Diagnostic and therapeutic potentials of microRNAs in cholangiopathies

Abstract

Cholangiopathies are a group of rare, devastating diseases that arise from damaged cholangiocytes, the cells that line the intra- and extra-hepatic bile ducts of the biliary epithelium. Cholangiopathies result in significant morbidity and mortality and are a major cause of liver transplantation. A better understanding of the underlying pathogenesis that influences cholangiocyte dysregulation and cholangiopathy progression is necessary, considering the dismal prognosis associated with these diseases.

MicroRNAs are a class of small, non-coding RNAs that regulate post-transcriptional mRNA expression of specific genes. The role of microRNAs has expanded to include the initiation and development of many diseases, including cholangiopathies. Understanding microRNA regulation of cholangiopathies may provide diagnostic and therapeutic benefit for these diseases. In this review, the authors primarily focus on studies published within the last five years that help determine the diagnostic and therapeutic potential of microRNAs in cholangiopathies.

1. Introduction

The liver is a heterogeneous tissue whose functions include the production of bile acids, detoxification of toxins and xenobiotics, cholesterol synthesis, and glucose storage, among other activities [1]. At least 15 different cell types have been identified in the liver, but the primary cell type is the hepatocyte (∼70% of the nuclear cell population) [2]. Other liver cells include sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, portal fibroblasts and cholangiocytes [2]. Cholangiocytes make up 3%–5% of the liver cell population and are the epithelial cells that line the intra- and extra-hepatic bile ducts of the biliary epithelium [1]. Cholangiocytes are a heterogeneous cell type: (i) small cholangiocytes are cuboidal in shape, line small bile ducts (lumen diameter < 15 mm) and proliferate in response to IP₃/Ca²⁺ signaling; and (ii) large cholangiocytes are more columnar in shape, line large bile ducts (lumen diameter ³ 15 mm) and proliferate in response to cAMP signaling [1, 3-8]. Under normal conditions, cholangiocytes are mitotically dormant and primarily participate in the modification of canalicular bile by secreting water and electrolytes, including Cl^- and HCO₃^- [6, 7, 9, 10]. However, following injury, cholangiocytes begin to proliferate in an attempt to maintain biliary homeostasis [1, 8, 11]. The proliferating cholangiocytes display a neuroendocrine phenotype defined by enhanced synthesis and secretion of various neuroendocrine factors that regulate biliary and liver pathogenesis by both autocrine and paracrine mechanisms [1, 8, 11, 12].

Cholangiopathies are a group of rare diseases whose primary or secondary target is the cholangiocyte [13-15]. The onset and subsequent development processes of cholangiopathies are extremely diverse, with causes that are genetic, neoplastic, immune-mediated, idiopathic, infectious, and toxin- or drug-induced [13-15]. Cholangiopathies contribute to significant morbidity and mortality and are one of the main causes of liver transplantation in both pediatric and adult patients [16-18]. In the early stages, cholangiopathies can primarily be characterized by enhanced biliary inflammation and apoptosis that can lead to cholestasis and biliary proliferation [3, 19, 20]. If unmanaged, these hepatic insults can further progress to destruction of cholangiocytes, extensive fibrosis, cirrhosis and possibly cholangiocarcinoma [21]. As previously stated, cholangiocytes begin to proliferate and secrete neuroendocrine factors in response to damage, and this cellular function has been largely characterized during the progression of cholangiopathies [1, 8, 10]. Recently, however, microRNAs (miRNAs) have been recognized as initiators and/or drivers of some cholangiopathies [21-23].

MiRNAs are short, non-coding RNAs, around 18–23 nucleotides in length, that regulate post-transcriptional gene expression of specific mRNAs. In mammals, miRNAs regulate the expression of approximately 60% of genes [24]. In the nucleus, primary-miRNA (pri-miRNA) is transcribed from DNA by Pol II, and then pri-miRNA is recognized and cleaved by the RNase III endonuclease Drosha to release precursor-miRNA (pre-miRNA) [25]. This pre-miRNA (60–90 nucleotides in length) is transported to the cytoplasm in a RAN-GTP-dependent manner via Exportin-5; once in the nucleus it is ultimately processed by another RNase III endonuclease, Dicer, into a miRNA duplex (18–23 nucleotides in length) [26, 27]. Next, the miRNA duplex is inserted into the miRNA-associated RNA-induced silencing complex (RISC) and separated into a functional guide strand and a passenger strand [25].Once in the cytoplasm, the guide strand, or mature miRNA, will recognize and bind to complementary sequences in mRNAs, primarily within the 3′ untranslated region (3′ UTR) [28]. This interaction will either result in transcriptional suppression or degradation of the mRNA, depending on binding efficiency [24].

