Serum microRNA microarray analysis identifies miR-4429 and miR-4689 are potential diagnostic biomarkers for biliary atresia

This study aimed to investigate pathogenesis and novel diagnostic biomarkers of biliary atresia (BA). Serum samples from infants with BA and non-BA neonatal cholestasis (NC) were collected for miRNA microarray analysis, and then differentially expressed miRNAs were screened. Differentially expressed miRNAs were validated by qRT-PCR using an independent serum samples from infants with BA and NC. Diagnostic utility of validated miRNAs was further analyzed using serum samples by receiver-operating characteristic curve analysis. Totally, 13 differentially expressed miRNAs were identified including 11 down-regulated and 2 up-regulated ones. Target genes of hsa-miR-4429 and hsa-miR-4689 were significantly involved in FoxO signaling pathway. Eight differentially expressed miRNAs were chosen for validation by qRT-PCR analysis, and four miRNAs (hsa-miR-150-3p, hsa-miR-4429, hsa-miR-4689 and hsa-miR-92a-3p) were differentially expressed. The area under the curve of hsa-miR-4429 and hsa-miR-4689 was 0.789 (sensitivity = 83.33%, specificity = 80.00%) and 0.722 (sensitivity = 66.67%, specificity = 80.00%), respectively. Differentially expressed miRNAs including hsa-miR-4429 and hsa-miR-4689 might play critical roles in BA by regulating their target genes, and these two miRNAs may have the potential to become diagnostic biomarkers.

Biliary atresia (BA), a rare but serious cholestatic disorder in newborn infants, is caused by obstruction of extrahepatic or intrahepatic bile ducts with an occurrence rate of about 1/12,000 cases in United States and a higher incidence in Asia 1,2 . If not recognized and treated, BA will develop to progressive biliary cirrhosis and liver failure which could cause death within two years 3 . Kasai operation can reestablish bile flow in up to two-thirds of BA patients when performed earlier than 60 days of age 2 . However, a majority of patients will need liver transplant for survival if significant fibrosis and cirrhosis still exist after surgical intervention. Therefore, early identification and timely surgery are crucial for better prognosis. Unfortunately, definitive diagnosis of BA requires invasive and time-consuming diagnostic procedures, such as intra-operative cholangiogram and liver biopsy 4 . Consequently, it is urgently necessary for identification of noninvasive and convenient diagnostic biomarkers which may be helpful in distinguishing BA from the other neonatal cholestatic diseases.
MicroRNAs (miRNAs) are an abundant class of endogenous small and noncoding RNAs of about 22-nucleotides that post-transcriptionally regulate gene expressions 5 . Many studies have reported that miRNAs play important roles in experimental BA. The up-regulated expression of miR-29 in liver of murine BA model could lead to dysregulations of Igf1 and Il1RAP which are respectively responsible for cholangiocyte survival and modulation of inflammation 6 . In addition, miR-133a/b, miR-30b/c, miR-200a, miR-195, miR-365 and miR-320 have regulatory roles in pathogenesis of BA according to the miRNA expression profiles of extrahepatic bile ducts and gallbladder from murine BA model 7 . Shen et al. have reported that miR-222 was highly expressed in the extrahepatic bile ducts which might be responsible for liver fibrosis in the murine BA model 8 . The PI3K/Akt signaling pathway is activated in BA by elevated expression of miR-200b through suppressing FOG2, leading to increased growth and migration of human hepatic stallate cells 9 . Although understanding of BA pathogenesis has been improved, BA still remains to be a significant challenge and needs to be further investigated 10 . Therefore, it is of great importance to focus on the pathogenesis and diagnosis of BA. Expression patterns of serum miRNAs can detect various diseases and distinguish similar disorders because serum levels of miRNAs are reproducible, stable and consistent among individuals of the same species 11,12 . What's more, circulating miRNAs have been proposed as novel noninvasive biomarkers with encouraging diagnostic utility of BA. Zahm  In order to screen more differentially expressed miRNAs, human microRNA microarrays from Agilent Technologies containing probes for 1523 miRNAs were adopted in this current study,. Meanwhile, advanced bioinformatics analysis including prediction of target genes, identification of interaction relationships between target genes and functional enrichment analysis was also conducted for better understanding of the molecular mechanisms of BA. Moreover, the differentially expressed miRNAs were further validated in a larger and independent cohort of 45 infants with BA and 30 controls with non-BA neonatal cholestasis to find potential serum miRNA biomarkers.

