Whole-exome sequencing （WES）, also known as targeted exome capture sequencing, refers to a genomic analysis method that utilizes sequence capture technology to isolate and enrich the exonic regions of the whole genome, followed by high-throughput sequencing. Exome capture sequencing targets only the sequences within exonic regions, making it more cost-effective compared to whole-genome resequencing. It allows for the acquisition of substantial useful information with relatively small data volumes, offering significant advantages for studying SNPs, InDels, and other genetic variations.

1.Utilization of cutting-edge exome capture sequencing technology—Ion Proton Ampliseq Whole Exome Capture Sequencing—enables more uniform coverage of the same gene fragments, leading to more accurate data analysis results.
2.Extremely low sample input requirement: as low as 10 ng, compatible with FFPE samples.
3.Ultra-high sequencing depth: capable of detecting both common variants and rare variants with frequencies below 1%.
4.Integration of Liebing Technology’s years of bioinformatics analysis expertise and high-impact publication experience, offering customized analysis solutions tailored to different biological research questions.

1.DNA Samples:Total genomic DNA ≥ 1 µg; electrophoresis must show a distinct band >10 kb, with no degradation (no obvious smear below the main band) and no contamination from proteins or RNA. For FFPE DNA samples:total DNA ≥ 1 µg, with >50% of fragments >1000 bp. Dissolve in TE, AE, or ddH₂O, store at -20°C, and ship on dry ice.
2.Fresh Tissue:Tissue weight 50–100 mg (approximately the size of a soybean). Flash-freeze in liquid nitrogen, store at -80°C (may also be stored in RNAlater; do not store in Trizol), and ship on dry ice (samples stored in RNAlater may be shipped with ice packs).
3.Peripheral Blood:2–3 ml of peripheral blood collected in EDTA anticoagulant tubes (purple cap). Store at -80°C, avoid repeated freeze-thaw cycles. Ship on dry ice.
4.FFPE Tissue Sections:Thickness 8–10 µm, tissue area >1 cm² (HE staining should show >50% tumor cell content). Provide 10–20 sections. Store at room temperature or 4°C. Ship at ambient temperature.
5.Needle Biopsy Tissue:Two biopsy cores, diameter 1–2 mm, length >0.5 cm. Flash-freeze in liquid nitrogen, store at -80°C. Ship on dry ice.
6.Paraffin-Embedded Tissue Blocks:Tissue surface area >1 cm². Store at room temperature or 4°C. Ship at ambient temperature.
7.Cell Samples:1–4 × 10⁶ cells (tumor cell cultures are best limited to within three passages). After harvesting, wash cells by centrifugation to remove culture medium, transfer to a 1.5 ml RNase-free EP tube, flash-freeze in liquid nitrogen, and store at -80°C. Ship on dry ice.
8.Saliva Samples (containing exfoliated oral epithelial cells):Rinse mouth with drinking water 30 minutes beforehand; prior to saliva collection, refrain from eating, drinking, smoking, etc. Scrape the upper and lower palate repeatedly with the tongue, and gently scrape the tongue with the teeth if possible to ensure sufficient exfoliated cell count. Collect >1 ml saliva using a saliva collection tube. Store at -80°C, avoid repeated freeze-thaw cycles. Ship on dry ice.

Experimental Groups：
Hepatocellular carcinoma (HCC) tissue sample group (n=106): includes primary HCC tissues and paired adjacent non tumor tissues; some samples were from patients with hepatitis B virus (HBV) infection (HBV+).
Control group (n=12): includes dysplastic nodules (DNs) and non tumor liver tissues (including cirrhotic tissues).
Cell experiment group: two HCC cell lines (Huh7 and MHCC-97H) were used, with UBE3C knockdown (shRNA) versus control (scramble) experiments.

Main Technical Approaches：
Whole-exome sequencing （WES）;Sanger sequencing validation; immunohistochemistry (IHC);Western blotting; etc.

Analytical Methods：
Somatic mutation identification and filtering; gene expression and clinical correlation analysis; Kaplan-Meier survival analysis; Cox proportional hazards regression model; protein structure modeling and functional prediction; etc.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide and is strongly associated with chronic hepatitis B virus infection. The development of HCC involves multistep genetic and epigenetic alterations; however, the key genes driving HCC progression remain poorly understood. The ubiquitin ligase E3 family plays important roles in protein degradation, cell signaling, and tumorigenesis, but its role in HCC has not been systematically investigated.

To explore the genetic evolution during HCC development, the research team performed whole-exome sequencing on dysplastic nodules, two HCC foci, and peripheral blood samples from the same patient, systematically screening for somatic mutations associated with HCC occurrence, and focusing on the function and clinical significance of the UBE3C gene.

Targeted sequencing of 106 HCC samples revealed somatic UBE3C mutations in 16.0% of HCC patients, predominantly located in the HECT domain. The Glu959Lys mutation was found in 7 patients, suggesting its potential functional importance.

2.UBE3C Promotes HCC Progression *Knockdown of UBE3C in HCC cell lines significantly reduced cell proliferation, migration, and invasion.
*In animal experiments, UBE3C knockdown markedly suppressed tumor growth and lung metastasis.
*UBE3C promotes HCC metastasis by inducing epithelial-mesenchymal transition, characterized by downregulation of E-cadherin and upregulation of vimentin, N-cadherin, and Snail1.

In a tissue microarray of 323 HCC patients, high UBE3C expression was significantly associated with shorter overall survival and early recurrence. Multivariate analysis indicated that UBE3C is an independent prognostic factor for HCC recurrence.

Besides HBV-related HCC, UBE3C mutations were also observed in HCV-related and non-viral HCC, suggesting that its mutation spectrum may be influenced by etiology.

Structural modeling showed that mutations in the HECT domain (e.g., Glu959Lys) may affect substrate binding or catalytic activity, thereby contributing to tumorigenesis.

This study, through whole-exome sequencing and systematic functional validation, reveals for the first time that UBE3C is a frequently mutated candidate oncogene in HCC. It promotes HCC progression by inducing EMT, and its high expression is significantly correlated with poor patient prognosis. These findings not only deepen the understanding of HCC pathogenesis but also provide a theoretical basis for developing UBE3C-targeted therapeutic strategies for HCC. NovelBio contributed to the whole-exome sequencing and bioinformatics analysis in this study, providing crucial sequencing and data analysis support for the discovery and validation of UBE3C.

Original Article Link：DOI 10.1002/hep.27012