Mature miRNAs play major roles in various cell processes in both physiological and pathological conditions; these include, but are not limited to, proliferation, apoptosis, metabolism, inflammatory response and fibrotic reaction [29-31]. These effects can be regulated in an autocrine manner or through paracrine signaling by exosome-mediated release of miRNAs [32]. In addition, different types of liver injury can elicit different miRNA secretion patterns and concentrations. Therefore, identifying disease-specific miRNAs could be used as potential diagnostic and therapeutic tools. In this review, we will focus primarily on human studies conducted in the past five years that identify miRNA patterns during cholangiopathies and the possible role that these miRNAs may play during pathogenesis.

2. Primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease that primarily affects the biliary epithelium [33]. While PSC is a fairly rare disease, its incidence and prevalence has recently been increasing [34]. PSC primarily affects middle-aged men, but cases have been seen in females as well as pediatric patients [35]. PSC patients typically present with chronic inflammation surrounding the biliary epithelium that eventually leads to bile duct strictures that can obstruct bile flow and lead to hepatic cholestasis [35]. Eventually, this cholestatic injury can induce surrounding resident liver cells to increase liver fibrosis, which can result in cirrhosis. PSC patients are also at a higher risk of developing cholangiocarcinoma (CCA), a highly malignant and devastating disease [36]. Research on the role of miRNAs during PSC pathogenesis has been insufficient. Therefore, the analysis of miRNA expression patterns should become an increased area of interest for the diagnosis of or therapeutic intervention for patients with PSC.

Recent studies have analyzed the miRNA profile of serum and bile samples from patients with PSC and patients with CCA complicating PSC. Overall, the miRNA expression of all analyzed miRNAs from both patients with PSC and patients with CCA complicating PSC was significantly higher compared with healthy samples [37]. Furthermore, miRNA concentrations in both serum and bile were increased in patients with CCA complicating PSC compared to levels in patients with PSC alone. The researchers also analyzed whether miRNA profiles differed between PSC alone and CCA complicating PSC patients and found that there were increased serum levels of miR-1281, miR-126, miR-122, miR-26a and miR-30b in PSC patients compared to CCA complicating PSC patients. These miRNAs, aside from miR-122, are mostly unstudied in liver pathology, but some studies have been reported on their functions. MiR-1281 is decreased during bladder cancer; miR-126 inhibits non-small cell lung carcinoma invasion; miR-122 aids in hepatocyte differentiation to reduce hepatocellular carcinoma formation; miR-26a inhibits the proliferation and invasion of cervical cancer cells; and miR-30b suppresses the formation of colorectal cancer [38-42]. Interestingly, these upregulated miRNAs appear to all play a role in the inhibition of cancer formation, which may explain why they are upregulated in PSC alone patients versus CCA complicating PSC patients.

Examination of the expression of miRNAs in bile revealed significantly increased concentrations of miR-412, miR-640, miR-1537 and miR-3189 in PSC alone patients compared with CCA complicating PSC patients. Similar to the upregulated miRNAs in serum, these miRNAs are largely unstudied in the liver. However, previously published data identifies miR-412 as upregulated in patients with ischemic hepatitis and patients with acetaminophen hepatotoxicity; miR-640 decreases angiogenesis in endothelial cells; miR-1537 is decreased in neuroblastoma; and miR-3189 plays an anti-tumoral role during glioblastoma [43-46]. Similar to the above miRNAs, miR-1537 and miR-3189 may act as tumor suppressors, which explains why they are upregulated in PSC alone patients versus CCA complicating PSC patients. However, the role of miR-640 and miR-412 in these patient subsets is a bit unclear.

