Introduction
Biliary atresia (BA) is a rare but severe neonatal disease characterized by an inflammatory and progressive fibrotic obstruction of the extrahepatic bile ducts resulting in cholestasis and subsequent hepatic failure if left untreated (1). Rapid liver fibrosis is a major outcome for children with BA even though they might appear normal at birth (2,3). Knowledge on the pathogenesis of liver fibrosis in BA is still limited. Considering the complicated etiologies, the identify potentially modifiable factors in BA associated liver fibrosis is an urgent need (2).
The microarray and RNA-seq technique can help to identify signatures of predominant transcriptions of the liver during fibrosis progressive in BA, which could be beneficial for uncovering new molecular mechanisms to improve the prognosis, diagnosis and treatment of this disease (4). This technique has been successfully applied for liver tissues from BA and CC to investigate the potential underlying mechanism (5-8). A transcriptional analysis of liver tissues from BA and CC identified 877 differentially expressed genes (such as COX1, SCO2, and CYTB, which are involved in oxidative phosphorylation) and several pathways with immune and inflammatory responses involved in the pathogenesis of BA (such as the Wnt-signaling pathway and TGF-β pathway) (5) Previous studies have found that the NF-κB signaling pathway could regulate genes related to immune response and mediate liver fibrosis by initiating fibrosis factors and thus might serve a key role in the development of BA (9-11). Another RNA sequencing of livers from 6 BA and 6 CC found that Foxa3 may exert a role in the progression of hepatic fibrosis in BA, which may be a potential targeted treatment (6). Fas ligand mRNA (7), MMP7 and PCK1(8) can be used as potential biomarkers to predict the outcome of BA and the fibrosis progression. A comparison of mRNA expression level from normal, diseased control and end-stage BA livers identified 35 genes involved in cell signaling, transcription regulation, hepatobiliary development and fibrosis process (12). The expression level of genes regulating fibrosis in liver tissues increases in infants who survived within 2 years (13). Furthermore, cDNA array demonstrate two hepato-fibrogenesis-associated genes (4), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1).
Long noncoding RNAs (lncRNAs) exert specific regulatory functions in various cellular organization (14), which are involved in hepatic fibrosis by regulating gene or protein expression (15). LncRNAs can act as competing endogenous RNAs (ceRNAs) to participate in hepatic fibrosis (16). The expression level of long noncoding RNA H19 (lncRNA H19) is positively correlated with the severity of fibrotic liver injuries in BA patients, which serves a pivotal role in BA cholangiocyte proliferation and cholestatic liver injury as a sponge for let-7/HMGA2 axes and regulates S1PR2/SphK2(17). LncRNA-Annexin A2 pseudogene 3 (ANXA2P3) is identified to participate in the process of BA-induced liver fibrosis by regulating Annexin A2 (ANXA2) (18). Our previous study on lncRNA-adducin 3 antisense RNA1 was identified as participating in liver fibrosis by targeting ADD3 in vitro accelerating the proliferation and migration of LX-2 cells, which are the key cells involved in hepatic fibrosis (19). The construction and analysis of regulation network based on microarray and RNA-seq results can improve understanding of the mechanisms underlying BA associated hepatic fibrosis. More evidence will pave the way to fully understand hepatic fibrosis pathogenesis in BA patients (20).
The present study aimed to identify some key DEmRNAs and DElncRNA in liver tissues of BA patients through RNA-sequencing and also to discover some hub proteins via constructing PPI networks of DEmRNAs and DElncRNA-DEmRNA co-expression networks. Through the aforementioned approaches, it was expected to ascertain some lncRNAs, mRNAs and pathways that serve an important role in the pathogenesis of liver fibrosis with BA.
Discussion
Hepatic fibrosis usually develops in a progressive pattern in infants with BA. Even though these patients received Kasai portoenterostomy (KPE), the majority of them still have to undergo liver transplant due to severe fibrosis, cirrhosis and liver failure. However, the molecular network underpinning this expeditious fibrogenic process remains to be elucidated (23). In this regard, the present study performed RNA sequencing on liver samples of BA and CC to identify abundant DElncRNAs and DEmRNAs between two groups.