Discussion
Long-term survival can be achieved without liver transplantation when the Kasai procedure is conducted within the first 30-45 days of life 15 . Since early diagnosis and convenient screening means of BA haven't been well established, treatment for BA is inadequate due to delayed diagnosis and poor understanding of the pathogenesis. In this study, 13 differentially expressed miRNAs were identified using the Agilent miRNA microarray expression profiling. Moreover, eight differentially expressed miRNAs were selected for validation by qRT-PCR using an independent serum samples from infants with BA and NC controls. The validation analysis revealed that four miRNAs including hsa-miR-150-3p, hsa-miR-4429, hsa-miR-4689 and hsa-miR-92a-3p were differentially expressed.  In our study, functional enrichment analysis indicated that GNAI1 targeted by hsa-miR-4429 and GPR180 by hsa-miR-92a-3p were significantly related to the function of G-protein coupled receptor signaling pathway. It has been revealed that secretin can contribute to increased ductular choleresis after stimulation of G-protein-coupled receptor and cAMP/protein kinase A (PKA)-dependent signalling pathway 16 . Moreover, decreased expression of the type III InsP3R in the cholangiocytes of patients with biliary atresia is associated with impaired Ca2+ signaling and messenger molecule interacts with specific G protein-coupled receptors can induce activation of phospholipase C and formation of InsP3 to increase Ca 2+ 17,18 . Accordingly, hsa-miR-4429 and hsa-miR-92a-3p may be important for BA by targeting GNAI1 and GPR180 to influence the G-protein coupled receptor signaling pathway.
Moreover, target genes of hsa-miR-150-3p (IGF1, PRKAA2 and FOXO3), hsa-miR-4689 (SGK1), hsa-miR-4429 (MAPK1, AKT3, IGF1R and PTEN) and hsa-miR-92a-3p (NLK, KLF2 and PTEN) were significantly enriched in FoxO signaling pathway. BA is manifested by progressive inflammation and fibrosis of extrahepatic and intrahepatic bile ducts which can lead to cirrhosis 19 . It has been reported the FoxO3/Bim signaling pathway was obviously activated in patients with primary biliary cirrhosis 20 . Meanwhile, target genes of hsa-miR-4429 (AKT3, MAPK1, PTEN and TSC1) were significantly involved in the mTOR and PI3K-Akt signaling pathway. Increased expression levels of miR-200b in biliary atresia patients can acitvate PI3K/Akt signaling to accelerate migration and proliferation of hepatic stallate cells 9 . It has been reported that cystic proliferation of cholangiocytes of the polycystic kidney rat is associated with activation of the PI3K/mTOR pathway 21 . Therefore, it could be speculated thathsa-miR-4429 and hsa-miR-4689 might play important roles in BA by regulating their target genes that participate in these important signaling pathways.
Furthermore, the diagnostic utility of these four miRNAs (hsa-miR-150-3p, hsa-miR-4429, hsa-miR-4689 and hsa-miR-92a-3p) were analyzed in a larger independent sample set from another 35 infants with BA and 20 NC controls. The AUC of hsa-miR-4429 and hsa-miR-4689 was respectively 0.789 (95% CI, 0.590-0.921;  Table 4. The target genes with degrees not less than five in the protein-protein interaction network. It could be speculated the different results might be due to that samples obtained from different sources were investigated. However, there are some limitations in our study. Some characteristics of participates, such as ethnic and region, were not considered due to the limited sample size. Besides, the expression levels of import target genes were not studied. In conclusion, the differentially expressed miRNAs (especially hsa-miR-150-3p, hsa-miR-4429, hsa-miR-4689 and hsa-miR-92a-3p) might play important roles in the pathogenesis of BA by regulating their target genes. Furthermore, hsa-miR-4429 and hsa-miR-4689 might have promising diagnostic performance for BA. In our next study, expression levels of important target genes will be investigated by integrated miRNA and mRNA analysis to find more potential biomarkers for BA.

