High Olive Oil Diets Enhance Cervical Tumour Growth In Mice: Transcriptome Analysis For Potential Candidate Genes And Pathways

Objective: The aim of this study was to explore Hub genes associated with the high oive oil diet-induced tumour growth and to analyze their biological functions by using various bioinformatics analysis tools,providing valid biological information of those Hub genes for further investigation of cervical cancer.Methods: Part ? : Four-week-old athymic male BALB/c-nude mice were fed a control diet(CD;10 kcal % fat),or a high olive oil diet(OD;45 kcal % fat)ad libitum until the end of the study.All the animals were sacrificed 6 weeks after injection,and the tumours were excised,weighed and photoed.Then,the tumour tissue sections were stained with H&E and were incubated with PCNA primary antibodies at 4? overnight for immunohistochemistry.For growth curves,HeLa cells were seeded in 96-well plates and were imaged in an IncuCyte automated incubator microscope.Moreover,DNA synthesis was determined using a Cell-Light EdU Apollo 643 In Vitro Imaging Kit.Part ?: RNA-sequencing technology and comprehensive bioinformatics analyses were used to elucidate the molecular processes regulated by dietary fat.DEGs were identified and were functionally analyzed by GO enrichment analysis and KEGG pathway analysis using the online software DAVID.Then,we uploaded the up-regulated and down-regulated DEGs to online STRING database to construct PPI network with combined score > 0.7(high confidence score).Moreover,the PPI network was visualized by Cytoscape software(Version 3.5.1)and sub-PPI(module analysis)of the PPI network was explored by k-core scoring using the MCODE plugin in Cytoscape software.To evaluate the importance of nodes in the PPI network,the degree centrality of nodes was calculated and utilized in the present study using the CytoNCA plugin in Cytoscape software.The hub genes/proteins were chosen with a centrality degree > 8.Next,five DEGs(JUN,TIMP3,OAS1,OASL,and EGR1)were arbitrarily selected to confirm their differential expression levels by RT-qPCR.Results: Part ?: tumour growth over a 6-week time course was increased significantly in the OD group vs.the CD group and high olive oil diet also markedly increased the weight of the xenografts by more than 6-fold.Furthermore,OD group xenograft tissues exhibited nuclear hyperchromasia and increased nucleus-to-cytoplasmic ratios,representing poor differentiation and higher heterogeneity.Likewise,IHC analyses further uncovered a significant increase in PCNA-positive cells in the OD group(P<0.05).We observed a clear increase in cell proliferation following Oleic acid(OA)treatment(P<0.05)using the IncuCyte-automated incubator-microscope system.Next,the EdU assay showed that the EdU-positive cells were increased by OA treatment(P<0.05)compared with the negative control group.Part ?: Based on the transcriptome sequencing analysis of xenograft tumour tissues,a total of 648 DEGs obtained,including 155 up-regulated DEGs and 493 down-regulated DEGs.GO and pathway enrichment analysis revealed that some of the DEGs including EGR1 and FOXN2 were involved in the transcription regulation and others,including TGFB2 and COL4A3 in cell proliferation.According to the degree centrality of DEGs,15 hub genes were selected from the PPI network and the majority of hub genes are in the top 3 modules.Next,the qPCR results of the DEGs were correlated with the RNA-Seq.Conclusions: Our study suggests that a high olive oil diet aggravates CC progression in vivo and in vitro.We provide clues to build a potential link between dietary fat and cancerogenesis and identify areas requiring further investigation.