| Primary hepatocellular Carcinoma(HCC)is the most fatal malignancy threatening human health seriously.Although the ethiology,course,and underlying mechanisms of HCC remain elucidated,the frequent activation of Ras signaling pathway and the male prevalence are the distinct characteristics of HCC.Protein,the final product of gene expression,is the main carrier and direct executor of all life forms and activities.Therefore,the study of proteins is an important means to directly clarify the occurrence and development of diseases.From the perspective of proteomics,tumor disease is considered as a kind of protein defect disease.In recent years,proteomics and post-translational modification omics have become the main methods in tumor researches.Investigating the underlying molecular mechanisms of tumorigenesis and screening for early diagnostic and prognostic biomarkers by comparing and analyzing the differences in the overall expression levels of proteins and post-translational modifications among normal tissues,pre-cancerous and cancerous tissues,and using bioinformatics tools for in-depth analyses,may contribute to the clinical diagnosis,pathological research,and develop new therapeutic drugs for the tumor diseases.Therefore,proteomics and its post-translational modification omics provide a direct and ideal entry point to reveal the complex mechanisms of HCC.Laboratory animals are indispensable in vivo models for the study of human diseases.Various genetically modified mouse models not only help to explore the molecular mechanisms of the occurrence and development of HCC,but also can be used to screen new biomarkers and develop new therapeutic drugs,so as to accelerate the transformation of laboratory research results to clinical practice.Our group established a transgenic mouse model which specifically expressed Hras12 V oncogene in hepatocytes.This mouse model is characterized by a single etiology,a regular demonstration of the whole process of hepatocarcinogenesis,and the hepatic tumor formation in the mouse model showed a significant bias towards the males,which represents a valuable animal model for use in studies on the molecular mechanisms associated with the Ras-related and the striking male prevalence of HCC.In the present study,using the Hras12 V transgenic mice,we performed a high-throughput comparative proteomic and phosphoproteomic analyses which based on tandem-mass-tag(TMT)labeling,modification-specific enrichment techniques,combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS).The changes of protein types,content and phosphorylation sites during the development of gender-dependent HCC induced by Ras oncogene were revealed from a holistic and quantitative perspective.These results provided crucial databases for revealing the important proteins and phosphoproteins during the development of gender-dependent HCC induced by Ras oncogene,and provided new clues screening for new diagnostic biomarkers,novel therapeutic targets and new methods for preventing and treating HCC.PartⅠ.The Proteomics Study of Hepatic Tumorigenesis in Hras12 V Transgenic MiceObjective: The purposes of this study are a high-throughput screen to identify the differentially expressed proteins(DEPs)in tissues obtained from precancerous tissues and hepatocellular carcinoma of Hras12 V transgenic mice and normal liver of wild-type mice,therefore,to futher establish the global proteomics database regarding Hras12V-induced hepatocarcinogenesis,further explore the underlying mechanisms and screen for new tumor-specific markers,so as to provide the necessary theoretical basis for exploring the mechanisms of gender-dependent HCC induced by Ras oncogene,the clinical diagnosis,pathological research,screening and developing new therapeutic drugs.Methods: Normal C57BL/6J mice were used as the control group and Hras12 V transgenic mice were used as the experimental group.The precancerous tissues(P)and hepatocellular carcinoma(T)of Hras12 V transgenic mice(Ras-Tg)and normal liver(W) of wild-type mice(Non-Tg)were used for proteomic analysis.The protein samples were individually prepared for the study.1.The DEPs in W,P and T were screened and identified by high-throughput comparative proteomic analysis based on TMT labeling.2.Twenty three of the identified DEPs were randomly selected and evaluated by Western blot to verify the authenticity and reproducibility of the proteomic results.3.DAVID bioinformatics platform(http:// david.abcc.ncifcrf.gov/)was used for further GO and KEGG analyses on the identified DEPs.And the mechanisms of gender-dependent HCC induced by Ras oncogene were further explored.4.The particular variation trends of DEPs among T/P,T/W,and P/W in males and females were classified and analyzed.5.The specific tumor markers were screened and further validated by Real-Time PCR,Western blotting and immunohistochemistry.Results:1.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 analyses.2.The Western blot results showed that 23 DEPs were consistent with the obtained quantitative proteomics results which confirmed that the proteomics analyses are convincing.3.Among the quantified proteins,in males,77 proteins were up-regulated and 87 proteins were down-regulated in the peri-tumor tissues compared with normal tissues(P/W);392 proteins were up-regulated and 389 proteins were down-regulated in the tumor tissues compared with peri-tumor tissues(T/P);498 proteins were up-regulated and 546 proteins were down-regulated in the tumor tissues compared with normal tissues(T/W);in females,compared with normal tissues,59 proteins were up-regulated and 48 proteins were down-regulated in peri-tumor tissues(P/W);compared with peri-tumor tissues,348 proteins were up-regulated and 333 proteins were down-regulated in tumor tissues(T/P);compared with normal tissues,501 proteins were up-regulated and 465 proteins were down-regulated in tumor tissues(T/W).4.Subcellular localization analyses showed that the identified DEPs were found to be mainly located in the extracellular,cytoplasm,mitochondria,plasma membrane,nucleus,and endoplasmic reticulum.Notably,the proportion of subcellular located DEPs is obviously different between T/P(W)and P/W in both males and females,which indicates the particular changes in T.The obvious difference between males and females in P versus W indicates the gender-dependent responses to