Construction And Analysis Of HCC Gene Expression Profile And High Resolution Map Of Abnormal Alterations In HCC Genomic DNA

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and the second cancer killer in China, threatening Chinese people's health severely. Significant achievements have been made in basic and clinical research of HCC. However, as HCC is a kind of highly hetero-geneous disease, more studies are needed to deepen the cognition and un-derstanding of molecular mechanism of hepatocarcinogenesis. In this study, we try to elucidate the molecular mechanism more clearly by searching for genes differentially expressed in HCC as well as the abnormal alterations in HCC genomic DNA.In the first part, we applied SAGE and LongSAGE to obtain the gene ex-pression files of normal liver, HepG2 and HCC. The same HCC sample was further analyzed simultaneously using LongSAGE. By comparing gene ex-pression profiles of HepG2 and normal liver, we found that genes differentially expressed in HepG2 included those encoding ribosomal protein, blood coa-gulation factors, complement factors, as well as those involved in MAPK sig-naling pathway, cell cycles regulation, cell adhesion, calcium signaling pathway, and nicotinic acid/nicotinamide metabolism. Comparison of gene expression profiles of HCC and normal liver leaded to the identification of genes differen-tially expressed in HCC, which participated in biological processes of biosyn-thesis, cell proliferation, cell death, signal transduction, transport, blood coa-gulation, response to external stimulus, and cellular metabolism. Totally 12 genes (including PEG10, SGCE, ZNF83, PTPN12, TM4SF1 et al) differently expressed in HCC identified by SAGE were confirmed to be up-regulated in HCC compared to the corresponding nontumorous liver (p< 0.05). Among these 12 genes, it was worth mentioning that SGCE and PEG10 locate to-gether within 7q21, separated by only 115bp base pairs between the 5' ends of these two genes. Furthermore, similar expression patterns (up-or down-regulated simultaneously) of these two genes were observed in 75% of HCC cases detected, being up-regulated together in most cases.In the second part, we obtain a comprehensive and high resolution map of abnormal alterations in HCC genomic DNA with the help of Digital karyotyping technology and ultrahigh-throughput second generation sequencing system of Genome Sequencer FLX. The blunt-ended adaptors provided by Genome Sequencer FLX was modified to keep critical information contained in the ad-hesive ends of genomic tag concatemers, which in turn improved sequencing efficiency. Totally 111,585 reads were sequenced and 358,931 filtered tags were obtained from the Digital karyotyping library. By analyzing distribution of these filtered tags on each chromosomes, we found that genomic DNA ampli-fication in HCC were mainly occurred on chromosome 1,4,6,7,8,9,12,15, 17,19,21, X and Y. Genes involved in the amplified regions included BAGE, UTRN, DYNC1I1, SLC25A13, POTEB and CEACAM21 et al. The DYNC1I1 and SLC25A13 genes also locate on 7q21, very close to PEG10 and SGCE. Genomic DNA of DYNC1I1, SLC25A13, POTEB, CEACAM21, PEG10 and SGCE were detected to be amplified in 36%,52%,50%,36%,23%and 32% of HCC cases respectively. Gene expression level of DYNC1I1, SLC25A13, PEG10 and SGCE were also found to be increased in HCC compared to the corresponding nontumorous liver (p< 0.05). For DYNC1I1, SLC25A13, PEG10 and SGCE genes, amplification of genomic DNA accompanied by up-regulation of corresponding genes were observed in 55-69% of HCC cases. These results indicated that DYNC1I1, SLC25A13, PEG10 and SGCE genes located together within 7q21 may be involved in molecular mechanisms of development and progression of HCC, and genomic DNA amplification may play a role in their up-regulation in HCC.One of the most important biological characters of HCC is the improper activation and inactivation of protein tyrosine phosphatases which leads to abnormal alteration in the phosphorylation level of tyrosine amino acid. In the third part, we performed preliminary study on finding potential proteins inte-racted with PCP-2, a member of protein tyrosine phosphatases, using yeast two-hybrid technology. By screening the pre-transformed yeast library of human brain cDNA, the interaction between PCP-2 and theβ3A subunit of adaptor protein (AP)-3 was detected in yeast and further confirmed in mam-malian cells. Theβ3A subunit of AP-3 may interact with PCP-2 through rec-ognition of YXXΦand/or LL motifs contained within cytoplasmic domain of PCP-2, thus participating in biological processes of intracellular trafficking, lo-calization or recruitment of PCP-2.Our study on the transcriptome and genomic DNA abnormalities in HCC may lead to better understanding of molecular mechanism of hepatocarcino-genesis, providing more potential molecular markers for diagnosis, progres-sion, metastasis and prognosis of HCC, as well as more potential targeted genes for designing molecular drugs for HCC therapy.