Study On The Effect And Mechanism Of Hydnocarpin D In Inhibiting The Metastasis And Angiogenesis In Ovarian Cancer Via Regulating Hsa-miR-145-5p/Serpine1

[Objective]Ovarian cancer has the highest mortality rate among malignant tumors of the female reproductive system.The early treatment of ovarian cancer largely depends on tumor-reducing surgery and maintenance chemotherapy.For patients with inadequate tumor reduction or advanced ovarian cancer,additional anti-angiogenic drugs are typically used for maintenance treatment.However,current ovarian cancer treatments have many adverse effects,and there is an urgent need for safer and more effective drugs.The compound Hydnocarpin D(HD)is a flavonolignan,originally isolated from the Hydnocarpus anthelmintica Pierre,a member of the Chrysobalanaceae family.The compound HD can now be synthesized through a one-pot reaction that involves dehydration of silybin.Therefore,the present study aimed to investigate the effects of HD on ovarian cancer and its underlying molecular mechanisms.[Methods]1.Research on the anti-metastatic and anti-angiogenic effects of HD in vitro and in vivo:(1)MTT assay was used to detect the effect of HD on the viability of SKOV3,OVCAR4,and HUVEC cells;(2)Wound healing assay was used to investigate the effect of HD on the migration ability of SKOV3,OVCAR4,and HUVEC cells;(3)Transwell assay was used to evaluate the effect of HD on the migratory and invasive abilities of SKOV3,OVCAR4,and HUVEC cells;(4)Tube formation assay was used to detect the effect of HD on HUVEC tube formation;(5)Orthotopic transplanted ovarian cancer xenograft model were constructed.Live imaging was used to monitor the effect of HD on ovarian cancer metastasis,and H&E staining was used to detect the cardiac,hepatic,and renal toxicity of HD in vivo;(6)Chorioallantoic membrane(CAM)assay was used to examine the effect of HD on CAM angiogenesis;(7)Matrigel plug assay was used to investigate the effect of HD on micro vascular formation;(8)Aortic ring assay was used to assess the effect of HD on in vivo angiogenesis.2.Mechanistic study on the anti-metastatic and anti-angiogenic effects of HD targeting Serpine1 to regulate the Vitronectin/Integrin/FAK signaling pathway in ovarian cancer:(1)Transcriptomics was used to screen the differentially expressed genes before and after HD treatment in ovarian cancer cells and perform visualization analysis using bioinformatics;(2)qRT-PCR was used to validate the expression level of differentially expressed genes screened by transcriptomics;(3)TCGA database was used to analyze differential Serpine1 expression in tumor and paired samples;(4)Western Blotting was used to detect the expression level of Serpine1-translated protein PAI-1 in ovarian cancer cells after HD treatment;(5)Wound healing assay was used to investigate the effect of HD on the migration ability of SKOV3 and OVCAR4 cells after siSerpine1 transfection;(6)Western blotting was used to investigate the effect of HD on the Vitronectin/Integrin/FAK signaling pathway;(7)Immunohistochemistry(IHC)was used to detect the expression of PAI-1,CD31,and Ki-67 in ovarian cancer.3.Mechanistic study of the anti-migration effects of HD regulating has-miR-145-5p targeting Serpine1 in ovarian cancer cells:(1)Screening of miRNAs targeting Serpine1 in the four databases(miRDB,miRwalk,miRcode,Targetscan)and literature review;(2)qRT-PCR was used to validate the miRNAs regulated by HD;(3)The binding site between miRNAs screened by qRT-PCR and Serpine1 mRNA was computationally simulated and analyzed in the Targetscan;(4)Dual luciferase reporter gene assay was used to detect the binding of hsa-miR-145-5p with Serpine1 mRNA;(5)Western Blotting was used to investigate the effect of HD on PAI-1 expression in SKOV3 and OVCAR4 cells after transfection with hsa-miR-145-5p mimic/inhibitor;(6)Wound healing assay was used to investigate the effect of HD on the migration ability of SKOV3 and OVCAR4 cells after transfection with hsa-miR-145-5p mimic/inhibitor.[Results]1.HD significantly inhibited the migration and invasion of SKOV3 and OVCAR4 cells and suppressed HUVEC migration and tube formation.HD also significantly inhibited ovarian cancer metastasis in vivo,and suppressed angiogenesis in CAM,Matrigel plugs,and aortic rings.2.Differential gene of Serpine1 was selected based on transcriptome sequencing and literature research of ovarian cancer cells before and after HD treatment.HD inhibited metastasis and angiogenesis by serpinel based Vitronectin/Integrin/FAK signaling pathway in ovarian cancer.3.HD upregulated hsa-miR-145-5p in SKOV3 and OVCAR4 cells to suppress Serpine1 mRNA and inhibit the migration of SKOV3 and OVCAR4 cells.[Conclusion]HD inhibits the metastasis and angiogenesis in ovarian cancer both in vitro and in vivo,and its mechanism is related to upregulate hsa-miR-145-5p targeting Serpine1 mRNA and inhibiting the Vitronectin/Integrin/FAK signaling pathway.