Methods
Study population and sample collection. In our study, 45 infants with BA and 30 infants with non-BA neonatal cholestasis (NC) similar in age and sex distribution during the same time period were enrolled from the Children's Hospital of Fudan University (Table 7). These participants were enrolled based on the diagnoses of operative cholangiogram and liver pathology. The inclusion criteria for infants with BA were as follows: aged less than 90 days; type III BA according to the classification of BA phenotype 22 ; serum direct or conjugated hyperbilirubinemia (> 20% of total bilirubin and > 2 mg/dL). Infants were excluded when they had liver failure, malignancy, ischemic hepatopathy, hypoxia or shock within the preceding 2 weeks; treated with extracorporeal membrane oxygenation-associated cholestasis or prior hepatobiliary surgery. Meanwhile, infants with birth weight less than 1500 g, drug-or total parenteral nutrition-associated cholestasis, bacterial or fungal sepsis, or primary hemolytic disease were also excluded unless they were diagnosed with BA or another cholestatic disease definitively 23,24 .
Blood samples were collected from these participants within a few days of enrollment preoperatively when the parents had given voluntary informed consent for their children. Our studies have been reviewed and approved by the ethics committee of the Children's Hospital of Fudan University and all the experiments were carried out in accordance with relevant guidelines and regulations.
Sample processing and total RNA isolation. All whole blood samples collected from each participant were allowed to stand for about 1 h at room temperature. Then, these whole blood samples were separated into serum by centrifugation at 820 × g for 10 min at 4 °C, followed by further centrifugation at 16,000 × g for 10 min at 4 °C to completely remove cell debris. The supernatant serum was stored at − 20 °C until analysis. Total RNA was isolated from 400 μ l serum sample by using mirVana miRNA isolation kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The concentration and quality of total RNA were monitored  MF  15   KLF5, GATA2, MYB, NFIA, PBX1, SOX4, TCF21,  TFAP2B, BTG2, KLF4, ONECUT2, CREB5, EHF,  RIT1, MYOCD   0.012047   GO:0005515  Protein binding  MF  232   FOXO3, SMAD5, CREB5, KLF5, RNF4, HNF1B,  ROCK1, GATA2, MYB, PBX1, SOX4, TFAP2B,  BTG2, KLF4, CREB5, RIT1, MYOCD   0.015416   GO:0007156  Homophilic cell adhesion via plasma membrane  adhesion molecules  BP  22   CDH2, CDH10, DSC2, ROBO2, NPTN, PCDH11X,  CDH20, PCDHAC2, PCDHAC1, PCDHA13   Serum miRNA expression profiling and microarray analysis. Serum samples of 4 infants with BA and 4 NC controls were used for miRNA microarray analysis. Human miRNA microarrays from Agilent Technologies (8*60 K), containing probes for 1523 human miRNAs from the Sanger miRbase V18.0 database (http://www.sanger.ac.uk/Software/ Rfam/ mirna), were adopted. Total RNA (100 ng) extracted from each serum sample was used as inputs for sample labeling and hybridization preparation in accordance with the manufacturer's protocol (Agilent Technologies, Santa Clara, CA). The microarray image information was converted into spot intensity values using Scanner Control Software Rev. 7.0 (Agilent Technologies, Santa Clara, CA). The signal after background subtraction was exported directly into the GeneSpring GX version 12.5 software (Agilent Technologies, Santa Clara, CA) for quartile normalization. Then, differentially expressed miRNAs in serum were identified using the paired t-test with the cut-off criteria of P < 0.05 and |fold change|> 1.5. In order to ensure the screened differentially expressed miRNAs were accurately identified, hierarchical clustering analysis of samples was employed using heatmap.2 function of th gplots package in R 25 based on the expression values.
The target genes of differentially expressed miRNAs were predicted by at least two databases of the following five usual prediction databases: TargetScan (http://www.targetscan.org), miRanda (http://www.microrna.org/ microrna/home.do), PicTar (http://pictar.mdc-berlin.de/), MirTarget2 from miRDB (http://mirdb.org/miRDB/ download.html) and PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html). Moreover, the Gene Ontology (GO) functional and pathway enrichment analysis were conducted for the target genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tools 26 with the cut-off criterion of false discovery rate (FDR) < 0.05. The GO terms were identified in biological process (BP), cellular component (CC) and molecular function (MF) categories. The regulatory relationships for targets genes that simultaneously involved in significantly enriched functions and pathways were selected to constructe miRNA-target gene regulatory network. Protein-protein interactions (PPIs) for these target genes were revealed   Quantitative real-time PCR (qRT-PCR). The relative quantification of selected differentially expressed miRNAs was performed by qRT-PCR reaction with the miScript SYBR Green PCR Kit (Qiagen) using ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The miRNA specific primers were designed by Primer Express software (Version 2.0, Applied Biosystems) based on the miRNA sequences obtained from miRbase database (http://microrna.sanger.ac.uk/). Primer sequences are listed in the Table 8. Extracted total RNA (60 ng) from serum samples was reverse transcribed into cDNA using miScript Reverse Transcription Kit (Qiagen). Each reaction was performed in a 20 μ l volume system containing 1.5 μ l cDNA, 2 μ l of each primer and 1 × QuantiTect SYBR Green PCR Master Mix (Qiagen). MiR-1228 was used as a stable endogenous control for normalization since it functions as a housekeeping gene according to the study of Hu et al. 28 . All reactions were carried out in triplicate. The relative expression levels of miRNAs were calculated by the 2 −△△Ct method.

Statistics.
Receiver-operating characteristic (ROC) curve analysis was performed to determine the specificity and sensitivity of miRNA as a diagnostic biomarker. MedCalc (version 10.4.7.0; MedCalc, Mariakerke, Belgium) software was adopted to perform ROC analysis. Area under the ROC curve (AUC) was calculated as an accuracy index for evaluating the diagnostic performance of selected miRNA. The 95% confidence interval (CI) was used to reflect statistical significance.  Table 8. Primers used for quantitative real-time PCR (qRT-PCR).