机构地区:[1]湖北医药学院附属太和医院妇产中心,十堰442000
出 处:《医学研究杂志》2023年第4期53-58,共6页Journal of Medical Research
基 金:湖北省科技计划项目(2021CFB162)。
摘 要:目的 研究桃叶珊瑚苷调控miR-1294/酪氨酸3单加氧酶-色氨酸5单加氧酶激活蛋白ζ(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta, YWHAZ)分子轴对宫颈癌C-33A细胞增殖和迁移的影响。方法 采用DMEM细胞培养基配制桃叶珊瑚苷培养液,分别采用0、20、40、60、80、100μmol/L桃叶珊瑚苷处理宫颈癌C-33A细胞,噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide, MTT)实验分析C-33A细胞的增殖情况。将C-33A细胞分为对照组(0μmol/L桃叶珊瑚苷处理)和桃叶珊瑚苷组(80μmol/L桃叶珊瑚苷处理),以细胞划痕实验检测C-33A细胞的迁移情况。实时荧光定量聚合酶链反应(real-time quantitative fluorescence polymerase chain reaction, qRT-PCR)检测两组C-33A细胞中miR-1294的表达。采用MicroRNAdb数据库和双荧光素酶基因报告实验分析和验证miR-1294与YWHAZ之间的调控关系。qRT-PCR和Western blot法分别检测YWHAZ基因和Akt信号通路蛋白p-Akt、p-PRAS40、mTORC1、SGK1的表达。结果 0、20、40、60、80、100μmol/L桃叶珊瑚苷处理后宫颈癌C-33A细胞增殖活性(A)值分别为1.02±0.17、0.75±0.06、0.61±0.10、0.41±0.05、0.17±0.09、0.47±0.12,桃叶珊瑚苷能够明显抑制C-33A细胞的增殖活力(F=28.90,P<0.05)。对照组和桃叶珊瑚苷组C-33A细胞划痕愈合率分别为59.28%±9.93%和25.75%±9.29%,差异有统计学意义(t=4.93,P<0.05)。对照组和桃叶珊瑚苷组miR-1294的表达分别为1.01±0.62和10.14±2.02,差异有统计学意义(t=8.65,P<0.01)。miR-1294可靶向结合YWHAZ(t=8.76,P<0.01)。对照组和桃叶珊瑚苷组C-33A细胞中YWHAZ mRNA表达分别为4.74±1.22和1.01±0.22,miR-1294可负调控YWHAZ基因表达(t=6.00,P<0.01)。与对照组比较,桃叶珊瑚苷组YWHAZ蛋白和Akt信号通路蛋白p-Akt、p-PRAS40、mTORC1、SGK1表达明显降低。结论 桃叶珊瑚苷通过调控miR-1294/YWHAZ分子轴抑制宫颈癌C-33A细胞的增殖和迁移。Objective To study the effect of aucubin on the proliferation and migration of cervical cancer C-33A cells by regulating the miR-1294/tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta(YWHAZ)molecular axis.Methods DMEM cell culture medium was used to prepare the aucubin culture medium.Cervical cancer C-33A cells were treated with 0,20,40,60,80,100μmol/L aucubin,respectively,and the proliferation of C-33A cells was analyzed by methylthiazolyldiphenyl-tetrazolium bromide(MTT)experiment.The C-33A cells were divided into control group(0μmol/L aucubin treatment)and aucubin group(80μmol/L aucubin treatment).The migration of C-33A cells was detected by cell scratch assay.The expression of miR-1294 in the two groups of C-33A cells was detected by real-time quantitative fluorescence polymerase chain reaction(qRT-PCR).The regulatory relationship between miR-1294 and YWHAZ was analyzed and verified using the MicroRNAdb database and dual-luciferase gene reporter experiments.The expressions of YWHAZ gene and Akt signaling pathway proteins(p-Akt,p-PRAS40,mTORC1,SGK1)were detected by qRT-PCR and Western blot,respectively.Results The proliferative activity(A)values of cervical cancer C-33A cells treated with 0,20,40,60,80 and 100μmol/L aucubin were 1.02±0.17,0.75±0.06,0.61±0.10,0.41±0.05,0.17±0.09,0.47±0.12,aucubin can significantly inhibit the proliferation of C-33A cells(F=28.90,P<0.05).The scratch healing rates of C-33A cells in the control group and the aucubin group were 59.28%±9.93%and 25.75%±9.29%,respectively,and the difference was statistically significant(t=4.93,P<0.05).The expression of miR-1294 in the control group and the aucubin group was 1.01±0.62 and 10.14±2.02,respectively,and the difference was statistically significant(t=8.65,P<0.01).miR-1294 could be targeted to YWHAZ(t=8.76,P<0.01).The expression of YWHAZ mRNA in C-33A cells in the control group and the aucubin group was 4.74±1.22 and 1.01±0.22,respectively,and miR-1294 could negatively regulate the expression of YWHA
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