Evaluation Of Biosynthesis Of Terpenoid Quinones In Carya Cathayensis And The Molecular Mechanisms Of The Inhibition Of Juglone To Ishikawa Cancer Cell Proliferation

Carya cathayensis,Juglandaceae,is a widely cultivated woody oil tree species.C.cathayensis is popular because of its rich bioactive components such as oil,protein,fiber,phenolic acid,and terpenoids.In the recent years,several natural compounds have been studied for their anti-cancer activities.In this thesis,author first investigated the synthesis of terpenoid quinones after pollination of C.cathayensis,and combined transcriptomic and metabonomic methods to clarify the mechanism of terpenoid synthesis and metabolism during the development of C.cathayensis;Then,the inhibitory effect of juglone from C.cathayensis green peel on the proliferation of endometrial cancer Ishikawa cells was investigated,and the mechanism of inhibition of juglone on the proliferation of endometrial cancer(EC)Ishikawa cells was elucidated from the overall level by using mRNA-miRNA omics.Further,the mechanism of inhibition of proliferation and invasion of EC Ishikawa cells by juglone was clarified.The results of this dissertation provide theoretical support for the development of C.cathayensis terpenoid juglone as a functional food for particular therapeutic purposes.(1)Metabolic mechanism of terpenoids in C.cathayensis during development after pollination.90-105 days after pollination(P1-P2)is the key period for the formation of secondary metabolites.Related MENB and C4H genes were highly expressed in C.cathayensis embryos,with the highest transcriptome expression of671 and 1469,respectively,The high expression level of MENB may be an important reason for the high content of naphthoquinone in C.cathayensis embryos.By network co-expression analysis,a significant positive and negative correlation between six terpene quinone metabolites and 86 differentially expressed genes(r>0.8 or<0.8,P<0.05)was observed.The expression level of secondary metabolites was the highest in the early stage,which was basically consistent with the level of genes regulating their differential expression.(2)Juglone,an extract from C.cathayensis green peel,inhibited the proliferation of EC Ishikawa cells.After the ultrasonic assisted extraction process of crude C.cathayensis green peel extract and purification by macroporous resin and silica gel column,the content of compound was 3.31 mg/kg,and the purity was 98%.The component was identified as juglone by secondary mass spectrometry and ion fragment database.The IC₅₀of Ishikawa cells treated with juglone for 24 h was 20.81?mol/L.With different concentrations of juglone(0,10,15,and 20?M),morphological changes of Ishikawa cells were dose-dependent after 24 h treatment.(3)Transcriptomics and miRNA analysis showed that juglone could inhibit the proliferation of Ishikawa cells.After 24 h of juglone(20?M)treatment,the expression levels of 942 mRNA were significantly different.Among the differentially expressed mRNA,789 were up-regulated and 153 down regulated,and their enrichment analysis was in the pathways related to cell cycle and apoptosis.By analyzing the protein-protein interaction(PPI)of differentially expressed mRNA constructed by string database with cytohubba plug-in of Cytoscape,it was found that16 hub genes were mainly involved in the G1/S phase of cell cycle and HIF-1signaling pathway related to ferroptosis.The expression levels of 50 miRNAs in juglone treated Ishikawa were significantly changed,among which 24 were up-regulated and 26 were down regulated.Four key miRNAs(hsa-let-7i-5p,hsa-let-7g-5p,hsa-mir-148b-3p,and hsa-mir-148a-3p)were identified by constructing miRNA coexpression network,and their target genes were enriched in apoptosis related pathways.Mi RNA-mRNA regulatory network analysis showed that inhibition of proliferation induced by juglone in EC Ishikawa cells was closely related to iron death,cell apoptosis and cell cycle mechanism,and provided clues for further exploration of novel anti-cancer mechanisms of functional food ingredients.(4)Apoptosis of Ishikawa cells induced by juglone and its mechanism.Juglone could block Ishikawa cells in S phase through Cdc25A/CDK2/Cyclin A signaling pathway,which significantly promoted the onset of advanced apoptosis of EC cells.The apoptosis of Ishikawa cells induced by juglone involves both mitochondrial pathway and death receptor pathway.In the mitochondrial pathway,the expression of Bcl-2 and Bcl-xl was significantly down regulated,while the expression of bad and Bak was up-regulated.The combination of Caspase 3 inhibitor and juglone attenuated the inhibition to a certain extent,indicating that Caspase 3 in mitochondrial pathway plays a key role in juglone induced apoptosis of Ishikawa cells,which further confirmed that mitochondrial pathway was involved in juglone induced apoptosis.At the same time,in the death receptor pathway,the mRNA and protein expression of TNF-?,TNF-R1,TRADD,Fas,FADD,Caspase 8,Caspase 10,and DR3/5 was significantly up-regulated.When treated with Caspase 8 inhibitor(Z-IETD-FMK),the expression level of Caspase 8 decreased significantly;the addition of juglone reduced the inhibitory effect to a certain extent,which further indicated that death receptor pathway was involved in the juglone induced apoptosis of Ishikawa cells.(5)Juglone induced ferroptosis in EC Ishikawa cells and its mechanism.After treatment with juglone for 24 h,Fe²⁺accumulation,lipid peroxidation,GSH consumption and up regulation of HMOX1 were reported in EC Ishikawa cells,indicating that ferroptosis may be involved in juglone induced cell death.Beclin 1was down regulated in a dose-dependent manner in juglone treated Ishikawa cells.In addition,the expression of Beclin 1 in Ishikawa cells increased after treatment with juglone and ferroptosis inhibitor fer-1 for 24 h,indicating that juglone induced iron-autophagy.The expression of E-cadherin,MMP-9,and MMP-2 was significantly changed in juglone treated Ishikawa cells.These results suggest that juglone may inhibit the migration of Ishikawa cells by inhibiting epithelial mesenchymal transition(EMT).At the same time,treatment with fer-1 inhibitor and juglone further demonstrated that juglone induced Ishikawa cell death was related to ferroptosis.In addition,juglone induced endoplasmic reticulum stress in Ishikawa cells.