Molecular Mechanisms Of The Anti-Endometrial Cancer Potential Of Asparanin A From Asparagus Officinalis L.

Asparagus officinalis L.is a medicinal and edible vegetable which is rich in a variety of natural active compounds,and asparagus saponin is largely responsible for its biological activity.Endometrial cancer(EC)is one of the three most common malignant tumors in the female reproductive system.Dietary intervention and finding new therapeutic targets are important measures for its prevention and treatment.Asparanin A,a saponin is recognized with high anticancer activity in asparagus,its anti-EC activity and its mechanism has not been explored so far.This paper firstly analyzed and identified asparanin A in asparagus,and preliminarily investigated its anticancer activity in EC Ishikawa cells and tumor-bearing mice.Based on this,the target of asparanin A inhibiting EC was identified and its molecular mechanism was revealed from the multi-perspectives of miRNA-omics and transcriptome integrative analysis combined with molecular biotechnologies.This paper will lay a theoretical foundation for the development of foods for special medical purposes and may serve as a guide for healthy diets in human life,and it can also provide new ideas for cancer epidemiological research and prevention.(1)Preparation and anticancer activity of asparanin A from asparagus.The total saponins of asparagus were extracted by ultrasonic-assisted ethanol method,further purified by microporous resin and silica gel column chromatography,and identified by High performance liquid chromatography(HPLC),Ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)and nuclear magnetic resonance(NMR)analysis.The obtained compound was identified as asparanin A with a purity of 97.96%.Among the four asparagus saponins with similar structures,asparanin A had the strongest inhibitory effect on the proliferation of EC Ishikawa cells,and had a significant inhibitory effect on the three major gynecological cancer cells.Further,in vitro cell experiments showed that asparanin A significantly inhibited the proliferation of Ishikawa cells and altered the cell morphology in a concentration-and time-dependent manner,as well as affected the tumor growth in vivo.To summarize,it can be seen that asparanin A has significant anti-endometrial cancer activity.(2)The mechanism of asparanin A inhibiting endometrial cancer was analyzed based on miRNA-omics and transcriptomic analysis.After treatment with asparanin A(10μM),the expression of 5,281 genes and 289 miRNAs in EC Ishikawa cells were significantly changed,respectively.The regulatory target sites and biological processes of differentially expressed genes were mainly related to transcriptional regulation,cell cycle,DNA damage stress,protein processing and modification in the nucleus and cytoplasm.The KEGG enrichment analysis focused on signaling pathways related to cell cycle,apoptosis and autophagy,and migration.The target genes of differentially expressed miRNAs mainly regulated cell signal transduction,transcription regulation and other functions by affecting protein binding,metal ion binding and transferase activity at action sites such as cell membrane,cytoplasm and nucleus.The most enriched pathways were signal transduction pathways,among which PI3K-Akt,Ras,Rap1,p53,HIF-1,and AMPK signaling pathways had the highest significance,which preliminarily revealed their regulatory mechanisms at the overall level.(3)Asparanin A induced cell cycle arrest and apoptosis in Ishikawa cells and its mechanism.Through further screening and combined analysis of the two omics data,it can be found that the inhibitory effect of asparanin A on the proliferation of Ishikawa cells was closely related to cell cycle,apoptosis,autophagy,migration and other processes.Primarily,the effect and mechanism of asparanin A on Ishikawa cell cycle and apoptosis were investigated in vitro and in vivo.Asparanin A arrested the cell cycle in G0/G1 phase by regulating the expression of related genes and proteins such as p16,p21,p27,CDK2-Cyclin E and CDK4/6-Cyclin D.Asparanin A induced cell apoptosis by activating mitochondrial pathways,including dysregulation of Bak/Bcl-xl ratio,intracellularROS accumulation,up-regulation of Cytochrome C followed by decrease ofΔψ_m,and activation of Caspases family.In addition,asparanin A had a significant inhibitory effect on the PI3K/AKT/m TOR signaling pathway.The same results were confirmed in vivo by immunohistochemistry and Western blot assays.(4)Asparanin A triggered autophagy in Ishikawa cells through endoplasmic reticulum stress and DNA damage and its mechanism.Firstly,the significantly changed genes related to cell apoptosis and autophagy were screened based on miRNA-seq and mRNA-seq integrated analyses.The accuracy of the most relevant genes which selected from protein-protein interaction(PPI)relationship were verified byRT-q PCR.Secondly,the results of TEM and immunofluorescence demonstrated that asparanin A not only induced the apoptosis but also triggered the occurrence of autophagy in Ishikawa cells.Furthermore,the relationship between asparanin A-induced autophagy and apoptosis was found to be antagonistic by flow cytometry and Western blot assays.Finally,the p53 signaling pathway and PERK/ei F2α/ATF4/CHOP axis of PPER pathway were determined to be the main regulatory pathways viaRT-q PCR and Western blot techniques.Basis on this,the significantly different miRNAs and genes related to these two pathways were verified byRT-q PCR and their relationship were performed by co-network analysis,and miR-6236-p5 family was found to possess the highest correlation,suggesting that it has a key regulatory role.(5)Asparanin A inhibited the migration and invasion of Ishikawa cells and its mechanism.The KEGG enrichment analysis of the target genes of significantly different miRNAs revealed that among the signaling pathways related to cell migration,Ras signaling pathway,Rap1 signaling pathway and MAPK signaling pathway were significantly enriched.The cell cloning,cell wound healing,and Transwell assays further demonstrated that asparanin A inhibited Ishikawa cell migration and invasion in a dose-dependent manner.Further verification of these changes via immunohistochemistry and western blot assays in vitro and in vivo demonstrated that AA could suppress the expression of MMP2 and MMP9,and theRas/ERK/MAPK pathway was the main pathway.Furthermore,top three key functional miRNAs(miR-6236-p5,miR-12136＿R+8 and miR-6236-p3＿1ss24GC)and top four hub target genes(KITLG,PDGFD,KRAS and NRAS)were identified as the functional hub miRNAs and genes through miRNA-mRNA co-expression network analysis.(6)Molecular mechanism of miRNA-6236-p5＿4 mediating asparanin A against endometrial cancer.On the basis of previous studies,miR-6236-p5＿4 was selected and transfected into Ishikawa cells,and it was found to serve as a tumor suppressor in the anti-EC effect of asparanin A.Overexpression of miR-6236-p5＿4 significantly enhanced the effects of asparanin A on cell proliferation,cell cycle,apoptosis,autophagy,and migration,whereas downregulation of miR-6236-p5＿4 attenuated the anticancer activity of asparanin A.In addition,CDK6,PIK3CB and KRAS were found to be the directly targeted regulatory genes of miR-6236-p5＿4,and the PI3K-Akt signaling pathway,p53signaling pathway,Ras andRap1 signaling pathways were proved to be the vital relevant pathways.