Paradoxical Reactions Of β-Catenin To TNF-α And Fas-induced Acute Liver Failure

Part I Paradoxical Reactions of β-Catenin to TNF-a-and Fas-induced Acute Liver FailureBackground & Objective:Acute liver failure (ALF) is a life threatening clinical syndrome associated with high mortality rate worldwide. Mutiple organ failure is one of the common complicates. Lacking of specific effective treatment, the prognosis of ALF is often dissatisfactory. Orthotopic liver transplantation (OLT) remains the main effective option of treatment for ALF. However, most of patients are unable or reluctant to have OLT due to organ shortage, high surgery risk, high cost of treatment, and lifelong anti-rejection medication.The liver injury mechanisms of ALF can be classified into two groups. Firstly, directly damage of cellular organs by toxic substances, which lead to apoptosis. Secondly, secondary damage induced by immune system response. Consequently, eliminating apoptosis could be a new strategy for ALF treatment. Lipopolysaccharide (LPS) plus D-galactosamine (GalN) treatment of mice is a well-established experimental mode of ALF. TNF-α is the predominant mediator that induces hepatocyte apoptosis. Fas-mediated acute hepatocyte apoptosis participated in many liver diseases. The liver is very sensitive to Fas-mediated apoptosis because Fas antigen is highly expressed on hepatocytes. When mice are injected with anti-Fas antibody (Jo2), the cause of death is secondary to liver failure.β-Catenin is the chief downstream effector of the canonical Wnt signaling pathway, which plays essential role in regulating cell growth, regeneration, differentiation, and apoptosis. The aberrant alternations of Wnt/β-Catenin pathway, especially constitutively activation of β-Catenin have been described in various human cancers. However, the role in regulation apoptosis of β-Catenin is quite complicated. In our previously job, liver specific β-Catenin knockout mice alleviated GalN/LPS-induced liver injury, but aggravated Jo2-induced liver injury. The mechanism of the dual role of β-Catenin in two different liver injury process still unclear.This study uses hepato-specific β-Catenin knockout mice and Hepal-6 cell line overexpressed β-Catenin as tools to further validate the role of β-Catenin in acute liver injury in vivo and vitro. The mechanism of the dual role would be analyzed.Materials&Methods:1. Breed hepatocyte specific β-Catenin knock out mice. Genotype was identified via PCR;2. Mice liver injury models were established by intraperitoneal injection of Ga1N/LPS or Jo2. Liver samples were collected after 6hr administration;3. Hepatocytes apoptosis were assayed by TUNEL staining. Western blot were used to detect expression of apoptosis associated molecules, including Caspase 3 and PAPR.4. si-RNA technique was used to knockdown β-Catenin in Hepal-6 cell line. Knockdown efficiency was assayed by qRT-PCR and western blot. Cytotoxic effect of TNF-α and Jo2 on β-Catenin knockdown cells was detected by CCK-8 assay.5. Nuclear translocation of p65 was detected via immunohistochemistry. Reporter gene was used to assay transcription activity of NF-κ B.6. Expression of downstream target gene of NF-κ B (SOD2 and A20) were assessed by qRT-PCR.7. The interaction of β-Catenin and p65 was showed by Co-immunoprecipitation assay.8. Hepal-6 cells were infected with mutant active β-Catenin (Ad-S37A) overexpression adenovirus to establish β-Catenin overexpression cells. The overexpression efficiency was assayed by western blot, reporter gene, and qRT-PCR.9. P65 nuclear translocation after β-Catenin overexpression was observed by immunofluorescent assay.10. MDA detection Kit was used to check the oxidative stress status induced by TNF-a in Hepal-6 cells transfected with Ad-S37A.11. Fas expression in liver tissue was detected by qRT-PCR and Western blot.12. The data were analyzed by GraphPad Prism 5.0 statistics software.Results:1. Hepatocyte-specific knock-out of β-Catenin significantly reduced GalN/LPS-induced apoptosis. Cell death induced by TNF-a was reduced after silence β-Catenin in Hepal-6 cells, but increased in β-Catenin overexpression cells.2. β-Catenin physically