Overall, this study identified differences in miRNA expression and concentration between PSC alone and CCA complicating PSC. Identifying different miRNA expressions may be a possible diagnostic tool to identify between these two diseases; however, the data showing that different miRNAs were noted in serum versus bile pose an interesting finding. Since this study only analyzed serum and bile samples, it is hard to identify where these miRNAs were initially transcribed and secreted. Identifying the primary source of these identified miRNAs will be beneficial to understanding the pathogenesis of PSC.

Similar research from another group analyzed serum miRNA levels in healthy control and PSC patient samples. Differential levels of 667 miRNAs were noted in diseased serum samples compared with healthy samples; however, no differences in miRNA expression were noted between different disease stages [22]. From the 667 differentially expressed miRNAs, receiver operating characteristic (ROC) curves were analyzed to identify useful biomarkers. Following ROC analysis, a total of 21 differentially expressed miRNAs were noted as potential biomarkers of PSC compared with healthy controls. Furthermore, miR-200c was found to be decreased specifically in serum from PSC patients versus healthy controls. The results also demonstrated upregulation of miR-193b, miR-122 and miR-885-5p in serum from PSC patients compared with healthy controls [22]. From this study, potential miRNA-based, PSC diagnostic tools were further identified.

Work from another group of researchers analyzed miRNA expression profiles between PSC patients and healthy controls, but looked specifically at hepatic expression. This publication noted increased hepatic expression of miR-378a-5p in PSC patients compared with healthy controls [47]. This paper further identified sulphotransferase 2A1 (SULT2A1), generally suggested to be a hepatoprotective protein, as a target of miR-378a-5p. Based on these findings, the paper concluded that increased levels of miR-378a-5p leads to decreased SULT2A1 expression and a subsequent increase in hepatic damage [47]. Unlike the previous studies, this publication aimed to understand the underlying cellular pathway that regulates PSC-associated hepatic damage. These results suggest that targeting miR-378-5p or SULT2A1 could provide therapeutic benefit to patients suffering from PSC.

3. Primary biliary cholangitis

Primary biliary cholangitis (PBC), previously known as primary biliary cirrhosis, is another rare but devastating immune-mediated cholangiopathy [48]. Similar to PSC, PBC is characterized by portal inflammation leading to fibrosis and eventually cirrhosis [49]. Distinct from PSC, advanced PBC is characterized by the destruction of small bile ducts leading to cholestatic injury and the subsequent hepatic damage associated with this event. Furthermore, PBC primarily affects middle-aged women, which is an opposite trend of that observed in PSC patients [50]. While identified as an autoimmune disorder, PBC is not considered a classical autoimmune disorder and patients usually do not respond to immunosuppressants [48]. Ursodeoxycholic acid (UDCA) was previously the only FDA approved treatment for PBC but tends to yield inconsistent results and a poor biochemical response [51]. Obeticholic acid is currently FDA approved for the treatment of PBC and tends to show favorable biochemical responses, but its direct effect on clinical outcome and quality of life have yet to be determined [52, 53]. Due to the varying efficacy of current treatment strategies, identifying other forms of therapy, such as miRNA-based therapeutics, could prove beneficial for patients suffering from PBC.

PBC can be broken down into three subtypes based on clinical course: (i) gradually progressive, (ii) portal hypertension, and (iii) hepatic failure [29, 54]. The authors of this study separated samples from patients who had received two years of UDCA treatment into each category and analyzed the obtained serum samples[54].The authors identified 97 miRNAs that were differentially expressed among the different subtypes. Furthermore, heat mapping showed that miRNA profiles from hepatic failure and portal hypertension subtypes were clustered differently than those from the gradually progressive subtype or controls. The authors then honed in on miR-139-5p due to the large amount of total short reads obtained for this miRNA, and since it was previously reported as increased in the serum of PBC patients [54]. The expression of miR-139-5p in lymphocytes that infiltrated the liver of PBC patients was significantly increased compared to controls, and in vitro work demonstrated that upregulation of miR-139-5p in hepatocytes correlated with increased tumor necrosis factor (TNF)-α secretion and repressed c-FOS gene transcription. Based on these findings, the authors concluded that miR-139-5p expression is enhanced during PBC and regulates inflammation via TNF-α and c-FOS regulation [29].

