Male prevalence is an outstanding characteristic of hepatocellular carcinoma(HCC),and the underlying mechanisms for this have remained largely unknown.In the present study,Hras12V transgenic mice,in which hepatocyte-specific expression of the ras oncogene induces male-biased hepatic tumorigenesis,were studied,and altered proteins were detected by two-dimensional fluorescence difference gel electrophoresis(2D-DIGE).Protein samples from hepatic tumor tissues(T)and peri-tumor tissues(P)of transgenic males and females and the corresponding normal liver tissues(Wt)of non-transgenic males and females were subjected to pairwise comparisons based on proteomic analysis.Among 2381 auto-detected protein spots,more than 1600 were differentially expressed based on a pairwise comparison(|ratio| >= 1.5,p <= 0.05).Of these,180 spots were randomly selected for matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry(MALDI-TOF/TOF MS)identification; finally,89 distinct proteins were obtained.Among these 89 proteins,7 and 50 proteins were further validated by Western blotting and literature investigation,respectively.Intriguingly,compared to Wt,the altered proteins were relatively concentrated in T in transgenic females but in P in transgenic males.Consistently,the levels of p-ERK and p-mTOR were significantly higher in the T of females compared to that of males.The pathway enrichment assay showed that 5 pathways in males but only 1 in females were significantly altered in terms of the up-regulated proteins in T compared to Wt.These data indicate that female hepatocytes are disturbed by oncogenes with great difficulty,whereas male hepatoctyes readily do so.In addition,33 proteins were genderdependently altered in hepatic tumorigenesis.Moreover,4%DNA packaging and 4%homeostasis-related functional proteins were found in females but not in males,and more nucleus proteins were found in females(8%)than in males(3%).In conclusion,the proteomic data and comparative analysis presented here offer crucial clues for elucidating the mechanisms that underlie the male prevalence in HCC.