasing information about the role of stem cells in the pathophysiology of biliary diseases[60]; in this regard, the potential role of miRNAs in the regulation of biliary stem cells during cholangiocyte injury, as well as the role of miRNAs in the development of cancer stem cells in CCA[61] need to be elucidated.