Exploring Genomic Variants And Genes In Hepatocellular Carcinoma By High-throughput Sequencing Technology

Background and objective:Hepatocellular carcinoma(HCC)is one of the most common malignancies worldwide with an annual incidence of 523,432 cases,56%of which are in China.The estimated mortality is 266,830 cases deaths a year in China.The risk factors for HCC include infection with hepatitis B virus(HBV)or hepatitis C virus(HCV),alcoholic liver disease,and most probably nonalcoholic fatty liver disease.At present,many studies have been performed to define the molecular mechanisms underlying hepatocarcinogenesis.There are many genomic alterations in liver cancer tissues,including changes in expression regulation,chromosome structure abnormalities,and single nucleotide variations.It has been found that gene mutations and expression regulation have an important relationship with the development of tumors.Now,the driver genes in HCC include:TP53,ARID1A,CTNNB1,NRAS,RBI,BCL9,CDKN2A and PTEN,but there are still many genes to be further confirmed,and the study of expression of somatic mutations at the level of transcriptome is not fully characterized.Methods:(1)We performed transcriptome sequencing in liver cancer and cirrhotic tissues of nine HCC patients.We identified differentially expressed genes(DEGs)and constructed a weighted gene co-expression network(WGCNA)for the DEGs.The relationship between DEG and the clinical features of HCC patients(such as tumor grade and alpha fetoprotein(AFP)levels)was analyzed to explore expression patterns between liver cancer and cirrhotic tissues;identify important modules associated with clinical characteristics;discovery new biological regulatory networks related to liver cancer;identify key hub genes in the networks and validate hub genes using quantitative reverse transcription-polymerase chain reaction(qRT-PCR).(2)We performed whole exome sequencing(WES)in 12 liver cancer tissues,matched cirrhotic tissues,and whole blood samples to identify somatic mutations and characterize mutation patterns in HCC.Subsequently,by combining with transcriptome sequencing data in nine liver cancer and matched cirrhotic tissues,we described the expression of somatic mutations and identified novel HCC candidate genes.We explored clinical significance of novel HCC candidate genes in the Cancer Genome Atlas(TCGA)and the potential roles of them in cancer signaling pathways.Results:(1)In total,747 DEGs were identified,and MEtan co-expression module was significantly associated with a-fetoprotein(AFP)level.Of note,we identified 15 hub genes in MEtan module associated with AFP level,and three(SPX,AFP and ADGRE1)of four hub genes were validated in an independent HCC cohort(n=78).(2)Among 1,554 non-silent mutations(i.e.,missense,nonsense mutations,splice sites and translation start sites)identified in 12 HCC patients,we found that T:A>A:T substitutions showed the highest percentage of non-silent mutations.We found that the majority of somatic mutations occurred in the stage from cirrhosis to HCC and the prevalence of non-silent mutations from blood(i.e.,surrogate for normal liver tissues)to cirrhosis was significantly less than that from cirrhosis to HCC.By comparison of somatic mutations identified in liver cancer tissues and cirrhotic tissues,we found that somatic mutations were not accumulated in genes in HBV-associated cirrhotic liver tissues.Notably,two genes(TP53 and KRTAP4-3)were shown to be significantly mutated by MutSigCV analysis,suggesting that there was evidence for mutations of these genes contributing to the development of liver cancer.Subsequently,by combining with transcriptome sequencing data in nine liver cancer and matched cirrhotic tissues,we analyzed the transcriptional pattern of mutant allele and identified a novel HCC-related candidate gene(VPS35).Mutation and differential expression of VP S3 5 were both associated with poor survival in the TCGA HCC cohort(p=3.3E-2,2E-3).We found that VPS35 may be involved in RTK/Ras/PI(3)K pathway of HCC,and the expression of NRAS was regulated by both FGFR3 and VPS35.The RTK/Ras/PI(3)K pathway has pivotal roles in subtype-specific HCCs.Conclusions:High throughput sequencing technology has been broadly applied in the field of life sciences.Complex disease researches have promoted the development of analysis methods of Macro genomics.Our study identified hub genes for HCC clinical traits and characterized the patterns of somatic mutation expression,which provide new ideas and theoretical basis for further exploration of the pathogenesis of HCC.