combined with NF-κ B resulting in reduced NF-κ B transcription activity. After GalN/LPS administration, hepatocytes from β-Catenin knockdown mice showed more p65 nuclear translocation and higher A20 and SOD2 expression. Hepal-6 cells transfected with Ad-S37A showed less p65 nuclear translocation.3. Hepatocyte-specific knockout of β-Catenin reduced oxidative stress level in liver after GalN/LPS treatment. Conversely, overexpression of β-Catenin in vitro aggravated TNF-a-induced oxidative stress and cell death.4. The combination of β-Catenin and p65 affected Fas expression. Hepatocyte-specific knockout of β-Catenin exacerbated Jo2-mediated liver injury due to the higher expression of Fas, which sanitized hepatocytes to Jo2-induced cell death.Conclusions:We investigated the different role of β-Catenin in TNF-α and Fas induced acute liver failure in vivo and vitro. β-Catenin could aggravate TNF-α-induced hepatocyte apoptosis through combining with NF-κ B, which suppressed it’s transcription activity. In addition,β-Catenin could increase TNF-α induced oxidative stress in liver. Controlled by NF-κ B, Fas expression elevated in β-Catenin knockout hepatocytes, which lead to more severe liver injury.β-Catenin modulates TNF-α and Fas induced ALF through it’s interaction with NF-κ B.Part Ⅱ Detection of single nucleotide variants and indels within intraheptic cholangiocarcinoma exome dataBackground & Objective:Intrahepatic cholangiocarcinoma is a malignant tumor originated from peripheral (intrahepatic) biliary epithelial cells, which ranks the second common liver tumor. Lacking of specific biomarker and typical imaging features lead to difficulties in early diagnosis. Furthermore, ICC always accompany with early lympho node metastasis, as a result, the vast patients diagnosed with ICC are in advanced stage, which caused short survival after diagnosis. Patients diagnosed with ICC are increasing in the last two decade, which requires more attention from clinical and research workers.Exome is the group of whole exons of human genome functioned for coding proteins. 85% of disease-causing mutations originated from exons. Exome sequencing was widely used in tumor genome research for its high through-put and low cost in resent years. Through exome sequencing, mutations in protein coding region could be detected. By evaluate the effect of somatic mutation’s on protein coding genes, tumor-causing mutations could be found.By sequencing 24 genomes from 8 ICC cases, our study aimed at finding SNPs and InDels associated with ICC, which could provide new directions in finding ICC-causing gene.Materials&Methods:1. Collect liver samples from eight ICC cases which were verified by pathological tests. Each case includes three samples, tumor, paracancerous and normal liver.2. Genomes were extracted by QIAamp DNA Mini Kit. Exomes were captured by Roche NimbleGen V2 exome kit.3. Exome sequencing was applied on Illunima Solexa 2000 platform with a 2xl00bp paired-end strategy. Sequencing depth is 45x.4. After quality control by FastQC, sequencing reads were aligned to human reference genome (GRCh37.63) by BWA and further filtered by SAMtools.5. Variants were detected by VarScan, in-house Perl Script was used for further filtration.6. Extract germline mutations from tumor samples. Variants reported in dbSNP and 1000 Genome Project was also excluded.7. CooVar was used to predict the functions of somatic mutations on protein coding genes.8. Genes mutated in at least two cases were reported as candidate ICC driver genes.Results1. Sequencing reads from all samples have passed FastQC check.2. After alignment,70% of reads were perfectly aligned to human reference genome. The average read depth with coding region is 47.2x, around 90.2% of coding region were reached more than 10 read depth.3. After variant detection, filtration, and function prediction,2550 mutations were found to change amino acid coding.23 genes were found to be mutated in at least two cases.ConclusionIn our study,24 genomes from 8 ICC samples were sequenced by exome sequencing. We detected the mutations in ICC genome for the first time. Mutations in 23 genes could be associated with ICC.