Genome-wide and differential proteomic analysis of hepatitis B virus and aflatoxin B1 related hepatocellular carcinoma in Guangxi, China

Abstract

Both hepatitis B virus (HBV) and aflatoxin B1 (AFB1) exposure can cause liver damage as well as increase the probability of hepatocellular carcinoma (HCC). To investigate the underlying genetic changes that may influence development of HCC associated with HBV infection and AFB1 exposure, HCC patients were subdivided into 4 groups depending upon HBV and AFB1 exposure status: (HBV(+)/AFB1(+), HBV(+)/AFB1(-), HBV(-)/AFB1(+), HBV(-)/AFB1(-)). Genetic abnormalities and protein expression profiles were analyzed by array-based comparative genomic hybridization and isobaric tagging for quantitation. A total of 573 chromosomal aberrations (CNAs) including 184 increased and 389 decreased were detected in our study population. Twenty-five recurrently altered regions (RARs; chromosomal alterations observed in ≥10 patients) in chromosomes were identified. Loss of 4q13.3-q35.2, 13q12.1-q21.2 and gain of 7q11.2-q35 were observed with a higher frequency in the HBV(+)/AFB1(+), HBV(+)/AFB1(-) and HBV(-)/AFB1(+) groups compared to the HBV(-)/AFB(-) group. Loss of 8p12-p23.2 was associated with high TNM stage tumors (P = 0.038) and was an unfavorable prognostic factor for tumor-free survival (P =0.045). A total of 133 differentially expressed proteins were identified in iTRAQ proteomics analysis, 69 (51.8%) of which mapped within identified RARs. The most common biological processes affected by HBV and AFB1 status in HCC tumorigenesis were detoxification and drug metabolism pathways, antigen processing and anti-apoptosis pathways. Expression of AKR1B10 was increased significantly in the HBV(+)/AFB1(+) and HBV(-)/AFB1(+) groups. A significant correlation between the expression of AKR1B10 mRNA and protein levels as well as AKR1B10 copy number was observered, which suggest that AKR1B10 may play a role in AFB1-related hepatocarcinogenesis. In summary, a number of genetic and gene expression alterations were found to be associated with HBV and AFB1- related HCC. The possible synergistic effects of HBV and AFB1 in hepatocarcinogenesis warrant further investigations.

Figure 1. Whole-genome profiles and frequency plots of chromosomal alterations in HCC patients (z-scoring:2.5).

(A) The genomic alterations in tumor samples from each HCC patient (n = 32) are illustrated in individual vertical lanes. A total of 573 gene copy number alterations were mapped and ordered by chromosomal position from 1pter to Yqter using the workbench Lite Edition 6.5.0.18. Tumor versus the reference intensity ratios (in log₂ ratio) for individual tumor samples are plotted in different color scales reflecting the extent of increases (red) or decreases (green) in copy number. The detail chromosomal alterations were shown in 1B (chromosome 1 to chromosome 12) and in 1C (chromosome 13 to chromosome 22, X chromosome and Y chromosome, respectively). Nine chromosomes showed increased chromosomal DNA (1q, 5p, 6p,7q, 8q, 17q, 20p, 20q and X) and 16 chromosomes had deceased chromosomal DNA (1p, 4q, 8p, 9p, 10q,13q, 14q, 16p, 16q ,17p ,18q, 19p ,19q, 21q, 22q and Y) that were repeatedly observed in > 20% of tumor samples.

Figure 2. Representative comparison of incidence of RAR:4q12-q35.2、RAR:13q12.1-q21.1 and RAR:7q11.2-q35 in the 4 groups based on HBV and AFB1 status.

(%). Red bar: the loss of chromosome; Green bar: the gain of chromosome. Yellow frame: Recurrently Altered Regions (RARs).

Figure 3. Venn diagram depicting the overlap of differentially expressed proteins quantified in the 4 groups based on HBV and AFB1 status.

The number in parentheses indicates the number of differentially expressed quantified proteins in each group.

Figure 4. The AKR1B10 gene location and its expression levels validated by Reverse Transcriptase PCR and Western Blot.

(A) AKR1B10 is located on RAR: 7q11.2-q35 (position 7q33.1) a chromosomal region that showed a high instance of instability in HBV(+)/AFB1(+) ,HBV(+)/AFB1(-) and HBV(-)/AFB1(+) groups. (B) Representative RT- PCR and Western blot images of AKR1B10 in the 4 groups. T1–T4 ,T5-T8, T9-T12 and T13-T16 were HCC samples obtained from HBV(+)/AFB1(+) , HBV(+)/AFB1(-), HBV(-)/AFB1(+) and HBV(-)/AFB1(-) groups, respectively. Human GAPDH was used as an internal control. (C) The regression analysis showed a significant correlation between AKR1B10 expression level (both mRNA level and protein level) and AKR1B10 copy number. (D) RT-PCR and Western blot analysis of AKR1B10 expression. Mean mRNA and protein expression levels of AKR1B10 were calculated by RT-PCR and Western blotting for the 4 subgroups. Human GAPDH was used as an internal control. * indicated a significant difference (P<0.05) as compared to HBV(+)/AFB1(+), †indicated a significant difference (P<0.05) as compared to HBV(+)/AFB1(-), ‡ indicated a significant difference (P<0.05) as compared to HBV(-)/AFB1(+).