MicroRNAs and extracellular vesicles in cholangiopathies

Contents lists available at ScienceDirect BBA - Molecular Basis of Disease journal homepage: www.elsevier.com/locate/bbadis Review MicroRNAs and extracellular vesicles in cholangiopathies ☆ P. Olaizola a,1 , P.Y. Lee-Law a,b,c,1 , A. Arbelaiz a , A. Lapitz a , M.J. Perugorria a,b,d , L. Bujanda a,b,d , J.M. Banales a,b,d,⁎ a Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute –Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain b National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, “Instituto de Salud Carlos III”), Spain c Department of Gastroenterology and Hepatology, Radboud University Medical Centre, Nijmegen, Netherlands d IKERBASQUE, Basque Foundation for Science, Bilbao, Spain ARTICLE INFO Keywords: microRNAs Extracellular vesicles Cholangiopathies Diagnosis Pathogenesis Therapy ABSTRACT Cholangiopathies encompass a heterogeneous group of disorders affecting biliary epithelial cells (i.e. cholangiocytes). Early diagnosis, prognosis and treatment still remain clinically challenging for most of these diseases and are critical for adequate patient care. In the past decade, extensive research has emphasized microRNAs (miRs) as potential non-invasive biomarkers and tools to accurately identify, predict and treat cholangiopathies. MiRs can be released extracellularly conjugated with lipoproteins or encapsulated in extracellular vesicles (EVs). Research on EVs is also gaining attention since they are present in multiple biological fluids and may represent a relevant source of novel non-invasive biomarkers and be vehicles for new therapeutic approaches. This review highlights the most promising candidate miRs and EV-related biomarkers in cholangiopathies, as well as their relevant roles in biliary pathophysiology. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen. Research strategy: PubMed search (April 2017) was done with the following terms: “microRNA”,“miRNA”, “miR”,“extracellular vesicles”,“EV”,“exosomes”,“primary biliary cholangitis”,“primary biliary cholangitis”, “PBC”,“primary sclerosing cholangitis”,“PSC”,“cholangiocarcinoma”,“CCA”,“biliary atresia”,“BA”,“polycystic liver diseases”,“PLD”,“cholangiopathies”,“cholestatic liver disease”. Most significant articles in full-text English were selected. The reference lists of selected papers were also considered. http://dx.doi.org/10.1016/j.bbadis.2017.06.026 Received 16 May 2017; Received in revised form 27 June 2017; Accepted 28 June 2017 ☆ This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen. ⁎ Corresponding author at: Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute –Donostia University Hospital, Paseo del Dr. Begiristain s/n, E20014 San Sebastian, Spain. 1 Both authors contributed equally to this work. E-mail address: [email protected] (J.M. Banales). Abbreviations: 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; ADPKD, autosomal dominant polycystic kidney disease; ADPLD, autosomal dominant polycystic liver disease; AE2/ SLC4A2, CI − /HCO 3 – anion exchanger 2; ALP, alkaline phosphatase; AMA, anti-mitochondrial antibody; ARPKD, autosomal recessive polycystic kidney disease; Ars2, arsenic resistance protein 2; ASGPR1, asialoglycoprotein receptor 1; AUC, area under the curve; BA, biliary atresia; CA19-9, cancer antigen 19-9; CCA, cholangiocarcinoma; CCL2, C-C motif chemokine ligand 2; Cdc25A, cell division cycle 25A; CDH6, cadherin-6; CDK6, cyclin-dependent kinase 6; CHEK2, checkpoint kinase 2; c-Myc, myc proto-oncogene protein; CXCL1, chemokine (C-XC motif) ligand 1; eCCA, extrahepatic cholangiocarcinoma; EGFR, epidermal growth factor receptor; ELK1, ETS domain-containing protein Elk-1; EMT, epithelial mesenchymal transition; ERK, extracellular signal-regulated kinases; EV, extracellular vesicle; FDA, food and drug administration; FIBG, fibrinogen gamma chain; FOXA1, forkhead box protein A1; FOXO1, forkhead box protein O1; FXR, farnesoid X receptor; GSK3β, glycogen synthase kinase-3 beta; HCC, hepatocellular carcinoma; HDAC4, histone deacetylase 4; HSC, hepatic stellate cell; iCCA, intrahepatic cholangiocarcinoma; IGF1R, insulin-like growth factor-1 receptor; IL, interleukin; IL1β, interleukin-1 beta; InsP3R3, type III inositol 1,4,5-triphosphate receptor; ITGB4, integrin beta-4; MAPK, mitogen activated protein kinase; MAP3K8, mitogen-activated protein kinase kinase kinase 8; MBD2, methyl-CpG-binding domain protein 2; Mcl-1, induced myeloid leukemia cell differentiation protein Mcl-1; MDR1, multidrug resistant protein 1; miR, microRNA; MMP, matrix metalloproteinase; MSC, mesenchymal stem cell; mTOR, mechanistic target of rapamycin; MVB, multivesicular body; NCAM1, neural cell adhesion molecule 1; NDRG2, N-myc downstream-regulated gene 2; NUAK1, (nua) family kinase 1; PBMC, peripheral blood mononuclear cells; PBC, primary biliary cholangitis; pCCA, perihilar cholangiocarcinoma; PDC-E2, pyruvate dehydrogenase complex-E2; PDCD4, programmed cell death protein 4; Per1, period circadian protein homolog 1; PLD, polycystic liver disease; PSC, primary sclerosing cholangitis; PSMD10, proteasome 26S subunit non-ATPase 10; PTEN, phosphatase and tensin homolog; PTPN14, tyrosine-protein phosphatase non-receptor type 14; RB, retinoblastoma protein; RECK, reversion-inducing cysteine-rich protein with Kazal motifs; RhoC, ras homolog family member C; ROC, receiver operating characteristic; Smad4, small mothers against decapentaplegic homolog 4; SULT2A1,sulphotransferase 2A1; TET1, ten-eleven translocation 1; TGFβR2, transforming growth factor beta receptor 2; TIMP3, metalloproteinase inhibitor 3; TNFα, tumor necrosis factor alpha; TRAIL, TNF-related apoptosisinducing ligand; UDCA, ursodeoxycholic acid; VEGF, vascular endothelial growth factor; XIAP, X-linked inhibitor of apoptosis protein BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 