| Objective:Dysregulation of metabolism is critically involved in hepatocellular carcinoma(HCC).Further,male predilection and Ras signaling pathway hyperactivation are distinct characteristics of HCC.However,mechanisms underlying their connections remain unknown.The effects of different etiologies on lipid metabolism of HCC are complex and difficult to distinguish,and there is a lack of reliable early diagnostic markers,these realities make it difficult to collect clinical samples of a single etiology that reflect the early stages of HCC development.Therefore,animal models have become an indispensable tool for alternative studies of human HCC.The aim of the present study was to perform a comprehensive non-targeted lipidomic analysis of normal liver tissues(W)of wild-type mice,HCC(T)and peri-tumor tissues(P)of Hras12V transgenic mice(Ras-Tg,a mice model of HCC induced by hepatocyte-specific Ras pathway activation and characterized by male predilection and a disrupted lipid metabolism),so as to screen out and identify differentially expressed lipids(DELs)associated with sex,Ras oncogene expression,and hepatocellular carcinogenesis,and to investigate the alterations in lipid metabolism,and to provide important clues for further investigation of the molecular mechanism and discovery of potential early diagnostic markers and therapeutic targets of HCC occurrence and development,and also to provide new insights into the clinical treatment strategy of HCC.Methods:Lipidomics analysis was performed on W,P,and T tissues from 9-month-old male and 15-month-old female Ras-Tg mice(C57BL/6J mice served as control)with similar degrees of HCC development:1.Identification and quantification of lipids in tissue samples using the untargeted lipidomics strategy of ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).2.An overall analysis of the content and proportions of major lipid species and their subclasses,as well as the lipid-bound saturated fatty acids(SFA),monounsaturated fatty acids(MUFA),and polyunsaturated fatty acids(PUFA)was performed.3.DELs for longitudinal(between W,P,and T tissue samples from single-sex)and lateral(between samples of the same type of tissue from different sex)comparisons were screened out by univariate(t test for data with normal distribution and Wilcoxon rank sum test for data with non-normal distribution)and multivariate(orthogonal projections to latent structures discriminant analysis,OPLS-DA)statistical analyses.Lipids based on the criteria of p < 0.05,foldchange > 1.5(elevation)or foldchange < 0.667(reduction)and variable importance of projection(VIP)> 1 were considered as DELs.4.The DELs of longitudinal comparison were classified into specific patterns describing the trends of lipids positively/negatively correlated with HCC/Ras oncogene expression in hepatocellular carcinogenesis,respectively.Next,the change patterns of male and female DELs were subjected to cross-matrix analysis to further screen out the DELs with common and characteristic patterns between the sexes.5.Based on the results of cross-matrix analysis,the mRNA expression of key enzymes in the metabolic pathway was detected by RT-qPCR for lipids with common change patterns in males and females to further investigate the mechanisms of dysregulation in lipid metabolism during hepatocellular carcinogenesis.Results:1.A total of 710 lipids were identified and quantified in six groups of tissue samples by UPLC-MS/MS.Principal component analysis and clustering heat map indicated good clustering within groups and significant differences in lipid profiles between groups.2.The results of longitudinal comparisons showed significant variations in the proportion and content of lipid species.Compared to W,glycerolipid levels were significantly/obviously reduced in both male and female P tissues,but phospholipid and sphingolipid levels were significantly elevated only in male P tissues,suggesting that phospholipid and sphingolipid metabolism in male and female hepatocytes respond differently to Ras oncogene expression.In addition,compared to P,both male and female T tissues showed significantly elevation of glycerolipids and significantly/obviously reduction of phospholipids and sphingolipids,suggesting a convergent variation of lipid metabolism in male and female hepatic tumor cells after hepatocellular carcinogenesis.3.In the T vs.P comparison,the content and proportion of lipid-bound monounsaturated fatty acid(MUFA)were significantly/obviously elevated in both male and female mice.In particular,the proportion of MUFA in phosphatidylcholine(PC),a major component of the plasma membrane,was significantly elevated.The mRNA expression of key enzymes of the MUFA synthesis metabolic pathway in T vs.P comparison were detected using RT-qPCR,and the results indicated that enhanced MUFA synthesis led to MUFA accumulation in HCC.4.Longitudinal comparisons yielded 132 DELs in male mice and 96 DELs in female mice.In the P vs.W and T vs.P comparisons,the amount of DELs was much higher in male mice than in female mice,suggesting that more changes in lipid metabolism occurred during hepatocellular carcinogenesis in male mice.5.By cross-matrix analysis of the change patterns of DELs between male and female mice during hepatocellular carcinogenesis,the DELs with common and characteristic change patterns between the sexes were selected.Generally,DELs positively associated with HCC were mainly glycerolipids,while DELs negatively associated with HCC were mainly phospholipids.6.The mRNA expression of key enzymes of the glycerolipid and phospholipid metabolic pathways in T vs.P comparison were detected using RT-qPCR,results from male and female mice consistently showed that enhanced synthesis and uptake of fatty acids promoted the accumulation of glycerolipids in HCC,while inhibited phospholipid synthesis led to significantly reduced levels of phospholipids in HCC.7.The lateral comparisons between male and female mice showed a significant convergence between the sexes during hepatocellular carcinogenesis.Conclusion:1.The present study provides the first comprehensive and systematic non-targeted lipidomic database of Ras oncogene-induced hepatocellular carcinogenesis with sex disparity,and reveals the common and characteristic lipid profile alterations between male and female mice during Ras oncogene-induced hepatocellular carcinogenesis,and provides important clues for further investigation of the molecular mechanism and discovery of potential early diagnostic markers and therapeutic targets of HCC occurrence and development.2.The accumulation of MUFAs and glycerolipids,and the reduction of phospholipid and sphingolipid levels are closely associated with hepatocellular carcinogenesis.3.Phospholipid and sphingolipid metabolism responded differently to the Ras oncogene expression in the hepatocytes of male and female mice.More lipid metabolism variations occurred in male mice during hepatocellular carcinogenesis than in female mice.This evidence suggests that dysregulated lipid metabolism may be associated with a male predisposition to hepatocellular carcinogenesis.4.The obvious trend of convergence between male and female lipid components during hepatocarcinogenesis provides an experimental basis to explain the poor efficacy of gender-specific treatment strategies for HCC in clinical practice. |
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