Quantitative Proteomics Analysis Of Hepatitis B Virus Core Protein Induced Liver Cell Lipid Accumulation Via Transcription Factor MLX

Quantitive Proteomics is one of the most powerful tool to panoramicly identify and quantify proteins in cells or tissues. With the development of proteomics technology, SILAC (Stable Isotope Labeling with Amino acids in cell Culture) based quantitative proteomics has becomes the gold standard in this field because of higher precision and accuracy.Type B hepatitis caused by hepatitis B virus is one of the major infectious diseases in China. With the disease development, hepatitis, liver cirrhosis, liver failure or hepatocellular carcinoma (HCC) would be occurred progressively. The core protein (HBc), encoded by HBV genome, serve as a centrol role in HBV life cycle. With the assistance of HBc, the mature HBV virus genome can be transferred into the host cell nucleus from cytoplasm. However, the role of HBc protein in this process remains unclear.The HBc stable expression cell line and the control cell line were screened by G418 after transfection of p3×Flag CMV14/HBc plasmid to liver cancer cell line HepG 2. Phenotypic studies of selected cell lines were performed, oil red O stain experiment found that lipid was accumulated after the expression of HBc in HepG 2. CCK 8 cell proliferation test showed that the HBc protein might promote the proliferation of HepG 2. The cell scratch assay confirmed that the migration of HBc stable expression cell line was improved obviously compared to the control cells. The free cholesterol level of HepG 2 was not affected by HBc expression, but the total cholesterol was significantly increased, which indicate that the intracellular cholesterol ester content was highly raised in the HBc cell line. Under the guidence, I applied forward and reverse SILAC strategy to systematically compare the proteome of HBc transgenic cell line and its control. Totally, we identified and quantified xxx proteins. Among them,165 proteins displayed HBc-dependent changes in abundance including xxx upregulated proteins and xxx downregulated proteins. Bioinformatic analyses suggested that the biological proccess of differently expressed proteins were significantly enriched in bio-synthesis and accumulation of cholesterol and lipid, indicating that HBc may play roles in the process of fatty liver disease. To further understand the disregulating mechanism for the metablism of cholesterol and lipid, we investigated and confirmed that MLX, one of energy metabolism transcription factor, was interacting with HBc. By ChIP analysis following by qPCR for the upstream regulation sequence of these encoding genes, we confirmed that their binding had also been increased. With confocal microscope, we found that the 3 X Flag tagged HBc could co-translocated into nucleus with HA-tagged MLX. In conclusion, this research illuminates a molecular mechanism of the lipid synthesis and accumulation caused by HBc through protein-protein interaction of HBc and MLX.