Available online 13 July 2017 0925-4439/ © 2017 Elsevier B.V. All rights reserved. T 1. Introduction Bile duct epithelial cells (i.e. cholangiocytes) are important in health and disease. They represent a small proportion (3–5%) of the total liver cell population but, nonetheless, play essential roles for normal liver function including the alkalization and fluidization of the primary bile produced by hepatocytes. Biliary diseases, also termed as cholangiopathies, encompass a wide spectrum of etiologies comprising genetic, infectious, immune-mediated, drug-induced, vascular, neoplastic or idiopathic. Although their pathogenesis still remains obscure, chronic inflammation and cholestasis seem to be common events that exacerbate the wound-healing response leading to the development of liver fibrosis and cirrhosis. Most cholangiopathies lack valid diagnostic and/ or prognostic biomarkers, as well as adequate targets for therapy, demanding the need for further research. In the last years, the discovery of microRNAs (miRs) has represented a revolution and a paradigm shift (Fig. 1), postulating them as promising biomarkers and targets/tools for therapy. These small (18–23 nucleotides) endogenous non-coding RNAs play significant roles in most physiological and pathological cellular events including proliferation, differentiation, migration, senescence and survival, by regulating post-transcriptional gene expression [1,2]. Cholangiopathies display aberrant miR signatures in cholangiocytes, immune cells, liver tissue and biological fluids among others, evidencing their potential value in diagnosis, prognosis and therapy [3–5]. MiRs can be released into the extracellular medium associated with lipoproteins or encapsulated in extracellular vesicles (EVs), thus participating in intercellular communication (Fig. 2)[6]. EVs are small lipidenclosed spheres secreted by many cell types and found in multiple biological fluids including bile, blood and urine. There are different types of EVs according to their origin, size, molecular composition and biological function. Regarding their origin, EVs are classified into exosomes, plasma membrane-derived vesicles and apoptotic bodies. Besides miRs, they can also contain other nucleic acids, lipids and proteins. To date, the most studied EVs are exosomes, which are released extracellularly upon exocytic fusion of endosome-derived multivesicular bodies (MVBs) with the plasma membrane of the cells. Then, the exosomal content can be further delivered into recipient cells where it can regulate gene expression and cellular functions. Changes in the transcriptomic and proteomic EV content have been reported in different cholangiopathies, pointing out their potential value as non-invasive biomarkers (Fig. 1)[7]. This review provides current knowledge on the role of miRs and EVs in the pathogenesis of biliary diseases, and their potential therapeutic value. Moreover, the most promising miRs and EV-related biomarkers as new non-invasive diagnostic and prognostic tools, and their therapeutic value will be discussed. Finally, future directions on basic and clinical investigations will be highlighted. 2. MicroRNAs in cholangiopathies 2.1. Fibro-inflammatory cholangiopathies Primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and biliary atresia (BA) are cholangiopathies characterized by chronic inflammation, cholestasis and biliary fibrosis. Along the disease progression, cirrhosis, portal hypertension and, ultimately, liver failure may arise in these three disorders. 2.1.1. Primary biliary cholangitis (PBC) PBC is a chronic cholestatic liver disease of unknown etiology linked to autoimmune processes targeting small and medium intrahepatic bile ducts. Without treatment, PBC may progress to liver fibrosis, cirrhosis and, ultimately, liver failure [8]. PBC mainly affects middle-aged women (~90%) and has been associated with environmental toxins, infectious agents and certain genetic factors [9]. Diagnosis is based on clinical and serological parameters such as elevated levels of alkaline phosphatase (ALP) and the presence of specific anti-mitochondrial antibodies (AMAs) against pyruvate dehydrogenase complex-E2 (PDC-E2) in up to 95% of patients [10,11]. Moreover, a high proportion of PBC patients also presents serum anti-nuclear antibodies (ANAs) [12]. However, individuals without these serological features require a liver biopsy to determine the diagnosis. The choleretic bile acid Fig. 1. Trends of miRand EV-related articles since 2000. A) Exponential growth in the number of miR-related articles (upper left panel) and total number of articles related to each type of cholangiopathy (upper right panel). B) Exponential growth in the number of EV-related articles (lower left panel) and total number of articles related to each cholangiopathy (lower right panel). Red lines indicates the trends. P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1294 ursodeoxycholic acid (UDCA) is the mainstay treatment, which is administered daily and chronically. Nonetheless, depending on age, up to 50% of PBC patients lack an adequate response to UDCA treatment and have lower long-term survival than the general population [13,14]. For those UDCA non-responders, the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has recently been approved by the food and drug administration (FDA) as monotherapy or combination with UDCA. OCA improves some markers of cholestasis but also induces side effects like pruritus [15]. Therefore, it is fundamental to identify accurate biomarkers for specific and early diagnosis, prognosis and response to therapy, as well as new therapeutic targets for individualized patient care in PBC. Several studies have investigated the expression of miRs in serum [16,17] and liver tissue [18], as well as in specific cell types including