Metabolomic And Transcriptomic Profiling Of Hepatocellular Carcinomas In Hras12V Transgenic Mice

Objective: To explore the ras oncogene induced the development of hepatic tumor,the characteristic of changes in the level of metabolomics and transcriptome,and provide useful clues for the study of the pathogenesis of liver tumors and the clinical diagnosis and treatment.Methords:1.Parts of the hepatic tumor from Hras12 V oncogene mouse and wild-type liver tissues of control mouse were cut into ~ 1 cm3 tissue blocks,rinsed in cold saline,and immediately flash frozen in liquid nitrogen for collecting total RNA and GC-MS analysis in further.10% formalin fixation the remains liver tissues,and the tissues were used for the further experimental process after the morphological diagnosis.2.All of the samples were prepared for GC-MS analysis,and were subjected to two derivatization steps for GC-MS analysis,and mixed well for GC-MS analysis.Then begin the GC-TOF-MS analysis;the chroma TOF4.3X software of the LECO Corporation and LECO-Fiehn Rtx5 database were used for the extraction of raw peaks,the filtering of data baseline,the calibration of the baseline,peak alignment,deconvolution analysis,peak identification and integration of the peak area,the generated peak lists were all imported into Pirouette software(Infometrix,Woodinville,WA)for multivariate statistical analysis.Briefly,all metabolites from the GS-MS array dataset were entered into MSEA as a list of metabolites.The mouse pathway library was chosen,and the ORA algorithm was selected hypergeometric test for metabolite set enrichment.3.Different genes expression of the two groups were detected.The samples which will be tested total RNA,prepared samples for RNA-Seq analysis were performed using the Tru Seq RNA LT Sample Prep Kit v2.The samples were sequenced by Illumina Hi Seq2000 instrument.Read alignment to the UCSC Mouse Reference Genome(http://genome.ucsc.edu/)was performed using Tophat v1.3.2.Transcript assembly and quantification were performed by Cufflinks v2.0.2.DAVID v6.7 was used to interpret the sequence data and KEGG enrichment assay.4.Our laboratory tested some important metabolites with kits,including NADPH,triglyceride,cholesterol,pyruvate,lactate,bile acid,GSH,and ROS.The all above metabolites were quantified by using corresponding kits strictly according to the directions of the instruction books.All of the tests were measured in three coupies.Results:1.Identify with the phenomenon of riched lipid droplets in hepatoma carcinoma,the lipid biosynthesis in liver tumor tissues was significantly added;2.Acetyl-Co A from glycolysis and citrate shuttle activity was improved;a sufficient quantity supply of NADPH was from pentose phosphate pathway activity;3.Upregulation of key enzymes was related to lipid de novo biosynthesis;but downregulation of key enzymes was associated with bile acid biosynthesis;4.Primary carcinoma of the liver contrast with normal liver tissues,NADPH,pyruvate,triglyceride,cholesterol,and glutathione was significantly elevated,but lactic acid was significantly reduced,however,ROS was contributed into a normal level.Conclusion: Our results suggest that the lipid metabolism,glycolysis,phosphate pathway activity,tricarboxylic acid cycle,citrate shuttle activity,bile acid synthesis,and redox homeostasis in the HCC induced by ras oncogene are significantly perturbed,and these altered metabolic processes may play crucial roles in the carcinogenesis,development,and pathological characteristics of HCC.