the Ras oncoprotein in hepatocytes.However,the largely similar distribution of DEPs in T/P(W)between sexes indicates similar pathways towards T and similar characteristics of T in males and females.5.Bioinformatics analyses showed the common and unique cluster-enriched items between sexes,indicating the common and gender-disparate pathways towards HCC.6.The HCC and Ras oncogene positively and negatively associated proteins were obtained by pattern analysis.7.Activation of the Ras oncogene gradually reduced the response of hepatocytes to sex hormones,thereby narrowing the sex difference between males and females,which may be responsible for the little effect targeting sex hormone systems for clinical HCC patients.8.The proteomic analysis showed FABP5 was up-regulated in the tumor tissues.which was further validated by Western blotting and immunohistochemistry.Conclusion:1.This study presents,for the first time,global proteomics data regarding Hras12 V induced hepatocarcinogenesis with gender disparity.2.For the first time,the common,unique,and systemic signatures in the protein expression profiles of male-biased hepatocarcinogenesis were found,which will provide crucial clues for further revealing the underlying mechanisms.3.Especially,the common and unique DEPs detected in precancerous hepatocytes and hepatoma cells of male and female Ras-Tg offer important candidate biomarkers for detecting the activation of Ras signaling pathway and HCC in clinical diagnosis.4.The convergent trend during hepatocarcinogenesis between males and females indicates the homogeneity and the loss of sex hormones responses of HCC which reflects the little effect for therapeutic strategies targeting sex hormone systems for clinical HCC patients.5.FABP5 was found to be a more sensitive biomarker for clinical diagnose and prognosis prediction of HCC.PartⅡ.The Phosphoproteomics Study of Hepatic Tumorigenesis in Hras12 V Transgenic MiceObjective: Based on the proteomics results of section 1,the purposes of this study are a high-throughput screen to identify the differential phosphorylation sites(p-sites)and phosphoproteins in tissues obtained from precancerous tissues and hepatocellular carcinoma of Hras12 V transgenic mice and normal liver of wild-type mice,therefore,to further establish the global phosphoproteomics database regarding Hras12V-induced hepatocarcinogenesis,so as to provide the necessary theoretical basis for exploring the mechanisms of gender-dependent HCC induced by Ras oncogene,the clinical diagnosis,pathological research,drug screening and developing new therapeutic drugs.Methods: Normal C57BL/6J mice were used as the control group and Hras12 V transgenic mice were used as the experimental group.The precancerous tissues(P)and hepatocellular carcinoma(T)of Hras12 V transgenic mice(Ras-Tg)and normal liver(W)of wild-type mice(Non-Tg)were used for proteomic analysis.The protein samples were individually prepared for the study.1.A high-throughput comparative proteomic analysis based on TMT labeling combined with IMAC-based phosphopeptide enrichment and LC-MS/MS on W,P and T were performed.2.DAVID bioinformatics platform was used for further GO and KEGG analyses on the identified differentially expressed phosphoproteins.And the mechanisms of gender-dependent HCC induced by Ras oncogene were further explored.3.The particular variation trends of the identified differentially expressed phosphoproteins among T/P,T/W,and P/W in males and females were classified and analyzed.4.Protein kinases and phosphatases with different phosphorylation levels were analyzed.Results:1.In total,6517 p-sites of 2805 phosphoproteins were identified and a total of5410 p-sites from 2427 proteins were quantified.2.The differentially expressed p-sites and phosphoproteins(quantified with 2observations of 3 replicates;|ratios| ≥ 1.5,p < 0.05)were selected for further analyses.3.Among the quantified phosphoproteins,in males,192 p-sites of 164 phosphoproteins were up-regulated and 110 p-sites of 86 phosphoproteins were down-regulated in P/W;625 p-sites of 426 phosphoproteins were up-regulated and 122p-sites of 92 phosphoproteins were down-regulated in T/P;846 p-sites of 578 phosphoproteins were up-regulated and 201 p-sites of 141 phosphoproteins were down-regulated in T/W;in females,39 p-sites of 31 phosphoproteins were up-regulated and 108 p-sites of 100 phosphoproteins were down-regulated in P/W;540 p-sites of 389 phosphoproteins were up-regulated and 154 p-sites of 115 phosphoproteins were down-regulated in T/P;607 p-sites of 435 phosphoproteins were up-regulated and 287p-sites of 219 phosphoproteins were down-regulated in T/W.4.Subcellular localization analyses showed that the identified differentially expressed phosphoproteins were found to be mainly located in the nucleus,cytoplasm,plasma membrane and mitochondria.The proportion of subcellular localization was different between males and females.5.Bioinformatics analyses showed the common and unique cluster-enriched items between sexes,indicating the common and gender-disparate pathways towards HCC initiated by Hras12 V oncogene.6.The HCC and Ras oncogene positively and negatively associated phosphoproteins were obtained by pattern analysis.7.Two hundred and eighty five p-sites of 135 kinases were identified and 114p-sites of 80 kinases were differentially expressed.Sixty three p-sites of 36 phosphatases were identified and 27 p-sites of 20 phosphatases were differentially expressed.Conclusion:1.This study presents,for the first time,global phosphoproteomics database regarding Hras12 V induced hepatocarcinogenesis with gender disparity.2.For the first time,the common,unique,and systemic signatures of the phosphoproteins expression profiles in Ras oncogene-induced hepatocarcinogenesis between males and females were found,which suggests that hepatocarcinogenesis is closely associated with abnormal phosphorylation modifications in which male-and female-specific differentially phosphorylated protein kinases and phosphatases may contribute to sex-differentiated hepatocarcinogenesis.3.Especially,the common and unique differentially expressed phosphoproteins detected in P and T of male and female Ras-Tg offer important candidate biomarkers for detecting the activation of Ras signaling pathway and HCC in clinical diagnosis. |
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