peripheral blood mononuclear cells (PBMCs) [19,20] and biliary epithelial cells [21] from PBC patients. The serum from PBC patients was characterized by an altered miR profile [17,22], with upregulation of both miR-122-5p and miR-141-3p, and downregulation of miR-26b-5p that conferred a higher diagnostic value than the serum levels of ALP and ANAs (Table 1)[16]. In addition, deep sequencing analysis of serum samples from PBC patients identified miR-139-5p to be downregulated in patients with advanced PBC vs healthy controls and tended to be lower in advanced PBC patients vs early PBC patients [23], suggesting its potential value to predict the disease progression. The role of miRs as biomarkers to predict the response to treatment in PBC was highlighted by the fact that overexpression of miR-299-5p in serum was associated with non-response to UDCA treatment compared to both PBC responders and healthy controls [20]. On the other hand, alterations in the miR expression profile were also reported in PBMCs from PBC patients [22]. In particular, downregulation of miR-181a, miR-181b, miR374b, and miR-425 was found in CD4 + T cells from PBC patients compared to healthy controls. Of note, the downregulation of miR-425 in CD4 + T cells induced inflammatory cytokines production [24]. Cholestasis is one of the main hallmarks of PBC and is caused, partially, by downregulation of both the CI − /HCO 3 – anion exchanger 2 (AE2/SLC4A2) and the type III inositol 1,4,5-triphosphate receptor (InsP3R3) in cholangiocytes [25–29]. Remarkably, the etiopathogenic role of AE2 in PBC was also highlighted by the fact that Ae2 −/− mice spontaneously developed different PBC-like features, including serum specific AMAs [30,31]. One of the triggering causes for both AE2 and InsP3R3 downregulation in PBC cholangiocytes is miR-506, which was found overexpressed in these cells and directly targeted both mRNAs leading to cholestasis [21,32].Different pro-inflammatory cytokines found overexpressed in PBC livers such as interleukins (ILs) 8, 12, 17, 18 and tumor necrosis factor alpha (TNFα) enhanced the transcriptional activity of miR-506 gene promoter in cholangiocytes, subsequently leading to altered expression of proteins involved in several biological processes, particularly in mitochondrial energy metabolism [33]. MiR-506 induced PBC-like features in cholangiocytes including cell dedifferentiation, stress, susceptibility to bile-salt induced apoptosis, dysregulation of mitochondrial metabolism and PDC-E2 overexpression, ultimately promoting the activation and proliferation of PBMCs from PBC patients when co-cultured [33]. These data point out the relevant role of miR-506 in the etiopathogenesis of PBC and its potential therapeutic regulatory value. 2.1.2. Primary sclerosing cholangitis (PSC) PSC is a chronic cholestatic liver disease affecting both intraand extrahepatic large bile ducts [34,35]. Most patients are middle-aged men (~60%) and up to 80% also present inflammatory bowel disease, most commonly ulcerative colitis [8,34,35]. Importantly, PSC patients have a 400-fold increased risk for developing cholangiocarcinoma (CCA) compared to the general population [36]. Similar to PBC, hereditary and environmental elements are associated with PSC, but the etiopathogenesis remains unclear. There is no effective medical treatment to alter the disease course and liver transplantation is the only curative option [34,35,37]. In PSC, studies on the pathogenesis and role of miRs as biomarkers or tools for therapy are still limited. A report from 2016 pointed out the relevance of serum miRs as potential biomarkers for the diagnosis of PSC and CCA (Table 1)[38]. Fig. 2. Biogenesis and release of miRs. Primary miRs (Pri-miRs) are synthesized by RNA pol II or III (1). This initial RNA form, with 5′cap and poly-A tail, is then cleaved by the microprocessor complex formed by DROSHA/DGCR8 generating the hairpin-shaped Pre-miR (Pre-miRs) (2). The Pre-miRs are then exported to the cytoplasm via Exportin-5 (3), where they are processed by DICER/TRBP into a double-stranded mature miR (4). One strand of the miR duplex can enter the RISC assembling pathway (5) or be released by the cell, while the complementary strand is often degraded. In order to be released into the extracellular space, the mature miR strand can associate to AGO 1–4 proteins (6) or to lipoproteins (HDL, LDL) (7). On the other hand, miRs can be loaded into exosomes that are extracellularly released upon exocytic fusion of MVB with the plasma membrane (8) or into microvesicles formed by the blebbing of the cellular plasma membrane (9). Additionally, miRs can be discharged in apoptotic bodies along with other cellular-derived material (10). RNA pol II: RNA polymerase II; Pre-miR: precursor miRNA; RISC: RNA-induced silencing complex; AGO: Argonaute protein; HDL: high density lipoprotein; LDL: low density lipoprotein; MVB: multivesicular body. P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1295 In a discovery phase, 21 miRs were found differentially expressed in PSC, 33 in CCA and 26 in both groups compared to healthy controls, as well as 24 miRs in PSC vs CCA with area under the receiver operating characteristic (ROC) curve (AUC) > 0.700 [38]. After a validation phase in a second cohort of patients, miR-200c was confirmed to be downregulated in PSC vs healthy controls, whereas increased levels of both miR-483-5p and miR-194 were found in CCA vs healthy controls as well as both miR-483-5p and miR-222 in CCA vs PSC [38]. Combination of these particular miRs further improved the specificity and accuracy of diagnosis. In terms of liver pathophysiology, and particularly in cholestasis, pregnane X receptor (PXR) induced the expression of sulphotransferase 2A1 (SULT2A1) to convert lithocholic acid into a less toxic form and, thus, prevented liver injury. However, PSC patients are characterized by disease-specific impairment of SULT2A1 expression following PXR activation. In PSC, SULT2A1 expression might be regulated by miR378a-5p, which was found overexpressed in PSC vs PBC livers and was predicted by bioinformatics tools to target SULT2A1 gene expression [39]. On the other hand, it was reported that miR-21 promotes biliary hyperplasia [40] and miR-7a enhances cholangiocyte proliferation [41] in animal models of cholestasis and sclerosing cholestasis, respectively, suggesting a potential role of these miRs in the pathogenesis of PSC. However, the role of these or other miRs in the PSC etiopathogenesis remains still unknown. 