A recent study aimed to evaluate the potential roles that miRNAs may play during PBC progression [55]. The authors performed microarray analysis on healthy and PBC plasma samples and identified sixteen miRNAs that were differentially expressed in PBC plasma samples versus healthy controls. Their results also revealed that miR-92a was significantly downregulated in the plasma of PBC patients compared with healthy samples. In focusing specifically on peripheral blood mononuclear cells (PBMCs), the authors found that the expression pattern of miR-572 and miR-92a in PBMCs correlated with the decreased expression pattern seen in the plasma. Furthermore, miR-92a expression inversely correlated with the number of IL-17-producing T helper (Th17) cells, and the expression of miR-92a was highly co-localized with interleukin (IL)-17A in PBMCs isolated from PBC patients, suggesting a direct regulation of IL-17A by miR-92a. There was no correlation between the expression of other miRNAs and the Th17 population. Overall, these findings suggest a unique miRNA profile seen in the plasma of PBC patients that may be used as a diagnostic tool. However, this paper also identifies the possible role that miR-92a may play during the progression of PBC via regulation of Th17 cells [55]. While this paper does not identify the exact role that miR-92a plays in Th17 cells, the authors hypothesized that miR-92a may regulate Th17 cell differentiation, based on other publications [56, 57]. Considering that Th17 cells are a pro-inflammatory cell type, modulation of miR-92a may be a therapeutic option for ameliorating the inflammatory phenotype associated with PBC [58].

The Cl^-/HCO₃^- anion exchanger 2 (AE2) is found on cholangiocytes and regulates intracellular pH homeostasis and stimulates bicarbonate secretion [59]. Previous reports have revealed that PBC patients show decreased biliary AE2 expression [60], and researchers hypothesized that this decrease in AE2 expression may be regulated by miRNA expression. According to miRBase database analysis, miR-506 was predicted to potentially target the 3′ UTR of human AE2 mRNA, and miR-506 was also found to be highly upregulated in human PBC liver samples compared with healthy controls by microarray [61]. In situ hybridization verified that the increased miR-506 hepatic expression noted in human PBC was largely seen in cholangiocytes compared with other liver cell types. To confirm the activity of miR-506 on AE2, miR-506 was overexpressed in normal human cholangiocytes and decreased AE2 expression and activity were observed. The ability of miR-506 to target AE2 was validated in vitro using luciferase assay and site-directed mutagenesis in the 3′ UTR of AE2. To analyze miR-506 and AE2 expression in human PBC, the authors used primary cultures of isolated human PBC cholangiocytes obtained from a liver explant from a transplanted female patient. From this primary cell line, the authors found that the cultured human PBC cholangiocytes showed increased miR-506 expression, accompanied with decreased AE2 expression and activity, and transfection of the human PBC cholangiocytes with anti-miR-506 restored AE2 activity [61]. Overall, this paper indicates hepatic levels of miR-506 as a diagnostic tool. Considering AE2 is a key player in biliary function, and decreased AE2 activity is noted in PBC patients, targeting miR-506 may provide a much-needed therapeutic target for patients with PBC.

4. Biliary atresia

Biliary atresia (BA) is a neonatal liver disease that is characterized by inflammation and obliteration of the biliary tree leading to cholestasis and hepatic fibrosis [62]. If untreated, this damage can cause progressive conjugated hyperbilirubinemia, cirrhosis and hepatic failure [63]. The incidence of BA is approximately one in 10,000 births worldwide, but without therapeutic intervention, BA can be fatal within 2 years, with a median survival rate of eight months [64]. Considering the rarity of the disease, it is imperative that proper diagnostic tools are developed. Curative treatments have yet to be established for BA. The Kasai procedure is primarily performed to help restore bile flow and hopefully manage symptoms of the disease [62]. One-third of patients who undergo the Kasai procedure will survive more than 10 years without liver transplantation, while another third will have proper bile drainage but require liver transplantation prior to age 10 due to complications with cirrhosis; the last third of patients will have an inadequate response to the surgery and further develop progressive fibrosis and cirrhosis [64].

