摘要
目的研究知母皂苷B-Ⅱ抑制人胃癌细胞株BGC-823和MGC-803增殖及迁移的作用机制。方法使用终浓度为50 ng/m L的知母皂苷B-Ⅱ处理BGC-823和MGC-803细胞48 h,用q PCR和蛋白质印迹实验分别检测细胞内清道夫受体A5(SCARA5)的mRNA含量及蛋白表达;采用生物信息学方法预测hsa-miRNA-766-3p与SCARA5 3′UTR区的结合位点,并通过荧光素酶报告基因实验进行验证;转染hsa-miRNA-766-3p mimic或siRNA-SCARA5至BGC-823、MGC-803细胞,24 h后使用50 ng/m L知母皂苷B-Ⅱ处理细胞48 h,用q PCR和蛋白质印迹实验检测细胞内hsamiRNA-766-3p相对含量和SCARA5蛋白表达,MTT法检测细胞的增殖活性及迁移能力。结果 50 ng/m L知母皂苷B-Ⅱ处理BGC-823、MGC-803细胞48 h能增强肿瘤细胞中SCARA5蛋白表达(P<0.01),而SCARA5 mRNA相对含量变化差异无统计学意义(P>0.05)。与报告基因表达载体单独转染比较,hsa-miRNA-766-3p mimic和hsamiRNA-766-3p inhibitor与野生型荧光素酶报告基因共转染可分别抑制和增强细胞内荧光素酶活性(P<0.05,P<0.01);hsa-miRNA-766-3p mimic和hsa-miRNA-766-3p inhibitor转染对突变型荧光素酶报告基因载体荧光素酶活性无明显影响(P>0.05)。50 ng/m L知母皂苷B-Ⅱ处理BGC-823、MGC-803细胞48 h,细胞内hsa-miRNA-766-3p含量均降低,SCARA5蛋白表达则升高(P<0.01);hsa-miRNA-766-3p mimic转染+皂苷处理与单纯皂苷处理比较,细胞内hsa-miRNA-766-3p含量上升(P<0.01)、SCARA5蛋白表达降低(P<0.01);siRNA-SCARA5转染+皂苷处理与单纯皂苷处理比较,细胞内hsa-miRNA-766-3p含量无明显变化(P>0.05),但SCARA5蛋白表达降低(P<0.01)。细胞增殖及迁移实验检测结果表明,与细胞对照或溶媒对照比较,经知母皂苷B-Ⅱ处理的胃癌细胞BGC-823和MGC-803增殖活性及迁移能力均降低(P<0.01),siRNA-SCARA5转染+皂苷处理的细胞增殖活性和迁移能力与单纯皂苷处理的细胞相比增强(P<0.01)。结论知母皂苷B-Ⅱ能够抑制hsa-miRNA-766-3p的表达,进而上调其靶基因SCARA5的表达,最终抑制胃癌细胞BGC-823和MGC-803的增殖和迁移。
Objective To explore the inhibitory mechanism of timosaponin B-Ⅱagainst the proliferation and migration of human gastric cancer cell lines BGC-823 and MGC-803.Methods BGC-823 and MGC-803 cells were treated with timosaponin B-Ⅱ(50 ng/mL)for 48 h,and the mRNA and protein expressions of scavenger receptor A5(SCARA5)were measured by qPCR and Western blotting,respectively.The binding site of hsa-miRNA-766-3p in SCARA5 gene 3′UTR was predicted by bioinformatics,and was validated by luciferase report assay.After transfecting with hsa-miRNA-766-3p mimic or siRNA-SCARA5 for 24 h,the BGC-823 and MGC-803 cells were treated with timosaponin B-Ⅱ(50 ng/mL)for 48 h.The relative levels of hsamiRNA-766-3p and SCARA5 protein expression were detected by qPCR and Western blotting,respectively.The proliferation and migration abilities of cells were determined by MTT.Results The expressions of SCARA5 protein in BGC-823 and MGC-803 cells treated with timosaponin B-Ⅱ(50 ng/mL)for 48 h were significantly increased versus the control group(P<0.01),while no significant difference was found in relative mRNA level of SCARA5 between the timosaponin B-Ⅱtreated cell group and the control group(P>0.05).Compared with transfection of reporter gene expression vector alone group,the luciferase activity was significantly inhibited or enhanced in the cells co-transfected with hsa-miRNA-766-3p mimic or hsa-miRNA-766-3p inhibitor and wild-type luciferase reporter gene(P<0.05,P<0.01).No change was observed between the cells co-transfected with hsa-miRNA-766-3p mimic or hsa-miRNA-766-3p inhibitor and mutant-type luciferase reporter gene expression vector and the cells transfected with reporter gene expression vector alone(P>0.05).Hsa-miRNA-766-3p levels were significantly decreased and SCARA5 protein expressions were significantly increased in BGC-823 and MGC-803 cells treated with 50 ng/mL timosaponin B-Ⅱ(P<0.01).Compared with the timosaponin B-Ⅱtreatment group,hsa-miRNA-766-3p levels were significantly increased and SCARA5 protein expressions were significantly decreased in the BGC-823 and MGC-803 cells of the hsa-miRNA-766-3p mimic transfection+timosaponin B-Ⅱtreatment group(P<0.01).There were no differences in the hsa-miRNA-766-3p levels between the hsa-miRNA-766-3p mimic transfection+timosaponin B-Ⅱtreatment group and the timosaponin B-Ⅱtreatment group(P>0.05),but SCARA5 protein expressions were significantly decreased(P<0.01).Compared with cell control group and vehicle control group,the proliferation and migration abilities of BGC-823 and MGC-803 cells were significantly inhibited by timosaponin B-Ⅱ(P<0.01).Compared with the timosaponin B-Ⅱtreatment group,the proliferation and migration abilities of BGC-823 and MGC-803 cells were significantly increased in the siRNA-SCARA5 transfection+timosaponin B-Ⅱtreatment group(P<0.01).Conclusion Timosaponin B-Ⅱcan inhibit the proliferation and migration of BGC-823 and MGC-803 cells via suppressing hsamiRNA-766-3p and upregulating the target gene SCARA5.
作者
庞涛
陆文铨
陈小玲
陈万生
PANG Tao△;LU Wen-quan;CHEN Xiao-ling;CHEN Wan-sheng(Department of Pharmacy,Changzheng Hospital,Navy Medical University(Second Military Medical University),Shanghai 200003,China)
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2018年第4期380-387,共8页
Academic Journal of Second Military Medical University
基金
上海市科委科技支撑项目(15401972200)
上海市临床药学重点专科建设项目(2016-40044-002)
上海市卫生计生系统重要薄弱学科建设计划(2016ZB0303)~~
作者简介
共同第一作者:庞涛,硕士生,主管药师.E-mail:yckpangtao@smmu.edu.cn;共同第一作者:陆文铨,博士,副主任药师.E-mail:lwqp@smmu.edu.cn;通信作者:陈万生.Tel:021-65307133,E-mail:chenwansheng@smmu.edu.cn