2.1.3. Biliary atresia (BA) BA is a progressive and destructive cholangiopathy affecting the intraand extrahepatic bile ducts of neonates and that causes severe cholestasis [42]. The etiopathogenesis of this disease remains still obscure but several genetic and environmental factors have been postulated to participate in its development [43]. Without treatment, BA patients seldom survive more than 2 years [42]. To date, the only effective therapy is the Kasai portoenterostomy, which restores the bile flow. However, early diagnosis and intervention are crucial for the prognosis if this disease. BA is diagnosed by operative cholangiography and/or liver biopsy, which are invasive and time consuming, highlighting the urgent need for non-invasive alternatives. Serum of BA patients may contain promising miR biomarkers with high sensitivity and specificity for the diagnosis (Table 1). Indeed, upregulation of miR-200a, miR-200b and miR-429 (miR-200 cluster) was reported in BA patients compared to other forms of neonatal hyperbilirubinemia (NH) [44]. On the other hand, miR-4429 downregulation and miR-4689 upregulation were also reported in another study with potential diagnostic value [45]. Additionally, by using next-generation sequencing, miR-140-3p was found downregulated in plasma of BA patients compared to cholestatic disease patients and healthy controls, showing diagnostic potential [46]. Interestingly, the same study tried to identify previously reported dysregulated miRs (i.e.miR-200 family [44], miR-21 [47,48], miR-29a [48,49], miR-222 [47,50,51]) implicated in human and experimental BA, however only upregulation of miR-200 family was validated [46]. Different dysregulated miRs in BA are believed to be associated with pro-inflammatory and pro-fibrotic processes (Table 2)[22]. MiR-19b, which was found downregulated in liver tissue of BA patients [52], Table 1 MiRs as diagnostic biomarkers for cholangiopathies. AUC, area under the curve; BA, biliary atresia; CA19-9, cancer antigen 19-9; CCA, cholangiocarcinoma; iCCA, intrahepatic cholangiocarcinoma; MiR, microRNA; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; SEN, sensitivity; SPE, specificity. Disease MiR Expression Source Number of patients SEN (%) SPE (%) AUC Reference PBC 122-5p + 141-3p + 26b-5p Up (122-5p +141-3p) Down (26-5p) Serum PBC (n = 82) vs healthy controls (n = 60) 80.5 88.3 0.905 [16] PSC 200c Down Serum PSC (n = 40) vs healthy controls (n = 40) ––0.740 [38] BA 200a Up Serum BA (n = 24) vs other forms of neonatal hyperbilirubinemia (n = 24) 83.3 83.3 0.862 [44] 200b Up Serum 79.2 79.2 0.807 429 Up Serum 70.8 91.7 0.806 140-3p Down Plasma BA (n = 44) vs cholestatic disease controls (n = 20) and healthy controls (n = 20) 66.7 79.1 0.750 [46] 4429 Down Serum BA (n = 35) vs non-BA neonatal cholestasis (n = 20) 83.3 80.0 0.789 [45] 4689 Up Serum 66.7 80.0 0.722 CCA 9 Up Bile CCA (n = 7) and gallbladder cancer (n = 2) vs choledocholithiasis patients (n = 9) 88.9 100.0 0.975 [67] 145 Up Bile 77.8 100.0 0.975 942 Up Bile 77.8 100.0 0.765 302c Up Bile 88.9 100.0 e 199a-3p Up Bile 88.9 100.0 e 222 Up Bile 88.9 100.0 e 105 Up Bile 77.8 100.0 e 21 Up Serum iCCA (n = 74) vs healthy controls (n = 74) 87.8 90.5 0.908 [68] 21 Up Plasma iCCA (n = 25) vs healthy controls (n = 7) ––0.940 [71] 26a a Up Serum CCA (n = 66) vs healthy controls (n = 66) 84.8 81.8 0.899 [73] 106a a Down Serum CCA (n = 103) vs healthy control (n = 20) 81.6 85.0 0.890 [76] 150 Up Plasma iCCA (n = 15) vs healthy controls (n = 15) 80.6 58.1 0.791 [75] 192 Up Serum O.viverrini CCA (n = 10) vs healthy controls (n = 32) 74.0 72.0 0.809 [79] 21 + 192 Up Urine O.viverrini CCA (n = 22) vs healthy controls (n = 21) 81.8 71.4 0.849 [80] 483-5p + 194 Up Serum CCA (n = 40) vs healthy controls (n = 40) ––0.810 [38] 483-5p + 222 Up Serum CCA (n = 40) vs PSC (n = 40) ––0.770 1281 Down Serum CCA (n = 31) vs PSC (n = 40) 55.0 90.0 0.830 [81] 126 Down Serum 68.0 93.0 0.870 26a Down Serum 52.0 93.0 0.780 30b Down Serum 52.0 88.0 0.780 122 Down Serum 32.0 90.0 0.650 412 Up Bile PSC/CCA (n = 12) vs PSC (n = 52) 50.0 89.0 0.810 [81] 640 Up Bile 50.0 92.0 0.810 3189 Up Bile 67.0 89.0 0.800 1537 Up Bile 67.0 90.0 0.780 1537 + CA19-9 Up Bile 73.0 93.0 0.910 a Also prognostic biomarker. P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1296 directly targeted transforming growth factor beta receptor 2 (TGFβR2) gene expression and is believed to indirectly induce downstream TGFβ signaling, which is involved in hepatic stellate cells (HSCs) activation [52]. Additionally, increased miR-222, miR-200b and miR-21 in liver tissue of BA patients may also stimulate HSCs activation promoting fibrosis [47,50,53]. On the other hand, the pro-inflammatory cytokine IL6, upregulated in liver of BA patients, has been reported to enhance cholangiocyte proliferation through miR-124 and miR-200 family [54]. 