A recent study analyzed the efficacy of circulating miRNAs as markers of disease severity and etiology in pediatrics with cholestatic liver disease [65]. Analysis of serum levels revealed that BA patients, as well as pediatrics with other cholestatic liver diseases, had increased levels of miR-21 compared with healthy controls. Notably, BA patients had increased levels of miR-21 compared to pediatrics with other cholestatic liver diseases, indicating miR-21 as a possible BA-specific marker to rule out other pediatric cholestatic diseases. However, circulating miR-21 levels did not correlate with the degree of BA-associated hepatic fibrosis [65]. These results suggest that circulating miR-21 levels may serve as a useful diagnostic marker to identify BA from other pediatric cholestatic diseases, but it may not be suitable to determining the severity of BA-associated liver fibrosis. These findings were supported by another publication that found that hepatic expression of miR-21 was upregulated in BA patients compared with healthy controls [66].

Another study collected serum samples from BA and non-BA neonatal cholestasis and performed microarray analysis that identified 13 differentially expressed miRNAs [67]. The authors performed qRT-PCR analysis on 8 of the 13 differentially expressed miRNAs and verified that miR-92a, miR-4689 and miR-150-3p were upregulated, while miR-4429 was downregulated in BA samples. No significant change in expression was noted in the other analyzed miRNAs Functional enrichment analysis showed that miR-4689 and miR-4429 targeted genes were involved in the forkhead box O (FOXO)3 signaling pathway. Furthermore, area under the curve analysis showed that miR-4689 and miR-4429 had the potential to be useful biomarkers [67].

Considering BA is associated with enhanced biliary inflammation, it is important to recognize the role that miRNAs may play during the inflammatory process. One study looked at the role that miR-124 and miR-200 play during interleukin(IL)-6/signal transducer and activator of transcription (STAT)3 signaling[68]. IL-6 has been recognized as a key regulator of cholangiocyte proliferation [69]. miRNA analyses revealed 84 miRNAs that were decreased and 169 miRNAs that were increased in liver samples from BA patients compared with controls. Out of these miRNAs, miR-124 expression was decreased approximately 5-fold and members of the miR-200 family were increased approximately 5- to 10-fold. These results were confirmed by qRT-PCR. Moreover, expression of miR-124 was inversely correlated with STAT3, and expression of miR-200a inversely correlated with forkhead box (FOX)A2. Binding efficiency between these miRNAs and their respective mRNAs were predicted by TargetScan analysis and confirmed by luciferase assay [68].

In continuing study of other members of the miR-200 family, a second report found that BA patients had increased hepatic miR-200b expression that positively correlated with the degree of hepatic fibrosis [70]. Furthermore, in vitro studies indicated that miR-200b increased human hepatic stellate cell proliferation, migration and fibrotic reaction through modulation of PI3K/AKT signaling and matrix metalloproteinase 2 expression [70]. Combined, these studies allude to the diagnostic value of hepatic expression miR-200 family members and further suggest that therapeutic intervention of these miRNAs may play a role in fibrotic progression.

5. Polycystic liver disease

Polycystic liver disease (PLD) refers to a group of rare, inherited disorders in which structural changes in the biliary tree cause multiple cholangiocyte-derived cysts to develop within the liver [71, 72]. The most common complications associated with PLD include hypertension, back pain, abdominal distension, dyspnea, gastroesophageal reflux, cyst hemorrhage and infection [73]. PLD can occur on its own (autosomal dominant polycystic liver disease [ADPLD]) or develop as a consequence of autosomal dominant polycystic kidney disease (ADPKD) or autosomal recessive polycystic kidney disease (ARPKD) [73]. These three PLD-causing diseases arise from different inherited genetic mutations; development of ADPLD is due to mutations in protein kinase C substrate 80K-H (PRKCSH) and/or translocation protein SEC63 homolog (SEC63) genes; ADPKD is caused by mutations in the polycystin-1 and -2 (PKD-1 and PKD-2) genes; and ARPKD is due to mutations in the polycystic kidney and hepatic disease-1 (PKHD-1) gene [74]. Further perturbation of PLD occurs by malformation of the ductal plate and abnormal cell signaling and function [23]. Currently, therapeutic intervention relies heavily on the use of somatostatin analogs, and while these treatments tend to significantly decrease liver volume, there is only a slight improvement in symptoms [75]. Surgical procedures, such as fenestration of cysts and liver resection, tend to significantly relieve symptoms but have a high recurrence rate of cyst formation and symptoms [76, 77]. Very limited information exists regarding miRNA initiation, regulation, or expression during PLD.

