Transcriptomic Analysis Of Hepatic Tumorigenesis With Gender Disparity In Hras12V Transgenic Mice

Objective:The genesis and development of hepatocellular carcinoma（HCC）are closely related to Ras signal activation and with a characteristic of strikingly male prevalence.However,the underlying mechanisms still remain elucidation.The aim of this study was to explore the transcriptomic changes during the tumorigenesis and development of HCC induced by Ras oncogene,and to provide important evidence for researches on the common and gender-specific mechanisms as well as clinical diagnosis and treatment of HCC.Methods:In the present study,a systemic transcriptomic database was established by using the next generation sequencing technology on six composite samples originated from HCC（T）and their adjacent precancerous liver tissue（P）of Hras12V transgenic mice and normal liver tissue（W）from C57/BL6 wild-type mice of 9-month males and15-month females.Each of the composite sample was equally mixed from the six individuals of the same group thanks to the pure Hras12V transgenic features of the inbred mice.The samples are FW,FP,FT,MW,MP,MT,respectively.1.Total RNA was extracted from the tissues.Paired-end libraries were synthesized by using the TruSeq^?RNA Sample Preparation Kit,then sequenced on Illumina^?HiSeq Xten.Splicing-mapping was applied to get the sequencing results.2.The differentially expressed genes（DEGs）following the criteria of fold change≥2 and q<0.05 were filtered.Among them,21 genes were randomly selected for RT-qPCR to verify the reliability of the sequencing.3.Profiling the male and female mice transcriptomes during Ras oncogene induced hepatic carcinogenesis,and bioinformatics were performed to obtain the GO and KEGG pathways enrichment analysis.4.The expression patterns were established and the DEGs were classified into different categories according to the trends between the groups of male and female mice.Enrichment analysis to obtain the common and unique KEGG pathways that are related to HCC/Ras oncogene activation.5.Validations of the glutathione metabolism pathways were performed,including both the glutathione assay and the expression of related genes in the pathways.Results:1.In total,2708 DEGs were screened out for further bioinformatic analysis.Longitudinal comparative analysis within W,P,T and further correlation expression pattern analysis showed common and unique cluster-enriched items between sexes,indicating the common and gender-disparate pathways toward HCC.2.the numbers of DEGs in female W/P/T were much higher than those in the males and tumor suppressor genes,such as p21^Waf1/Cip1 and C6,are significantly higher in P of female,implying higher sensitivity of female hepatocytes to Ras oncogene than males and therefore difficult to develop HCC.3.the same convergent tendency showed in principal component analysis;hierarchical clustering analysis;horizontal comparative between sexes;and expression levels of sex hormone receptors and their related target genes,suggesting the underlying mechanisms for loss of effectiveness in sex hormone therapies.4.Expression pattern analysis showed HCC/Ras oncogene positive or negative DEGs and their relevant pathways,providing useful information for further mechanism elucidation and biomarker selection.5.The vital roles of GSH metabolism for HCC were further confirmed,supporting its useful target for HCC therapy.Conclusion:The systemic transcriptomic database of Ras oncogene induced HCC with gender disparities would provide valuable clues for underlying mechanisms investigation,diagnosis biomarker selection,and clinical therapy in HCC.