2.2. Polycystic liver diseases (PLD) PLD comprise a heterogeneous group of congenital cholangiopathies inherited in dominant [i.e. autosomal dominant polycystic liver disease (ADPLD) or autosomal dominant polycystic kidney disease (ADPKD)] or recessive form [i.e. autosomal recessive polycystic kidney disease (ARPKD)] and characterized by progressive development of multiple fluid-filled biliary cysts (> 10), which are the main cause of morbidity [55–57]. PLD can be found isolated (e.g. ADPLD) or associated with renal cystogenesis (e.g. ADPKD and ARPKD). Current surgical and/or pharmacological treatments do not improve the prognosis of these diseases, and liver transplantation remains the only curative option. Hepatic cystogenesis in PLD is characterized by functional alterations in bile duct epithelial cells [58] that include miRs dysregulation [59,60]. To date, there is only a previous report highlighting the important role of miRs in PLD pathophysiology [59,60]. In this study, an abnormal miR expression profile was found in cholangiocytes isolated from an animal model of ARPKD (i.e. the PCK rat), which has the same human orthologous gene mutated (i.e.PKHD1). Of note, most of the dysregulated miRs were found downregulated in PCK cholangiocytes [59].Of particular interest is miR-15a, which was found highly downregulated in both rat and human cystic cholangiocytes [59]. MiR-15a directly targets cell division cycle 25A (Cdc25a) promoting cystic cholangiocyte cell proliferation [59]. Interestingly, experimental targeting of miR-15a with specific anti-sense oligonucleotides inhibited cystic cholangiocytes growth [59]. 2.3. Cholangiocarcinoma (CCA) CCA includes a heterogeneous group of malignancies with biliary differentiation features that may arise from different liver cell types including mature cholangiocytes. CCA is the second most frequent liver tumor accounting for 10–20% of all primary liver neoplasms [61,62]. Attending to their anatomical location, CCAs are classified as intrahepatic (iCCA), perihilar (pCCA) and extrahepatic (eCCA) [63]. Epidemiological studies indicate that CCA worldwide incidence has been rising in the last decades [63,64] ranging from 0.30 to 8.75 per 100.000 individuals depending on the geographical area [61,63,64]. Several risk factors, including PSC, cirrhosis, viral hepatitis B and C, hepatolithiasis, congenital biliary malformations as well as the hepatobiliary flukes endemic in East Asia (i.e.Opisthorchis viverrini and Clonochis sinensis), have been identified for CCA [64]. However, the etiology of most CCAs still remains unknown [63,64]. Since CCAs are often asymptomatic in early stages, diagnosis is usually conducted when the disease is advanced and widespread [63,64]. The current diagnostic strategy comprises a combination of imaging methods, nonspecific tumor biomarkers in serum [i.e. carbohydrate antigen 19-9 (CA19-9)] and histological analyses of tumor biopsies. Late diagnosis compromises the potential curative options, which are mainly based on surgery, leading to poor prognosis [63,64]. Furthermore, the responsiveness of CCA to current chemotherapies is very limited [63,64]. Therefore, early detection of these tumors is crucial for those patients with risk factors and for those that present recurrence after surgery. During the last decade, a significant number of research articles have been published on the role of miRs in CCA (Fig. 1A) pointing out their relevant value as non-invasive biomarkers (Table 1) and potential targets for therapy. 2.3.1. MicroRNAs as biomarkers To strengthen the diagnosis of CCA and monitor tumor progression, elevated CA19-9 levels in serum are commonly used as a complementary approach to imaging methods. However, the sensitivity and specificity of this non-invasive biomarker is modest, particularly in early stages of the disease [65,66]. Increasing evidence points out the relevance of miRs as biomarkers for CCA. Indeed, several dysregulated miRs have been described in serum, plasma, urine or bile from CCA patients, showing high AUC values for diagnosis (Table 1). High-throughput real-time PCR-based assays performed in human bile samples showed that miR-9, miR-105, miR-145, miR-199-3p, miR-222, miR-302c and miR-942 levels were higher in both CCA and gallbladder cancer compared to patients with choledocholithiasis [67] and acknowledged miR-9 as a reliable diagnostic indicator for biliary tract cancer [67]. In addition, different studies revealed miR-21 as a potential biomarker candidate for the diagnosis of iCCA [68,69],as it was found overexpressed in both serum and plasma from these patients compared to healthy individuals [70,71]. Clinical stage and tumor differentiation degree were reported to correlate with the levels of miR-21 in CCA tissue, and high miR-21 expression has been linked to poor overall survival, evidencing its promising role as a prognostic biomarker [70,72]. Similarly, levels of miR-26a were shown to be increased in CCA tissues, cell lines and Table 2 MiRs involved in non-tumor cholangiopathies. AE2, CI − /HCO 3 – anion exchanger 2; BA, biliary atresia; Cdc25a, cell division cycle 25A; FOG2, friend of Gata 2; FOXA2, forkhead box protein A2; IGF1: insulin-like growth factor 1; IGF1R, insulin-like growth factor 1 receptor; IL6R, interleukin 6 receptor; InsP3R3, type III inositol 1,4,5-triphosphate receptor; Ngn-3, neurogenin-3; N-Ras, neuroblastoma RAS viral oncogene homolog; PBC, primary biliary cholangitis; PLD, polycystic liver disease; PPP2R2A, protein phosphatase 2 regulatory subunit B alpha; PSC, primary sclerosing cholangitis; PTEN, phosphatase and tensin homolog; Smad, small mothers against decapentaplegic; STAT3, signal transducer and activator of transcription 3; TGFβ, transforming growth factor beta; TNFα, tumor necrosis factor alpha. Disease MiR Expression Target Function Sample Reference PBC 506 Up AE2, InsP3R3 Secretion Cell lines, tissue [21,33] 139-5p Down c-FOS, TNF-αInflammation Tissue, serum [23] 425 Down N-Ras Inflammation Serum [24] PSC 7a Up Ngn-3 Proliferation Animal model [41] 21 Up Smad Fibrosis, proliferation Tissue, animal model [40] BA 21 Up PTEN Fibrosis Tissue [47] 29a