First, the results were analyzed for mRNA and lncRNA that were significantly upregulated or downregulated, including GPC3, AFP, CYP1A2, CCL20, 11βHSD1, AKR1D1, GNMT, JPX and MIR4435-2HG.
Glypican-3 (GPC3) a heparan sulfate proteoglycan attached to the cell membrane, serves a role in the regulation of the signaling activity involved in numerous growth factors (24), including Wnt/β (25), Hedgehogs (26), bone morphogenetic proteins and fibroblast growth factors (27). The Wnt/β-catenin signaling pathway has a key role in the regulation of cellular functions such as biliary cell fate (28), thereby raising a potential participation in BA etiology via GPC3-mediated Wnt/β-catenin signaling. Sirisomboonlarp et al (29) found that circulating GPC3 protein is significantly elevated in BA patients and positively correlated with liver stiffness, indicating that serum GPC3 could be a biomarker to evaluate hepatic function and to predict prognosis for BA patients following Kasai surgery. Notably, from the results of the present study, GPC3 was also identified to be the second most over-expressed DEmRNAs in BA liver tissues.
Serum α-fetoprotein (AFP) was strongly associated with epithelial liver tumors (30). It has a sensitivity of >90 and >70%, for hepatocellular carcinoma and hepatoblastoma (HB), respectively (31). Amir et al (32) found that patients with BA are far more prone to suffer from HB and in such cases, serum AFP levels might not be specific to diagnose HB in such cases, though remaining sensitive. A previous study (33) identified that ~8% of BA children were affected by liver tumors and AFP level screening and regular imaging examinations were crucial for early diagnosis of malignant liver tumors in BA patients.
Distinguishing BA from other non-BA neonatal cholestasis is challenging (34). Shteyer et al (35) show that Continuous Breath 13C-methacetin breath test might be useful to distinguish BA from non-BA. Methacetin is a substrate that can evaluate liver metabolic function by being metabolized by cytochrome P450 1A2 (CYP1A2) (36), which is negatively correlated with hepatic fibrosis in patients with chronic hepatitis C viral infection (37,38) and is also found to be involved in other forms of hepatic diseases (39). Crawford et al (40) discovered that downregulated CYP1A2 could aggravate inflammatory response due to an increase of proinflammatory cytokines, such as TNF-α and IL-6. CYP1A2 is the most significant downregulated DEmRNAs in BA liver tissues, indicating that it might participate in this inflammatory fibrosclerosing disease and thus can be an indicator to differentiate BA. Therefore, further investigations into the intrinsic pathway of CYP1A2 on the etiology of progressive fibrosis in BA are of importance.
It is hypothesized that the discovery of more valuable biomarkers to monitor the progression of hepatic fibrosis following KPE may help improve clinical outcomes in patients and delay the need for liver transplantation (41), including lncRNA APTR (42), miR-21(43) and mRNA FN14(44). In the present study, the results also identified a number of valuable biomarkers related to liver fibrosis. Chemokine ligand-20 (CCL20) acted as a potent chemoattractant for immature dendritic cells, which can mediate the strong inflammatory responses that drive liver fibrosis (45). In vivo experiments show that a deficiency of 11β-hydroxysteroid dehydrogenase-1 (11βHSD1) could lead to the activation of hepatic myofibroblast and thus exacerbate liver fibrosis in mice (46). Expression level of steroid 5β-reductase (AKR1D1) in the liver tissue is negatively correlated with liver fibrosis and inflammation (47). Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and its reduction has a negative effect on the maintenance of normal liver functions progressing to fibrosis, cirrhosis and hepatocellular carcinoma (48).
Liver fibrosis is strongly associated with the activation of hepatic stellate cells. LncRNA XIST enhances eol-induced autophagy process and subsequently activates hepatic stellate cells through the miR-29b/HMGB1 signaling pathway (49). LncRNA JPX, a non-protein coding RNA transcribed by the X deactivated central gene, activates XIST transcription via an interaction with CCCTC-binding factor (50). From the sequencing data, the present study identified that JPX was an upregulated DElncRNA. Although the regulatory relationship between JPX and XIST was established, the role it serves in hepatic fibrosis and BA remains to be explored in the future.