To date, only one study regarding the role of miRNAs during hepatic cyst formation has been published [78]. While this is an older publication, published in 2008, the findings are worth discussing, based on the limited information on miRNAs during PLD. In whole liver sections from patients with PLD due to ARPKD or ADPKD, the expression of miR-15a was decreased in cholangiocytes lining cysts compared with normal human liver sections. Further analysis identified cell division cycle (CDC) 25A, which plays a major role in cell cycle progression, as a target of miR-15a. To verify that CDC25A is a target of miR-15a, staining was performed in liver sections and the results revealed that cholangiocytes lining liver cysts in patients with ARPKD or ADPKD had increased expression of CDC25A compared with normal human liver [78]. These findings suggest that suppression of miR-15a promotes hepatic cyst formation by increasing cholangiocyte proliferation via upregulated Cdc25A. Replenishing miR-15a biliary expression may decrease cyst growth in patients suffering from PLD. Further work regarding miRNA regulation of PLD is necessary.

6. Cholangiocarcinoma

Cholangiocarcinoma (CCA) is the second most common liver cancer and arises from damaged cholangiocytes lining the intra- and/or extra-hepatic bile ducts [79]. CCA is a devastating cancer that commonly has a poor prognosis and low five-year survival rate. While the diagnosis of CCA is fairly rare, its incidence has recently been rising [36]. Men are 1.5 times more likely to be diagnosed than women, and Asians are almost twice as likely to be diagnosed [36, 80]. Risk factors known to increase the likelihood of developing CCA include PSC, liver fluke infestation, hepatolithiasis, choledochal cysts and chronic liver infection; however, these risk factors cannot explain the current rise in CCA incidence [36]. Currently, surgical resection is the preferred method of treatment for patients with CCA; however, CCA patients who also have decreased liver function and/or underlying cirrhosis have poorer prognosis to this treatment [80, 81]. Based on the limited treatment options, it is imperative that new and effective treatment strategies are developed. While a large number of studies have been published on the role of miRNAs during CCA diagnosis, treatment and pathogenesis, this review will focus on those published within the past five years.

N-myc downstream-regulated gene 2 (NDRG2) plays a major role in cell cycle, growth and proliferation, and is mainly recognized as a tumor suppressor during carcinogenesis [82]. A recent study investigated the role of NDRG2 during CCA progression and its regulation by miR-

181c [83] Human CCA tumor samples showed decreased NDRG2 levels but increased miR-181c expression compared with adjacent non-tumor liver samples. Binding between miR-181c and NDRG2 was confirmed by luciferase assay and site-directed mutagenesis in the 3′ UTR of NDRG2 in cultured human CCA cells. Additionally, NDRG2-linked reporter activity was repressed by miR-181c mimic treatment in a dose-dependent manner, but reporter activity was greatly increased following anti-miR-181c treatment. Furthermore, downregulation of NDRG2 coupled with upregulation of miR-181c was significantly associated with a poorer prognosis, and in vitro work conferred increased chemoresistance and metastatic potential [83]. These results identify a novel pathway whereby miR-181c regulates NDRG2 to influence carcinogenesis and metastasis, and suggests the therapeutic potential of targeting miR-181c during CCA.