Up IGF-1, IGF-1R Cell death, inflammation Animal model [49] 200a, 200b, 200c Up FOXA2 Inflammation, proliferation Tissue, animal model [44] 200b Up FOG2 Proliferation, migration, fibrosis Tissue [53] 222 Up PPP2R2A Fibrosis Tissue, animal model [50,51] 19b Down TGFβFibrosis Tissue [52] 124 Down STAT3, IL-6R Inflammation, proliferation Tissue, animal model [54] PLD 15a Down Cdc25a Proliferation Cell lines, tissue [59] P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1297 serum from CCA patients compared to healthy controls [73,74]. Furthermore, miR-150 levels were found upregulated in plasma from iCCA patients [75] and its combination with CA19-9 improved the individual diagnostic capacity of both biomarkers [75]. In contrast, the expression of serum circulating miR-106a was reported downregulated in CCA compared to healthy controls, but its diagnostic value was lower than CA19-9 [76]. As previously mentioned for PSC-CCA, serum miR-483-5p and miR-194 were found upregulated in CCA patients compared to control individuals and their combination improved the diagnostic potential of each miR [38]. Remarkably, increased serum levels of miR222 and miR-483-5p were identified in CCA vs PSC patients [38].A different study provided a panel of 5 serum miRs (miR-1281, miR-126, miR-26a, miR-30b and miR-122) able to discriminate between PSC and CCA patients [77]. Furthermore, a distinct miR expression pattern was identified in bile from PSC-CCA vs PSC patients (miR-412, miR-640, miR-1537 and miR-3189) [77]. Interestingly, combination of miR-1537 and CA19-9 levels displayed higher diagnostic capacity than CA19-9 alone [77]. MiRs have also been postulated as prognostic biomarkers for CCA. Downregulation of miR-106a in serum from CCA patients was associated with higher risk of lymph node metastasis and inversely correlated with overall survival [76]. Likewise, downregulation of miR-1505p in plasma, bile and tumor tissue from CCA patients negatively correlated with CA19-9 levels and tumor grade [78]. On the other hand, increased serum levels of miR-192 were associated with metastasis and poor overall survival in liver fluke-associated CCA patients compared to healthy controls [79]. Of note, increased miR-192 and miR-21 levels were found in urine from liver fluke-associated CCA patients and their combination improved the diagnostic power of these two miRs alone [80]. The overall diagnostic capacity of miRs in CCA was emphasized in two independent meta-analysis, where the pooled of 11 miRs from 8 independent studies displayed AUC values of 0.900 and 0.880, respectively [81,82]. miRs in bile and serum showed higher diagnostic value (i.e. 0.957 and 0.957, respectively) than tissue (0.847) and urine (0.745) [81]. 2.3.2. MicroRNAs in CCA pathology Aberrantly expressed miRs in CCA tumor cells have been described to participate in pathological processes including cell proliferation, differentiation, survival, invasion/migration, epithelial mesenchymal transition (EMT), epigenetics and chemoresistance (Fig. 3)[22]. These miRs can function as oncogenes or tumor suppressors (Table 3). 2.3.3. Onco-microRNAs Several upregulated miRs in CCA cells function as onco-miRs promoting tumor growth. For instance, miR-21 stands out as a pivotal regulator of several pathophysiological processes such as cell proliferation, survival, EMT, invasiveness/metastasis and chemoresistance via direct targeting of different tumor suppressors including programmed cell death protein 4 (PDCD4), metalloprotease inhibitor 3 (TIMP3), reversion-inducing cysteine-rich protein with Kazal motifs (RECK), tyrosine-protein phosphatase non-receptor type 14 (PTPN14), 15-hydroxyprostaglandin dehydrogenase (15-PGDH), phosphatase and tensin homolog (PTEN) and phosphatidylinositol 3-kinase (PI3K) [68,70,83–86], among others [87]. In addition, arsenic resistance protein 2 (Ars2), involved in miR biogenesis, was found overexpressed in CCA tissue and cell lines leading to increased miR-21 expression, which in turn inhibited its downstream targets further contributing to CCA cell proliferation and oncogenesis [69]. The mRNA of different genes involved in the blockade of cell cycle such as p15, p21 and Cyclin E1 has been reported to be directly targeted by the oncogenic miR-224, leading to enhanced cell cycle progression and tumor growth [88]. Another miR with a potential role in cell cycle progression is miR-34a, which was found to directly inhibit period circadian protein homolog 1 (Per1) expression and its interaction with checkpoint kinase 2 (CHEK2), preventing cell cycle arrest [89]. Emerging data have demonstrated that miR-191 directly targets ten-eleven translocation 1 (TET1), which in turn demethylates and thereby represses p53 expression in CCA cells. Hence, upon miR-191 upregulation, the tumor suppressor activity of p53 is inhibited and leads to cell survival and proliferation [90]. Furthermore, miR-429 expression was reported to be increased in CCA, correlating with the hypomethylated status of its promoter [91]. Functional in vitro studies showed that miR429 directly targeted the tumor suppressor cadherin-6 (CDH6) inducing tumor cell growth [91]. Likewise, increased cellular proliferation and apoptosis evasion were shown in iCCA upon miR-31 upregulation through RAS p21 GTPase activating protein 1 (RASA1) direct inhibition [92]. RASA1 suppression led to increased levels of the active RAS form (GTP-bound) as well as to increased ERK1/2 phosphorylation further activating the MAPK signaling pathway and ultimately enhancing tumor growth [92]. MiR-421 was identified as another onco-miR in CCA that promoted tumor cell proliferation, migration and colony forming via FXR inhibition [93]. CCA cell survival was associated with miR-25, which protected tumor cells against TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via direct