Identification Of The Candidate Genes In Biliary Atresia And The Function Exploration In Vitro

Biliary atresia (BA) is a progressive obliterative process involving the extrahepatic and intrahepatic bile ducts in the newborn. It is characterised by worsening cholestasis, hepatic fibrosis, and cirrhosis, which lead to portal hypertension and a decline in hepatic synthetic function. The etiology of BA remains unknown. It has recently been proposed that the perinatal/acquired form may be caused by a biliary trophicviral infection, leading to an initial bile duct epithelial injury that triggers a persistent immune-mediated sclerosing process resulting in obstruction of extrahepatic bile ducts. However, the relations between different genes are complicated. Therefore, we undertook a large-scale gene-expression analysis to identify physiologically relevant genomic signatures of biliary atresia in the livers of affected infants. Meanwhile, bioinformatic analysis was applied to classify the different genes and find regulatory modules in the pathgenesis of BA. Furthermore reverse-transcription polymerase chain reaction (RT-PCR) was performed to confirm changes in gene expression. We also investigated the role of the most important regulatory modules by gene transfection in votro.Objective To investigate the gene expressing profile and find regulatory modules through large scale gene expression data analysis in BA (biliary atresia).Methods Using cDNA microarrays, we analyzed and compared gene expression profiles in the liver tissure of BA and CBD. Meanwhile, bioinformatic analysis including STC,STC-GO (Series Test Cluster of Gene Ontology),pathway-finder and Dynamic-GeneNet construction was applied to classify the different genes and find regulatory modules in the pathgenesis of BA.Results There are more than 1000 different genes between BA and CBD. After STC, we find two significant profile(profile21 and 23); By STC-GO (Series Test Cluster of Gene Ontology), the different genes of profile21 and 23 mainly include immune response, antigen processing and presentation of exogenous peptide antigen via MHC classⅡand multicellular organismal development and extracellular matrix synthesis, retrospectively. By pathway-finder, integrin-mediated cell adhesion,matrix metalloproteinases,inflammatory response pathway were identified as the significant pathway. ITGAM ITGB2,TNFSFR21,RGS19 and LAMC1,LAMA5,MMP7,TIMP2,MMP11 were identified as the key genes in profile21 and 23 retrospectively. By construction of dynamic-genenet, LDOC1 was found as the most important regulatory modules.Conclusion 1.Biliary atresia was caused by immune inflammation conducted by MHC classⅡmolecules and extracellular matrix synthesis.2. ITGAM,ITGB2,TNFSFR21,RGS19 and LAMC1,LAMA5,MMP7,TIMP2,MMP11 have important role in the pathogenesis of immune inflammation and extracellular matrix synthesis in biliary atresia, respectively.3. LDOC1 may be the most important regulatory module in the pathogenesis of biliary atresia.Methods The liver biopsy tissures from 22 cases of biliary atresia and 14 cases of CBD were collected. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to confirm the gene expression changes of ITGAM,ITGB2,TNFSFR21,RGS19,LAMC1,LAMA5,MMP7,TIMP2,MMP11.Real-time PCR was used to investigate the expression of LDOC1.Meawhile, screening for mutations of the ITGB2 (CD18) gene and LDOC1gene were performed on DNA extracted from whole blood of 10 biliary atresia patients. Polymerase chain reaction amplification was performed, followed by bidirectional semi-automated DNA sequencing analysis.Results The levels of the expression of ITGAM,ITGB2,TNFSFR21,RGS19,LAMC1,LAMA5,MMP7,TIMP2,MMP11and LDOC1 mRNA were obviously higher in biliary atresia liver tissues than that in CBD liver tissues(p<0.05). After DNA sequencing analysis, ITGB2 gene showed variations in 9 bilary atresia patients (9/10), genetic variations include three mutation:N212Y(2/10), Y544H(1/10), P752S(4/10) and 11 polymorphisms:-156T/C(5/10),117G/A(1/10),919G/A(5/10),1101C/A(4/10),1533C/T(1/10), 3'-UTR+123C/T(4/10),3'-UTR+140G/A(4/10),3'-UTR+148C/A(6/10), 3'-UTR+170C/T(8/10),3'-UTR+219T/C(1/10),3'-UTR+286C/T(3/10). LDOC1 gene showed variations in 3 bilary atresia patients (3/10) including 1 polymorphisms: 3'-UTR+822C/T.Conclusion 1.The expression of ITGAM,ITGB2,TNFSFR21,RGS19,LAMC1,LAMA5,MMP7,TIMP2,MMP11and LDOC1 elevated obviously in biliary atresia. These genes may have important role in the inflammation and liver fibrosis in biliary atresia. 2.The exons of ITGB2 gene have 3 mutations and 11 SNP variations and LDOC1 gene have 1 SNP variations in biliary atresia. The mutations may cause the constructional and functional changes of the ITGB2 protein that involved in the pathogenesis of biliary atresia. The SNP variations of LDOC1 and ITGB2 can probably be linked to a genetic predisposition to biliary atresia.Methods We constructed plasmid pIRES2-EGFP-LDOC1 carrying a full-length LDOC1 cDNA and transfected into Jurkat cell by adenovirus. We divided into three groups including empty,pAD-LDOC1-IRES2-EGFP and PAd-IRES2-EGFP. The westernblot method was used to detect the expression of LDOC1 protein between the groups. The TNF-α,IL-2,IFN-γlevel of resting and activated Jurkat cell in each group were detected by elisa.Results After transfection, the LDOC1 protein elevated remarkably in pAD-LDOC1-IRES2-EGFP than that of PAd-IRES2-EGFP. The expression of TNF-α,IL-2,IFN-γlevel of resting Jurkat cell were of no statistically difference between pAD-LDOCl-IRES2-EGFP and PAd-IRES2-EGFP,but when Jurkat cell was activated by PMA, the expression of TNF-α,IL-2,IFN-γlevel increased more obviously in pAD-LDOCl-IRES2-EGFP than that of PAd-IRES2-EGFP(p<0.05).Conclusion Overexpression LDOC1 could enhance the production of TNF-α,IL-2,IFN-γ, and it indicated that LDOC1 may promote the inflammation reaction in biliary atresia which aggravate the injury of biliary epithelium.