川楝素通过MMP9抑制肾癌细胞的增殖、迁移和侵袭

Toosendanin inhibits proliferation,migration and invasion of renal carcinoma cells by MMP9

目的:基于网络药理学方法探讨川楝素治疗肾癌的靶点及作用机制,并通过体外内实验进行验证。方法:通过SwissTarget数据库和TargetNet数据库获取川楝素的作用靶点,GeneCards数据库获取肾癌疾病相关靶点;采用DAVID数据平台对靶点进行GO分析;利用基因表达汇编(GEO)数据库的组织芯片分析靶点基因在肾癌组织的表达水平;基于肿瘤基因组图谱(TCGA)数据库分析靶点基因与肾癌临床表型的关系。采用CCK-8实验及异种移植实验观察川楝素对肾癌细胞体内外增殖的影响;采用Transwell侵袭和迁移实验观察川楝素对肾癌细胞侵袭和迁移的影响;构建基质金属蛋白酶9(MMP9)过表达质粒并转染人肾癌细胞系ACHN和769-P,观察上调MMP9表达对肾癌细胞增殖、侵袭及迁移的影响。结果:基于网络药理学分析结果,川楝素作用于肾癌的靶点包括MMP9在内共有13个靶点;TCGA数据库的临床数据表明,MMP9在肾癌组织中的表达较正常肾组织表达上调(P<0.05),且其高表达与肾癌临床分期晚、分化程度低、复发及预后差密切相关(P<0.05)。功能实验中,川楝素呈时间和剂量依赖性抑制MMP9蛋白表达(P<0.05),显著抑制肾癌细胞ACHN、769-P的增殖、迁移和侵袭(P<0.05),并能明显抑制裸鼠肾癌皮下移植瘤的生长(P<0.05),使用MMP过表达质粒上调MMP9表达则明显逆转川楝素的抑癌作用(P<0.05)。结论:MMP9在肾癌中表达上调,并与肾癌的恶性临床表型和不良预后密切相关,川楝素可通过抑制MMP9抑制肾癌细胞的增殖、迁移和侵袭。

Objective:To explore the targets and action mechanism of toosendanin in the treatment of renal carcinoma based on network pharmacology methods,and then to verify them by in vitro and in vivo experiments.Methods:The targets of toosendanin were obtained by SwissTarget database and TargetNet database.The related targets of renal carcinoma were obtained by GeneCards da‐tabase.GO analysis was performed on the targets by DAVID data platform.The expression levels of target genes in renal carcinoma tis‐sues were analyzed by tissue microarray of gene expression omnibus(GEO)database.The relationship between target genes and clini‐cal phenotypes of renal carcinoma were analyzed based on the cancer genome atlas(TCGA)database.The effects of toosendanin on proliferation of renal carcinoma cells in vitro and in vivo were observed by CCK-8 and xenograft assay.The effects of toosendanin on in‐vasion and migration of renal carcinoma cells were observed by Transwell invasion and migration assay.The plasmids with matrix me‐talloproteinase-9(MMP9)overexpression were constructed and transfected into human renal carcinoma cell lines ACHN and 769-P.The effects of up-regulating MMP9 expression on the proliferation,invasion and migration of renal carcinoma cells were observed.Results:The analysis based on network pharmacology showed that there were 13 targets(including MMP9)of toosendanin on renal carcinoma.The clinical data of TCGA database showed that the expression of MMP9 in renal carcinoma tissues was higher than that in normal renal tissues(P<0.05).The high expression of MMP9 was closely related to late clinical staging,low differentiation recurrence and poor prognosis of renal carcinoma(P<0.05).In functional experiments,toosendanin could inhibit MMP9 expression with time and dose dependence(P<0.05),significantly inhibit proliferation,migration and invasion of ACHN and 769-P(P<0.05),and significantly inhibit the growth of subcutaneously implanted tumors in nude mouse(P<0.05).The application of plasmids with MMP overexpression to up-regulate MMP9 expression could significantly reverse above-mentioned tumor suppressor effects of toosendanin(P<0.05).Conclusion:MMP9 expression is up-regulated in renal carcinoma tissues,which is closely related to malignant clinical phenotypes and poor prognosis of renal carcinoma.Toosendanin can inhibit the proliferation,migration and invasion of renal carcinoma cells by inhibiting MMP9.