Detection Of CircRNA Specifically Expressed In Biliary Atresia And Preliminary Exploration Of Its Clinical Signicance

Objective: To screen circRNA associated with biliary atresia.To analyze the expression level and mechanism of specific circRNA in liver tissue of patients with biliary atresia,and to provide a new biomarker for the early diagnosis of biliary atresia.Methods: In this study,we harvested liver tissue samples from 30 children who underwent laparoscopic surgery in our hospital from June 2017 to December 2017.There were 15 cases of liver tissue from patients with biliary atresia(BA group).In the control group,15 cases of liver tissue from patients with choledochal cyst(CC group)were selected.The children in the BA group were 45 to 107 days old,6 children were male and 9 children were female.The age of children in the CC group ranged from 3 months to 5 years,with 5males and 10 females.The expression profiles of circRNA were obtained by high-throughput RNA sequencing in liver tissue of 3 children with biliary atresia and 3normal liver tissue of control group.Six circRNAs with the most significant difference in expression were selected for RT-qPCR verification,including 3 circRNAs with low expression(hsa＿circ＿0081171、hsa＿circ＿0083234 and hsa＿circ＿0000374)and 3 circRNAs with high expression(hsa＿circ＿0004408、 hsa＿circ＿000909 and hsa＿circ＿0081828).The miRNAs that could bind to them and their downstream target genes were predicted by bioinformatics technology,and the KEGG signaling pathway enrichment analysis was performed to elucidate the potential pathogenesis of BA.Results:(1)The circRNA expression profiles of biliary atresia liver tissue and their control normal liver tissues were compared and analyzed by high-throughput RNA sequencing technology,and a total of 27 differentially expressed circRNAs were found,among which17 were highly expressed and 10 were low expressed in the biliary atresia group.(2)The6 circRNAs with the most significant differences in expression were verified by RT-qPCR,and the results showed that the level of hsa＿circ＿0009096 in BA group was higher than that in CC group(P < 0.0001);the expression level of hsa＿circ＿0083234 was down-regulated in BA group(P=0.0037);the expression level of hsa＿circ＿0081171 in BA group was lower than that in CC group,and the statistical results were insignificant(P=0.01986);the circulation number of hsa＿circ＿0081828 in RT-qPCR experiment was too high for statistical analysis.(3)Four miRNAs were obtained by crossing the predicted target miRNAs with BA related miRNAs(hsa-mi R-145-5p,hsa-mi R-222-3p,hsa-mi R-221-3p 和 hsa-mi R-432-5p).Based on these two circRNAs(hsa＿circ＿0009096、hsa＿circ＿0083234)and four miRNAs,a circRNA-miRNA regulatory network was established.KEGG signaling pathway enrichment analysis based on miRNA downstream genes revealed that hippo,TGF-β,adhesions junction,focal adhesion,ECM receptor interaction and regulating pluripotency of stem cells may be related to the pathogenesis of BA.Conclusions: 1.The specific circRNA expression profile of biliary atresia was obtained.2.RT-qPCR demonstrated that hsa＿circ＿0009096 and hsa＿circ＿0083234 are misexpressed in biliary atresia liver tissue might be involved in the disease progression.Therefore,hsa＿circ＿0009096 and hsa＿circ＿0083234 can be used as a candidate marker for BA diagnosis,and the study of circRNA-miRNA may provide a new way for exploring the pathogenesis of BA.