Screening And Validation Of Fusion Gene Related To HCC Using RNA Sequencing

ObjectiveTo study the transcriptome changes in hepatocellular carcinoma（HCC）tumor tissues using RNA sequencing（RNA-Seq）by screening and validating the gene fusion related to HCC,and then find the new HCC molecular marker and explore its potential role in the tumorigenesis and development of HCC.Methods1.Screening and identification of HCC-related fusion genes based on RNA-Seq.The total RNA of 9 HCC patients’tumor and adjacent non-tumor liver tissues was extracted,and the RNA-Seq was performed on IlluminaHiSeq technology platform to detect the transcriptional activity at mononucleotide level.By comparing with the matched adjacent tumor tissues,the differentially expressed genes（DEGs）and gene fusion of tumor tissues were analyzed.Meanwhile,HCC related gene fusion was screened and identified,and then validated by Sanger sequencing.2.Validation of fusion genes in enlarged sample size.Acquisition of full-length cDNA of recurrent CRYL1-IFT88 and analysis of its bioinformatics.4 fusion genes identified by RNA-Seq were subjected to validation using RT-PCR and Sanger sequencing in enlarged sample size.CRYL1-IFT88 was found to be a recurrent fusion gene in HCC tumor tissues.The full-length of CRYL1-IFT88 cDNA sequence was obtained by 5’and 3’RACE（rapid amplification of cDNA ends）experiments,and the function of CRYL1-IFT88 was analyzed by bioinformatics.Results1.Screening and identification of HCC-related fusion genes by RNA-Seq.RNA-Seq was performed in tumor and adjacent non-tumor tissues of 9 HCC patients,and 35M reads equal to 4.38G bases（4.3G-4.5G）was generated in each sample.A total of 78.8G bases were obtained in 18 samples,average 93%（90.3%-94.5%）of reads were aligned to human genome,resulting in an average coverage of depth of 31X（30-33X）.Average 17686（16652-18347）expressed genes were validated in each sample,account for 70%of the amount of genes provided by UCSC HG19T（25265 species）,of which 12611（11663-13543）genes were FPKM≥1.By comparing with 9 pairs of tumor and adjacent non-tumorous tissues,we detected 1943 DEGs between groups,including 690 up-regulated and1253 down-regulated.The DEGs were enriched in ten pathweays,including cell cycle,DNA replication,p53 and complement and coagulation cascades pathway and several metabolism pathways.7 gene fusions were identified in 9 tumor tissues,including CRYL1-IFT88,BRD9-OR4N2,MPV17-TRIM54,BHMT-MTATP6P1,NOMO3-XYLT1（chr16:16372646;chr16:17318037）,NOMO3-XYLT1（chr16:16372646;chr16:17323802）and TNFSF14-C3,of which CRYL1-IFT88,BRD9-OR4N2,MPV17-TRIM54 and NOMO3-XYLT1（chr16:16372646;chr16:17318037）validated by RT-PCR and Sanger sequencing were consistent with that in RNA-Seq.2.Validation of fusion genes in extended sample size.Acquisition of full-length cDNA of recurrent CRYL1-IFT88 and analysis of its bioinformatics.2.1 4 fusion genes were validated in 54 HCC tumor and adjacent non-tumor tissues by using RT-PCR and Sanger sequencing,and then CRYL1-IFT88 fusion gene was found existed in 5 tumor tissues.The recurrent rate of CRYL1-IFT88fusion gene related to HCC tumor tissue was 9.52%（6/63）.However,compared with CYRL1-IFT88,the other 3 fusion genes including BRD9-OR4N2,MPV17-TRIM54 and NOMO3-XYLT1（chr16:16372646;chr16:17318037）were all negative in 54 pair of samples.2.2 The full-length of CRYL1-IFT88 fusion gene cDNA sequence,779bp,is obtained by performing both 3’and 5’RACE experiments,which open reading frame（ORF）was 279bp owning a coding product of 92 amino acids.2.3 According to bioinformatics analysis,the molecular weight,molecular formula and isoelectric point of CRYL1-IFT88 fusion protein were 9822.34,C₄₂₇H₇₀₁N₁₁₉O₁₃₃S₆,5.25,respectively.The function of CRYL1-IFT88 fusion protein was similar with CRYL1.The gene fusion between CYL1 and IFT88 changed the structure of IFT88,which was responsible for the depression of its tumor suppressor function.Conclusions1.Highly abnormal transcript level is present in the HCC tumor tissues.A large amount of DEGs are enriched in ten pathways,including cell cycle,DNA replication,p53 and complement and coagulation cascades pathway,etc.,and several metabolism pathways,of which cell cycle,DNA replication,p53 and complement and coagulation cascades pathways are closely related to HCC.Meanwhile,multiple gene fusions are present in HCC tumor tissues.2.CRYL1-IFT88 fusion gene is a recurrent transcript existed in HCC tumor tissues,and it would be a new HCC molecular marker.The gene fusion between CRYL1 and IFT88 might be involved in the tumorigenesis and development of HCC.