Inflammation favors tumorigenesis through enhanced angiogenesis, DNA damage, maintenance of cancer stem cells and cell proliferation [84]. Specifically, IL-6 has been recognized as a main cytokine produced by cholangiocytes during inflammatory processes such as cholangitis [85, 86]. One group studied miRNA and inflammatory differences between CCA tumors and adjacent non-tumor liver tissue [87]. In CCA samples, miR-122, miR-32, miR-101, let-7c, miR-99a and miR-125b were significantly downregulated, whereas miR-200c, miR-21 and miR-221 were significantly upregulated compared with adjacent non-tumor samples. To investigate whether these deregulated miRNAs played a role in CCA inflammation, predicted targeting relationships between these miRNAs and selected cytokines that are most commonly associated with CCA (IL-6, IL-8, insulin-like growth factor [IG]-1, transforming growth factor [TGF]-β1 and vascular endothelial growth factor [VEGF]) was performed using Starbase v2.0. The results demonstrated that miR-99a, let-7c and miR-125b (all of which were decreased in CCA) could regulate all of the aforementioned CCA-associated cytokines. Interestingly, these three miRNAs are found on the same cluster on chromosome 21q21, and it was hypothesized that the clustering of these three miRNAs could synergistically influence CCA-associated inflammatory processes. In vitro, reintroduction of miR-99a, let-7c or miR-125b inhibited human CCA cell migration and invasion, and reintroduction of the entire cluster significantly decreased human CCA cell ability to form cancer stem cell-like spheroids [87]. These results indicate that targeting miR-99a, let-7c and/or miR-125b may be a potential treatment strategy for targeting inflammation, cytokine production, metastasis, and cancer stem cell-like properties in CCA.

As found in the previous study, miR-101 was significantly downregulated in CCA tumors compared with adjacent non-tumor tissues and this was supported by work from another group [88]. This publication also found that miR-101 hepatic levels were decreased in CCA tumors compared with adjacent non-tumor tissues. Furthermore, the authors found that miR-101 levels were decreased in cultured human CCA cells compared with normal cultured cholangiocytes. The role of miR-101 during CCA pathogenesis was further characterized in vitro, and it was noted that VEGFC levels were inversely correlated with miR-101 expression. Overexpression of miR-101 decreased CCA migration and invasion, and administration of VEGFC reversed these effects [88]. This study provides therapeutic insight into the restoration of miR-101 on reversing CCA-associated angiogenesis.

Another study looked at the role of miR-21 during CCA proliferation [89]. The study found that in patients with CCA, serum miR-21 levels are drastically increased compared with serum levels in healthy controls, and this finding is partially corroborated by the previous study that found that hepatic miR-21 levels were significantly increased in CCA tumors compared with non-tumor liver tissue [87, 89]. Furthermore, this study showed that inhibition of miR-21 in vitro was able to reduce human CCA cell proliferation through modulation of the cell cycle and induction of apoptosis [89]. The authors also found that increased serum levels of miR-21 in CCA patients correlated with adverse clinical features, diminished survival and poor prognosis. These in vitro findings were also supported by another study that found that overexpression of miR-21 in human CCA cells promoted cell growth and migration, whereas inhibition of miR-21 decreased these activities [89]. Overall, these findings not only expand the role that miR-21 plays during CCA growth, but also highlights its usefulness as both a diagnostic tool and therapeutic target.

A recent publication analyzed the role of miRNAs during circadian rhythm oscillations and the expression of clock genes in CCA [90]. Circadian rhythm oscillations and the associated clock genes are primarily studied in the central nervous system, but these functions can be seen in peripheral tissues as well as single cells. However, aberrant circadian signaling can lead to tumorigenesis [91, 92]. PCR revealed that the gene expression of PER1, a clock gene, was decreased in cultured CCA cells compared to a normal cholangiocyte cell line, and this was supported by online microarray data showing similar results in human CCA compared with surrounding non-tumor tissue [90]. Protein expression of PER1 was also decreased in human CCA biopsies compared with normal human tissue. Conversely, CCA cells with Per1 overexpression showed decreased proliferation, arrested cell cycle progression and decreased cell migration. The authors hypothesized that Per1 expression may be regulated by miRNAs, and online analysis revealed that the 3′ UTR of PER1 could be targeted by miR-34a. Furthermore, miRNA microarray showed that miR-34a was upregulated in CCA cells compared with normal human liver samples. This interaction was verified by luciferase assay. However, when miR-34a was inhibited, CCA cells showed increased PER1 gene expression accompanied with decreased proliferation,arrested cell cycle and decreased cell migration [90]. These findings identify the role of miR-34a as an oncomir during CCA progression via decreased PER1 expression and increased cell proliferation and migration.