Kerola et al (51) found a decrease of the expression level of TGF-β1 after successful HPE, indicating that TGF-β1 might be involved in exacerbating liver fibrosis. LncRNA miR4435-2HG can promote prostate carcinoma (52), non-small-cell lung carcinoma (53) and ovarian carcinoma (54) by interacting with TGF-β signaling and thus lncRNA miR4435-2HG-mediated TGF-β1 signaling pathway might be involved in liver fibrosis of BA.
Second, Cytoscape was used to select the top 10 hub genes of PPI network for further analysis and it revealed that CXCL8, TLR8, CCR5, TLR7 and CYP3A4 were associated with BA, revealing that these hub genes may be involved in the occurrence of BA.
CXCL8 regulates inflammation and immune response via chemoattractance to neutrophils (55). Studies show that an elevation of serum CXCL8 together with its increase in liver may participate in inflammation and liver fibrosis in patients with BA and thus CXCL8 may be a potential biomarker for diagnosis, severity evaluation and outcome of BA (56,57).
The major inappropriate host immunological reactions against unknown ligands via the TLR cascades may trigger progressive inflammatory biliary destruction that manifests as BA in newborns or infants. TLR cascades may induce an excessive immune reaction against multiple ligands in the bile duct system, leading to a progressive destruction and inflammatory fibro-obstruction of biliary ducts seen in BA. miR expression level of TLR8 is significantly elevated in liver tissues in the early BA group (58). The activation of TLR7 signaling pathway is crucial for cholangiocytes proliferation in rhesus rotavirus (RRV)-infected mice models (59). In addition, TLR7 can recognize infectious pathogens, activated type 1 interferon and induce the expression of inflammatory response genes, including IL-8(60).
A previous study has shown that CC chemokines, including CCL2, CCL3, CCL5, stimulate THP-1 cells to increase MMP-9 protein levels in a dose-dependent manner, indicating that the activation of immune cells and MMP are closely related to the occurrence of tissue inflammation (61). Leonhardt et al (62) identified >40 significantly differentially-expressed genes in BA mice. Most of the upregulated genes in BA-positive mice were involved in immune response, such as CCL2, CCL5, CCR5, CXCL9, CXCL10 and IL1F5. To clarify the pathogenesis of BA, the immune reaction during the process of liver fibrosis should be further examined.
Finally, lncRNA-targets were analyzed. In order to fully illustrate how lncRNAs participate in a disease, it is widely accepted to perform a lncRNA-mRNA co-expression analysis to explore the biological functions of lncRNAs by evaluating and analyzing their co-expressed mRNAs. In the co-expression networks of the present study, CYP2E1 was a target for H19, H19 was upregulated and CYP2E1 was downregulated. Xiao et al (17) discovered that the levels of H19 in both liver and serum exosome increase in line with the severity of fibrosis in patients with BA. De Bock et al (63) found that CYP1A2, 2C19, 2E1 and 3A4 were at low activity levels in BA patients, which is consistent with the results of the present study. As for how they regulate each other in hepatic fibrosis and BA, this remains to be explored in the future.
A total of 2,086 DEmRNAs and 184 DElncRNAs were identified between liver tissues in BA and CC. DEmRNA and DElncRNA including GPC3, AFP, CYP1A2, CYP3A4, JPX and miR4435-2HG, might have an important role in the pathogenesis of liver fibrosis in BA. Furthermore, the hub genes of PPI network and the relation pairs in the lncRNA-target network were analyzed, including hub gene CXCL8, TLR7, TLR8, CCR5 and lncRNA-target pair H19 CYP2E1.
The mechanisms of fibrogenesis in BA remain to be elucidated, multiple pathways are simultaneous activated under cholestasis background. It is hoped that the RNA-sequencing results and bioinformation analysis in the present study could contribute some indications for description of this pathological process.
There are some limitation to the present study; first, as in any statistical analysis, a reliable result depends on large-scale samples. The sample size in the present study was relatively small, due to the rare incidence of this progressive and severe neonatal fibro-obstructive cholangiopathy. Second, two age-unmatched patients with choledochal cyst under normal liver function test was as control instead of normal liver tissues due to ethical issues. It is hoped that further studies can be performed to validate the findings of the present study by enrolling more clinical samples and then the possible mechanisms can be clarified more accurately. Third, experiments on these candidate hub genes and pathways of the potential relation pair and network crosstalking to elucidate the molecular mechanism of BA are required.