Proteomic analysis revealed common, unique and systemic signatures in gender-dependent hepatocarcinogenesis

Abstract

Hepatocellular carcinoma (HCC) is the most common liver cancer and is highly malignant. Male prevalence and frequent activation of the Ras signaling pathway are distinct characteristics of HCC. However, the underlying mechanisms remain to be elucidated. By exploring Hras12V transgenic mice showing male-biased hepatocarcinogenesis, we performed a high-throughput comparative proteomic analysis based on tandem-mass-tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the tissue samples obtained from HCC (T) and their paired adjacent precancerous (P) of Hras12V transgenic male and female mice (Ras-Tg) and normal liver (W) of wild-type male and female mice (Non-Tg). The further validation and investigation were performed using quantitative real-time PCR and western blot. Totally, 5193 proteins were quantified, originating from 5733 identified proteins. Finally, 1344 differentially expressed proteins (DEPs) (quantified in all examined samples; |ratios| ≥ 1.5, p < 0.05) were selected for further analysis. Comparison within W, P, and T of males and females indicated that the number of DEPs in males was much higher than that in females. Bioinformatics analyses showed the common and unique cluster-enriched items between sexes, indicating the common and gender-disparate pathways towards HCC. Expression change pattern analysis revealed HCC positive/negative-correlated and ras oncogene positive/negative-correlated DEPs and pathways. In addition, it showed that the ras oncogene gradually and significantly reduced the responses to sex hormones from hepatocytes to hepatoma cells and therefore shrunk the gender disparity between males and females, which may contribute to the cause of the loss of HCC clinical responses to the therapeutic approaches targeting sex hormone pathways. Additionally, gender disparity in the expression levels of key enzymes involved in retinol metabolism and terpenoid backbone/steroid biosynthesis pathways may contribute to male prevalence in hepatocarcinogenesis. Further, the biomarkers, SAA2, Orm2, and Serpina1e, may be sex differences. In conclusion, common and unique DEPs and pathways toward HCC initiated by ras oncogene from sexually dimorphic hepatocytes provide valuable and novel insights into clinical investigation and practice.

Keywords: Gender disparity; Hepatocellular carcinoma; Proteomics; Ras oncogene; Tandem-mass-tag (TMT).

Fig. 1

Analysis and validation of the quality of quantitative proteomics data. Unsupervised hierarchical clustering of all proteome datasets shows separation among the different groups. Protein expression levels are color-coded, showing higher and lower expression in red and blue, respectively. MW, normal liver tissues of non-transgenic 9-months-old males; MP, precancerous tissues of transgenic 9-months-old males; MT, hepatocellular carcinoma tissues of transgenic 9-months-old males; FW, normal liver tissues of non-transgenic 15-months-old females; FP, precancerous tissues of transgenic 15-months-old females; FT, hepatocellular carcinoma tissues of transgenic 15-months-old females. The numbers indicate different individuals or different composite samples

Fig. 2

Identification of the DEPs by LC-MS/MS and subcellular location analysis. a Number of DEPs in paired comparisons. b Subcellular location analysis. The paired comparison results in males and females are shown in the left and right panels, respectively. W, normal liver tissues of wild-type non-transgenic mice; T and P, HCC tissues and their paired adjacent precancerous tissues of Hras12V transgenic mice. Detailed information of DEPs among W, P, and T is shown in Additional file 5, Table S4. The slash symbol means “versus”. The abbreviation and symbol definitions as well as descriptions are the same as those in the caption for Fig. 1

Fig. 3

Validation of the DEPs identified by LC-MS/MS. The randomly selected twenty-three DEPs were validated in another set of samples from male (a) and female (b) of Non-Tg and Ras-Tg mice by Western blot. Non-Tg, wild type non-transgenic mice; Tg, transgenic mice. The different numbers represent different individuals. The level of GAPDH was used as a quantitative control. The abbreviation and symbol definitions as well as descriptions are the same as those in the caption for Fig. 1

Fig. 4

Bioinformatics analysis of DEPs during hepatocarcinogenesis in males and females. Heat map obtained from KEGG pathway analysis using DAVID bioinformatics. a For males and b for females. The slash symbol means “versus”. The abbreviation and symbol definitions as well as descriptions are the same as those in the caption for Fig. 1

Fig. 5

Common and unique DEPs detected during hepatocarcinogenesis in males and females. a Venn analysis for DEPs detected in the proteomics for males and females, respectively. The slash symbol means “versus”. b DEPs were divided into the different expression patterns in males and females, respectively, and then overlap the DEPs within the same pattern between males and females to determine common and unique DEPs correlated with hepatocarcinogenesis and/or the expression of the Hras12V oncogene. HCC positive-correlated (a) or negative-correlated (c) proteins represent that these proteins were significantly increased or decreased in T comparing with those in P and/or W, respectively. The Hras12V oncogene positive-correlated (b) or negative-correlated (d) proteins represent that these proteins were significantly increased or decreased in P and T when compared with those in W, respectively. In the schematic diagram of the expression pattern, the numbers 1, 2, and 3 represent the graded and significant expression levels. The colored columns represent the significantly changed expression levels comparing with at least one group in the same pattern, and the white column indicates no significant expression changes comparing with those of the other two groups. The deep red-colored boxes indicate completely overlapping DEPs between sexes in the same expression pattern. The light red boxes indicate DEPs with similar expression patterns. The blue boxes indicate unique DEPs between sexes. The abbreviation and symbol definitions as well as descriptions are the same as those in the caption for Fig. 1

Fig. 6

Converging trend during hepatocarcinogenesis between males and females of Hras12V transgenic mice. a Principal-component analysis (PCA) of all quantified proteins in samples (a, MW; b, MP; c, MT; d, FW; e, MP; f, MT). b mRNA levels of Ar and Esr1 in W, P, and T of males and females as detected by RT-qPCR. c The mRNA levels of female-prevalent enzymes CYP2B13 and SULT2A2 and male-prevalent enzymes CYP2D9, CYP7B1, UGT2B1, and HSD3B5 in W, P, and T of male and female mice as detected by RT-qPCR. The mRNA levels of the genes were normalized to Rp135a. The data are expressed as the mean ± SEM (n = 5–7).*p < 0.05; **p < 0.01.

Fig. 7

Gender disparity in metabolic pathways and biomarkers during hepatocarcinogenesis in Hras12V transgenic mice. a The mRNA levels of Cyp3a41 in W, P, and T of males and females as detected by RT-qPCR. The mRNA levels of the genes were normalized to Rp135a. The data are expressed as the mean ± SEM (n = 5–7). b The expression levels of key enzymes in terpenoid backbone biosynthesis and steroid biosynthesis pathways identified by TMT-based quantitative proteomic analysis. c The expression levels of SAA2, ORM2, and SERPINA1E identified by TMT-based quantitative proteomic analysis. *p < 0.05; **p < 0.01. The abbreviation and symbol definitions, as well as descriptions, are the same as those in the caption for Fig. 1