7. Conclusions and future perspectives

The complete determination of the cellular processes that regulate the initiation and progression of cholangiopathies is still widely unknown. Considering the major role that miRNAs play during mRNA expression and function, it is intuitive that these small RNAs could have key roles during cholangiopathy development. However, the specific role of individual miRNAs in the different cholangiopathies remains elusive. When looking at the miRNA expression profile for each cholangiopathy, there is little overlap in miRNA expression, implying that their expression may be cholangiopathy-specific (Table 1). However, some cholangiopathies do express the same miRNA. For instance, miR-21, a pro-fibrotic miRNA [31, 93], is upregulated in both BA and CCA, which are known to be fibrosing cholangiopathies [64, 79], explaining why miR-21 is increased in both BA and CCA. As well, miR-122 expression is upregulated in PSC but is downregulated in CCA. Since miR-122 has been identified as a marker of differentiation [94, 95] it makes sense that this miRNA is decreased in CCA as opposed to PSC. In contrast to miR-122 levels, miR-200c is downregulated in PSC but upregulated in CCA, and previous work has shown that miR-200c increases tumor growth and proliferation [96, 97], helping to explain this discrepancy in expression. Finally, miR-92a, another miRNA that supports tumor growth [98], shows decreased expression in PBC but increased expression in BA. This variation in expression may be dependent on the degree of hepatic damage and possible formation of liver cancer in the patients that were analyzed; however, this is not discussed in the referenced manuscripts.

Table 1.

Representation of the upregulated and downregulated miRNAs found in serum, bile or liver samples from patients with primary sclerosing cholangitis, primary biliary cirrhosis, biliary atresia, polycystic liver disease or cholangiocarcinoma.

Disease	sample type	upregulated	downregulated
	Serum	miR-1281	miR-200c
		miR-126
		miR-122
		miR-26a
		miR-30b
		miR-193b
Primary Sclerosing Cholangitis (PSC)		miR-885-5p
	Bile	miR-412
		miR-640
		miR-1537
		miR-3189
	Liver	miR-378a-5p
	Serum	miR-139-5p	miR-92a
Primary Biliary Cholangitis (PBC)			miR-572
	Liver	miR-139-5p
		miR-506
	Serum	miR-21	miR-4429
		miR-92a
Biliary Atresia (BA)		miR-4689
		miR-150-3p
	Liver	miR-200a	miR-124
		miR-200b
Polycystic Liver Disease (PLD)	Liver		miR-15a
	Serum	miR-21
		miR-181c	miR-122
		miR-200c	miR-32
		miR-21	miR-101
Cholangiocarcinoma (CCA)	Liver	miR-221	let-7c
		miR-34a	miR-99a
			miR-125b

Understanding and defining these miRNA profiles helps to uncover the clinical usefulness of miRNAs. Numerous human clinical trials are currently evaluating miRNA therapeutics during various liver diseases, such as targeting miR-122 during chronic hepatitis C infection [99, 100]. Aside from therapeutics, the development of diagnostic tools is of utmost importance, since these tools may be able to detect underlying liver injury that may not otherwise be noticed. However, the reliability and cost-effectiveness of screening for miRNAs pose a significant problem when developing miRNA-based diagnostic approaches. Based on the findings discussed in this review, it is evident that miRNA signatures are associated with diseased states, and that these expression patterns could provide important clinical benefit. These findings support the critical impact that these small RNAs have on cholangiopathy development.

Abbreviations

3′ UTR

3′ untranslated region

AE2

anion exchange protein 2

ADPKD

autosomal dominant polycystic kidney disease

ADPLD

autosomal dominant polycystic liver disease

ARPKD

autosomal recessive polycystic kidney disease

BA

biliary atresia

CCA

cholangiocarcinoma

(IL-6)

interleukin 6

MMP2

matrix metalloprotease 2

miRNA

microRNA

NDRG2

N-myc downstream-regulated gene 2

OCA

obeticholic acid

PBMC

peripheral blood mononuclear cell

PKD

polycystic kidney disease

PLD

polycystic liver disease

PBC

primary biliary cholangitis

pri-miRNA

primary miRNA

PRKCSH

protein kinase C substrate 80K-H

PSC

primary sclerosing cholangitis

qRT-PCR

quantitative real-time polymerase chain reaction

ROC

receiver operating characteristic

SULT2A1

sulfotransferase family 2A member 1

Th17

interleukin 17-producing T helper cell

TNFα

tumor necrosis factor-alpha

UDCA

ursodeoxycholic acid