The Mutations And Biological Role Of UBE3C In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common and aggressive human malignancies worldwide, the third most common cause of cancer-related mortality. An estimated748,300new liver cancer cases and695,900cancer deaths occurred worldwide. Half of these cases and deaths were estimated to occur in China. Although recent progress in early diagnosis, resection due to surveillance programs curative or liver transplantation has significantly improved the survival of patients with HCC, the overall survival of HCC patients is still poor. The mechanism of hepatocarcinogenesis remains unclear and HCC related susceptibility genes is still lacking. Thus, the discovery of novel HCC susceptibility genes will provide new targets for the development of new drugs and gene therapy.Tumorigenesis is a complex biological process. The activation of oncogenes or inactivation of tumor suppressor genes caused by gene changes is at its core. Similar to other solid tumors, HCC also accumulated a large amount of genetic variations in the development of hepatocarcinogenesis. Normal liver with increasing genetic variants gradually developed into precancerous lesions and eventually evolved into the neoplasm. Hepatocarcinogenesis is now known as a histological development process induced by multi-factor and multi-step. Most of HCC occurred from cirrhosis-inducing conditions, with a development process from chronic hepatitis B or C viral infection to dysplastic nodules (DN) and to HCC. Therefore, the earlier genetic variations associated with HCC will be found by comparing the difference of genetic variations among the normal cell genome, the DN genome and HCC genome.Recently, the progress of next-generation sequencing promotes the rapid development of the whole-genome sequencing and whole-exome sequencing which were widely used in susceptibility gene research of complex diseases such as human cancers. Exons represent the regions in genes that are translated into protein. It is estimated that the protein coding regions of the human genome constitute most of the disease-causing mutations. Exons constitute about1%of the human genome. Thus, exome sequencing is an efficient strategy to selectively sequence the coding regions of the genome as a cheaper but still effective alternative to whole genome sequencing. The goal of exome sequencing is to identify the functional variation that is responsible for both Mendelian diseases and complex diseases such as human cancers. The goal of this study is to look for the earlier genetic variations associated with HCC by sequencing the normal cell genome, the DN genome and HCC genome. The candidate gene(s) will be validated and investigated by functional studies. Part OneExome Sequencing to Identify Hepatocellular Carcinoma Related Genetic Variants AbstractPurpose:To identify novel hepatocellular carcinoma (HCC) related genetic variants by sequencing the whole exome of the normal cell genome, the dysplastic nodules (DN) genome and HCC genome from one selective HCC patient.Methods:Whole-exome capture sequencing was performed using the SureSelect Target Enrichment System (Agilent Technologies) in accordance with the manufacturer’s protocol with slight modifications. The novel HCC related genetic variants were selected by comparing the difference of genetic variations among the normal cell genome, the DN genome and HCC genome. All the candidate variations were validated by Sanger sequencing.Results:The5,72and182single nucleotide variations (SNVs) were found in DN, tumor1and tumor2, respectively. No gene was found between DN and two tumors. Two genes, UBE3C located at chromosome7and MUC16located at chromosome19, were found to be mutated in both tumors. The mutations were validated by Sanger sequencing. In addition, no small indel was found in DN. Three and nine novel small indels were found in tumor1and tumor2, respectively.Conclusions:No gene was found between DN and two tumors. The genes UBE3C and MUC16were found to be mutated in both tumors, indicating a relationship between these two genes and hepatocarcinogenesis. Part TwoThe Validation and Structure of the Candidate Gene UBE3C in Hepatocellular CarcinomaAbstractPurpose:To explore the mutations of ubiquitin ligase E3C (UBE3C) in hepatocellular carcinoma (HCC).Methods:All the exons of UBE3C were sequenced using Sanger sequencing in another independent set of105HCC patients. The relationship was analyzed between UBE3C mutations and HCC clinicopathological features.Results:There were17samples of all the106detected samples (including one sample used in exome sequencing) conferring allele change mutation in UBE3C. The frequency of gene mutations was16.0%. The mutation positions located at exon5,7,13,20and22. Among these mutations, the Asn929Ile, Glu959Lys and Thr1004Ala occurred in region of UBE3C HECT domain. And the Asn929, Glu959and Thr1004were highly conserved among different species. Statistical analysis showed that UBE3C mutations were not associated with sex, age, HBV DNA levels, liver cirrhosis, tumor size, tumor encapsulation, tumor differentiation, vascular invasion and tumor TNM stage.Conclusions:There were multisite and highly frequent mutations of UBE3C in HCC. The Asn929Ile, Glu959Lys and Thr1004Ala mutations may have an important impact on protein function of UBE3C.UBE3C may be involved in the occurrence and development process of HCC. Part ThreeThe Biological Role of UBE3C in Hepatocellular CarcinomaAbstractPurpose:To investigate the expression of UBE3C expression in HCC cell lines and clinical samples of HCC patients, and to explore the effect of UBE3C on human HCC proliferation and invasion.Methods:Western blot was used to detect the UBE3C protein expression in a normal hepatic cell line L-02and6HCC cell lines including SMMC-7721, Huh7, Hep3B, HCCLM3, MHCC97-H and MHCC97-L. Immunohistochemistry was performed to analyze the UBE3C expression in HCC tumor and adjacent tumor tissue. CCK8assay was used to detect the growth rates of HCC cell lines Huh7and MHCC97-H. The wound-healing assay and24-well Transwell test were used to evaluate the ability of cell migration. Cell invasion assays were performed using24-well Transwell precoated with matrigel.Results:The protein expression of UBE3C was mainly distributed in the cytoplasm and observed in almost all HCC cell lines, with relatively low expression in normal hepatic cell line L-02and relatively high expression in Huh7and MHCC97-H. In tissue samples, the UBE3C protein mainly existed in the cytoplasm and was found a higher expression in HCC tissues than in adjacent normal tissue. In HCC cell lines Huh7and MHCC97-H, knock-down UBE3C expression could inhibit cell proliferation, migration and invasion.Conclusions:UBE3C could promote HCC cell proliferation, migration and invasion and play a role in hepatocarcinogenesis, serving as a potential HCC related oncogene.