targeting of TRAIL Death Receptor-4 (DR4) [94]. The expression of the tumor suppressor N-myc downstream-regulated gene 2 (NDRG2) was found to be repressed by miR-181c, resulting in cell cycle progression, cell proliferation and chemoresistance [95]. Additionally, miR-181c correlated with the expression of mesenchymal markers N-cadherin and vimentin, and negatively correlated with the epithelial marker E-cadherin, therefore, promoting EMT [95]. Moreover, miR-221 was shown to target the tumor suppressor PTEN and promote migration and invasion in CCA cells through the β-catenin signaling pathway-mediated EMT, as it favored β-catenin translocation into the nucleus. Moreover, β-catenin could activate c-Jun, known to induce miR-221 expression. Thus, miR-221, β-catenin and c-Jun signaling pathways form a positive feedback loop through PTEN inhibition that enhances EMT [96]. Finally, miR-24 has been found to act as an onco-miR by partially repressing the tumor suppressor protein menin and, hence, stimulating proliferation, migration, invasiveness and angiogenesis in CCA tumors [97]. 2.3.4. Tumor suppressor microRNAs Different tumor suppressor miRs are downregulated in CCA cells leading to tumor growth. In particular, downregulation of miR-494 in CCA cells induced the expression of its direct target cyclin-dependent kinase 6 (CDK6) leading to cell cycle progression [98,99]. Similarly, miR-122 was able to inhibit the expression of genes involved in cell cycle progression including Cyclin G1 and insulin-like growth factor 1 receptor (IGF1R), and its baseline downregulation in CCA resulted in cell proliferation [100]. Regarding cell survival, decreased miR-29b levels in CCA cells promoted the expression of its target induced myeloid leukemia cell differentiation protein Mcl-1 (Mcl-1), an anti-apoptotic Bcl-2 family member that protects cells against TRAIL-mediated programmed cell death [101]. Similarly, decreased miR-410 expression in CCA cells resulted in the upregulation of its target, the X-linked inhibitor of apoptosis protein (XIAP), leading to tumor cell growth and invasiveness [102]. Likewise, reduced miR-212 levels in CCA cell lines led to the upregulation of its direct target forkhead box protein A1 (FOXA1), promoting increased tumor cell proliferation and invasion [103]. On the other hand, miR-145 was pointed out as another tumor suppressor found downregulated in CCA cells [104]. MiR-145 prevents the tumor growth, proliferation and invasion of iCCA cells by directly targeting novel (nua) family kinase 1 (NUAK1), which further negatively regulated Akt/Forkhead box protein O1 (FOXO1) pathway and matrix metalloproteinase (MMP) expression halting iCCA progression [104]. Moreover, decreased miR-150-5p levels in CCA cells promoted cell proliferation, migration and invasion via upregulation of its direct target the oncogenic ETS domain-containing protein Elk-1 (ELK1) [78]. Regarding EMT, migration and invasiveness, decreased miR-214 P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1298 levels were reported in metastatic iCCA tissues compared to non-metastatic ones, leading to decreased expression of the epithelial marker Ecadherin and induced expression of the EMT transcription factor Twist [105]. In addition, downregulation of miR-122 in human CCA has been suggested to be involved in tumor cell migration and invasion through dysregulation of MMP2, MMP9, RECK, E-cadherin and N-cadherin expression [100]. The activation of the ERK/MMP2/MMP9 signaling pathway and the consequent proliferation, migration and invasion were induced upon miR-138 downregulation in CCA cells via Ras homolog gene family member C (RhoC) upregulation [106]. Similarly, miR-200c was reported to mediate EMT in CCA cells via direct targeting of neural cell adhesion molecule 1 (NCAM1) [107]. On the other hand, the epidermal growth factor (EGF)-dependent migration of iCCA cells was associated with downregulated miR-376c expression via upregulation of its direct target growth factor receptor-bound protein 2 (GRB2). Additionally, interleukin-1 beta (IL1β) and MMP9 were suggested to be functioning downstream of GRB2 signaling [108]. Likewise, TGFβmay be responsible of the reduced miR-29a levels in CCA cell lines and tissue, leading to the stimulation of tumor growth and metastasis via activation of its direct target histone deacetylase 4 (HDAC4) [109]. Inflammation has been established as a relevant component of tumor progression. MiR-605 was found downregulated in iCCA tissue leading to overexpression of its direct target 26S proteasome ATPase regulatory subunit 10 (PSMD10), which prevented retinoblastoma protein (RB) from inhibiting interleukin 6 (IL6) [110]. Induction of the IL6/STAT3 pathway ultimately promoted cell proliferation and invasion in vitro and tumor growth in vivo through the upregulation of Cyclin D1, vascular endothelial growth factor (VEGF), MMP2 and MMP9 [110,111]. The IL6/STAT3 pathway has also been described as the target of the let-7/miR-99a/miR-125b cluster, which was found downregulated in CCA tissues [112]. Essentially, this miR cluster directly targeted central inflammatory elements including IL6, IL6R and IGF1R, which in turn activated STAT3 downstream signaling enhancing CCA progression [112]. Downregulation of miRs in CCA may be caused by hypermethylation of their promoters. Decreased miR-370 levels in CCA cells were linked to hypermethylation of its promoter via IL6-dependent activation of DNA methyltransferases. Consequently, the expression of miR-370 was reduced and the expression of its target the mitogen-activated protein kinase kinase kinase 8 (MAP3K8) was enhanced contributing to tumor growth [113]. In addition, the paternal allele of miR-370 was often found silenced through genomic imprinting in CCA patients [114]. Moreover, due to the overexpression of IL6 in Fig. 3. Aberrant miR expression in the pathogenesis of CCA. Dysregulated miRs in tumorigenic cellular events: cell cycle dysregulation and proliferation (A); EMT, migration and invasion (B); angiogenesis (C); cell death (D); inflammation (E); chemoresistance (F) and epigenetics (G). The central image corresponds to a human liver with CCA. Upregulated miRs are shown in blue color and downregulated ones in red color. CCA, cholangiocarcinoma; M1, macrophage type 1; M2, macrophage type 2; TRAIL, TNF-related apoptosis-inducing ligand; VEGF, vascular endothelial growth factor. P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1299 Table 3 miRs involved in biliary tumorigenesis. 