Differential Proteomic Analysis Of Gender-dependent Hepatic Tumorigenesis In Hras12V Transgenic Mice

Objective: Male prevalence is an outstanding characteristic of hepatocellular carcinoma(HCC),and the underlying mechanisms for this have remained largely unknown.The present study is aimed to identify the differential proteomic expression of gender-dependent HCC in Hras12 V transgenic mice and to investigate the underlying molecular mechanisms.Methods: By studying a mouse model of hepatic tumor induced by hepatocyte-specific expression of the H-ras12 V oncogene,to explore the molecular mechanisms of gender-dependent HCC in Hras12 V transgenic mice.1.H-ras12 V transgenic mice(Tg)and C57BL/6J wild type mice(Wt)(n=9 for each group)were subjected to autopsy,and the pathological changes of the liver were observed and counted.2.The liver tumor tissues and peri-tumor tissues of 10-month male and 15-month female H-ras12 V transgenic male mice and normal liver tissues of C57BL/6J male mice were sampled.One part of each sample was fixed in 10% formalin and undergone paraffin-embedded tissue section,HE staining,and pathological analysis.Another part of each sample was frozen in liquid nitrogen for extraction of total protein.The protein levels of p-MEK,p-ERK1/2,p-PI3 K,p-AKT and p-m TOR were detected by Western blot.3.Depending on the appearance and size of the liver and hepatic tumor in terms of gross anatomy,histopathological diagnosis,and signaling pathway analysis,3 representative individuals were selected in each group(18 protein samples were prepared from 12 individuals).The 2-D DIGE images were analyzed using De Cyder 5.0 software to objectively estimate the abundance of proteins in each sample and to generate quantitative data.4.Protein spots exhibiting significant changes were selected using a Spot Cutter and were subjected to in-gel digestion for MALDI-TOF/TOF MS analysis.In total,180 randomly selected protein spots were identified.5.To verify the authenticity and reproducibility of the 2D-DIGE proteomic results,seven of the identified differentially expressed proteins were randomly selected and were confirmed in samples from another set of males and females by Western blot analysis.6.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis for the identified proteins were performed with Uni Prot and David v.6.7.Results:1.Multiple and massive hepatic tumors were only present in transgenic 10-M and 15-F,but not in transgenic 10-F or any non-transgenic controls.Significant lower incidences of adenoma(<5 mm),carcinoma(> 5 mm),and total hepatic alterations were found in 15-F compared to 10-M transgenic mice.In addition,few nodules were found in transgenic 10-F.2.Intriguingly,although p-MEK levels did not obviously differ between the hepatic tumor tissues of transgenic 10-M and 15-F,p-ERK levels were obviously decreased in the hepatic tumor tissues of transgenic 10-M compared to transgenic 15-F.Moreover,although the p-PI3 K levels were significantly higher,the p-AKT and p-m TOR levels were significantly lower in the hepatic tumor tissues of transgenic 10-M compared to transgenic 15-F.In addition,the levels of p-m TOR and p-ERK were also obviously lower in the livers of non-transgenic 10-M compared to those of non-transgenic 15-F.3.The 2-D DIGE images were analyzed using De Cyder 5.0 software,and 2381 protein spots were auto-detected.Interestingly,significant differences were found between males and females regarding the numbers of protein spots that were differentially expressed among Wt,P,and T,regardless of whether the spots represented up-or down-regulated proteins.Specifically,in males,more spots were detected when comparing P and T versus Wt than when comparing P versus T.However,in females,more spots were detected when comparing T versus P and Wt,and fewer than 100 spots were differentially expressed between P and Wt.4.Protein spots exhibiting significant changes were selected using a Spot Cutter and were subjected to in-gel digestion for MALDI-TOF/TOF MS analysis.In total,180 randomly selected protein spots were identified.The same trend was observed as that for the summarized total protein spots.5.The expression changes of the selected proteins were consistent with the obtained 2D-DIGE results in both males and females,excepting PRDX6 in females that showed decreased protein levels in T compared with P and Wt,indicating that there were undetected PRDX6 isoforms.6.Most of the proteins that were expressed differentially and were classified into the HCC-positive or-negative related categories were biased toward females.However,most of the proteins that were expressed differentially and were classified into the Ras oncogene-positive or-negative related categories were biased toward males.Conclusion:1.Hepatic alteration in a transgenic mouse model in which the Hras12 V oncogene was specifically expressed in hepatocytes was male dependent.2.Ras oncogene expression in normal hepatocytes induces more changes in gene expression in males than in females,and this phenomenon reflects the significant male prevalence in hepatic tumorigenesis.