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; CCA, cholangiocarcinoma; CDH6, cadherin-6; CDK6, cyclin-dependent kinase 6; c-Met, tyrosine-protein kinase Met; c-Myc, myc proto-oncogene protein; DR4, death receptor 4; ELK1, ETS domain-containing protein Elk-1; EMT, epithelial mesenchymal transition; FOXA1, forkhead box protein A1; FXR, farnesoid X receptor; GRB2, growth factor receptor-bound protein 2; GSK3B, glycogen synthase kinase 3 beta; HDAC4, histone deacetylase 4; iCCA, intrahepatic cholangiocarcinoma; IGF1R, insulin-like growth factor 1 receptor; IL6, interleukin 6; IL6R, interleukin 6 receptor; KRT19, keratin 19; MAP3K8, mitogen-activated protein kinase kinase kinase 8; MBD2, methyl-CpGbinding domain protein 2; Mcl-1, induced myeloid leukemia cell differentiation protein Mcl-1; miR, microRNA; MMP2, matrix metalloproteinase 2; MMP9, matrix metalloproteinase 9; mTOR, mechanistic target of rapamycin; NCAM1, neural cell adhesion molecule 1; NDRG2, N-myc downstream-regulated gene 2; NUAK1, NUAK family kinase 1; PDCD4, programmed cell death protein 4; Per1, period circadian protein homolog 1; PIK3R, phosphoinsitide-3-kinase regulatory subunit 1; PSMD1, proteasome 26S non-ATPase regulatory subunit 1; PTEN, phosphatase and tensin homolog; PTPN14, tyrosine-protein phosphatase non-receptor type 14; PTTG1, pituitary tumor-transforming gene 1 protein; RASA1, RAS p21 protein activator 1; RECK, reversion-inducing cysteine-rich protein with Kazal motifs; RhoC, ras homolog family member C; Smad4, small mothers against decapentaplegic homolog 4; TET1, ten-eleven translocation 1; TIMP3, metalloproteinase inhibitor 3; TOP2A, DNA topoisomerase 2-alpha; Twist, twist basic helix-loop-helix transcription factor; WNT10B, Protein Wnt-10B; XIAP, X-linked inhibitor of apoptosis protein. ROLE IN TUMORIGENESIS MiR DISEASE TARGET FUNCTION SAMPLE REFERENCE Onco-miR 21 iCCA PTPN14, PTEN Proliferation, tumor growth, apoptosis [69] 21 O. viverrini iCCA PDCD4 Proliferation, tumour growth, migration, oncogenesis, model [72] 21 CCA PDCD4, TIMP3, E-Cadherin, N-Cadherin, Vimentin, PTEN, PDCD4, RECK, 15-PGDH chemoresistance Cell lines, tissue [68, 83-86] 24 CCA Menin Proliferation, migration, invasion, angiogenesis [97] 25 CCA DR4 Apoptosis Cell lines, tissue [94] 26a CCA GSK-3b Proliferation Cell lines, tissue [74] 31 iCCA RASA1 Proliferation, apoptosis Cell lines, tissue [92] 34a CCA Per1 Proliferation, cell cycle progression, tumor growth, invasion Cell lines, tissue [89] 181c CCA NDRG2 Proliferation, tumor growth, EMT, migration, invasion, chemoresistance, senescence Cell lines, tissue [95] 191 iCCA TET1 Proliferation, cell cycle progression, tumor growth, apoptosis, EMT, migration, invasion model [90] 221 CCA PTEN EMT, migration, invasion Cell lines, tissue [96] 224 CCA p15, p21, Cyclin E1 Cell cycle prograssion, tumour growth Cell lines, tissue [88] 421 CCA and gallbladder cancer FXR Proliferation, migration Cell lines, tissue [93] 429 CCA CDH6 Tumor growth Cell lines [91] Cell lines, tissue, serum Cell lines, tissue, animal Cell lines, animal model Cell lines, tissue, animal Proliferation, apoptosis, EMT, migration , invasion, oncogenesis, P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1300 Tumor suppressor let-7c1CCA IL6, IL6R Migration, invasion, inflammation, tumorigenicity, stemness Cell lines, tissue [112] 125b1CCA IL6R Migration, invasion, inflammation, tumorigenicity, stemness Cell lines, tissue [112] 99a1CCA IGF1R Migration, invasion, inflammation, tumorigenicity, stemness Cell lines, tissue [112] 26a CCA KRT19 Proliferation, tumor growth model [120] 29a CCA HDAC4 Tumor growth, EMT, migration, invasion Cell lines, tissue [109] 29b CCA Mcl-1, PIK3R1, MMP2 Apoptosis, chemoresistance Cell lines [101, 118] 34a CCA c-Myc, SMAD4, c-Met, CDK6, Cyclin D1 Tumor growth, EMT, migration, invasion, oncogenesis model [121-123] 122 CCA Cadherin Proliferation, migration, invasion model [100, 101] 138 CCA RhoC Proliferation, cell cycle progression, migration, invasion Cell lines, tissue [106] 145 iCCA NUAK Proliferation, tumour growth, invasion Cell lines, tissue [104] 150-5p CCA ELK1 Proliferation, tumour growth, migration, invasion [78] 199a-3p CCA mTOR Chemoresistance Cell lines [119] 200c iCCA NCAM1 EMT, migration, invasion Cell lines [107] 205 CCA -Chemoresistance Cell lines [118] 212 iCCA FOXA1 Proliferation, invasion Cell lines, tissue [103] 214 iCCA Twist EMT, migration, invasion Cell lines, tissue [105] 221 CCA PIK3R1 Apoptosis, chemoresistance Cell lines [118] 370 CCA MAP3K8, WNT10B Proliferation Cell lines, tissue [113] 373 pCCA MBD2 Epigenetics Cell lines, tissue [115] 376c iCCA GRB2 Migration Cell lines [108] 410 CCA XIAP Tumor growth, apoptosis, invasion Cell lines [103] 494 CCA CDK6, PTTG1, TOP2A Proliferation model [98, 99] 605 iCCA PSMD1/Gankyrin Proliferation, apoptosis, migration, invasion, inflammation model [110] Cell lines, tissue, animal Cell lines, tissue, animal Cell lines, tissue, animal Cell lines, tissue, serum Cell lines, tissue, animal Cell lines, tissue, animal Cyclin G1, IGF1R, MMP2, MMP9, RECK, E-Cadherin, N1 miR cluster. P. Olaizola et al. BBA - Molecular Basis of Disease 1864 (2018) 1293–1307 1301