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毛蕊异黄酮通过上调miRNA-1246表达对肾癌细胞增殖和迁移的抑制作用

Inhibitory effects of Calycosin on the proliferation and migration of renal cancer cell by up-regulating the expression of miRNA-1246
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摘要 目的通过观察毛蕊异黄酮对人肾癌769-P细胞增殖和迁移的影响,探究毛蕊异黄酮抗肾癌可能的作用机制。方法使用不同质量浓度的毛蕊异黄酮[0、12.5、25、50、100、200μmol/L,二甲基亚砜(DMSO)溶解]培养769-P细胞,CCK8法检测不同浓度的毛蕊异黄酮对769-P细胞活力的影响。以200μmol/L毛蕊异黄酮处理的769-P细胞作为毛蕊异黄酮组,以DMSO处理的769-P细胞作为对照组。细胞克隆形成实验、细胞划痕实验分别检测毛蕊异黄酮对769-P细胞增殖和迁移能力的影响。实时荧光定量聚合酶链反应(RT-qPCR)检测毛蕊异黄酮对769-P细胞中miRNA-1246和趋化因子受体4(CXCR4)表达的影响。Western blotting法检测毛蕊异黄酮对769-P细胞中CXCR4和细胞外信号调节激酶(ERK)通路蛋白表达的影响。计量资料以均数±标准差(x±s)表示,多组间比较采用单因素方差分析,两组间比较采用t检验。结果采用0、12.5、25、50、100、200μmol/L毛蕊异黄酮培养后,肾癌769-P细胞吸光度值分别为0.99±0.06、0.74±0.07、0.60±0.03、0.55±0.05、0.40±0.06、0.21±0.04,与0μmol/L比较,毛蕊异黄酮能够降低769-P细胞的存活率(P<0.05)。对照组和毛蕊异黄酮组769-P细胞克隆数量分别为(109.80±13.19)个和(60.66±11.22)个,毛蕊异黄酮组769-P细胞克隆数量降低,差异具有统计学意义(t=5.67,P<0.01)。对照组和毛蕊异黄酮组769-P细胞相对迁移率分别为(43.13±3.82)%和(14.27±3.25)%,毛蕊异黄酮处理769-P细胞后,细胞迁移能力减弱,差异具有统计学意义(t=5.71,P<0.05)。对照组和毛蕊异黄酮组769-P细胞中miRNA-1246的相对表达量分别为1.03±0.12和6.99±1.84,CXCR4 mRNA的相对表达量分别为7.17±2.96和0.98±0.06,毛蕊异黄酮能够上调769-P细胞中miRNA-1246的表达(t=3.24,P<0.01),下调CXCR4 mRNA的表达(t=4.18,P<0.01)。与对照组相比,毛蕊异黄酮能够下调769-P细胞中CXCR4蛋白和ERK通路蛋白表达。结论毛蕊异黄酮能抑制肾癌769-P细胞增殖和迁移,其机制可能与上调miRNA-1246表达进而阻断CXCR4/ERK通路有关。 Objective By observing the effects of Calycosin on the proliferation and migration of human renal cancer 769-P cell,to explore the possible molecular mechanism of Calycosin against renal cancer.Methods 769-P cell were cultured with different concentrations of Calycosin[0,12.5,25,50,100,200μmol/L,dissolved in Dimethyl sulfoxide(DMSO)],and the effects of different concentrations of Calycosin on the viability of 769-P cell was detected by CCK8 method.The 769-P cell treated with 200μmol/L Calycosin were used as the Calycosin group,and the 769-P cell treated with DMSO were used as the control group.The cell colony formation assay and cell scratch assay were used to detect the effects of Calycosin on the proliferation and migration of 769-P cell,respectively.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the effect of Calycosin on the expression of miRNA-1246 and chemokine receptor-4(CXCR4)in 769-P cell.Western blotting method was used to detect the effects of Calycosin on the expression of CXCR4 and extracellular signal-regulated kinase(ERK)pathway proteins in 769-P cell.Measurement data were expressed as mean±standard deviation(x±s),and one-way ANOVA was used for comparison between multiple groups,while t-test was used for comparison between two groups.Results After cultured with 0,12.5,25,50,100,and 200μmol/L of Calycosin,the absorbance values of renal cancer 769-P cell were 0.99±0.06,0.74±0.07,0.60±0.03,0.55±0.05,0.40±0.06,0.21±0.04,respectively;compared with 0μmol/L,the Calycosin could reduce the survival rate of 769-P cell(P<0.05).The number of clones of 769-P cell in the control group and the Calycosin group was 109.80±13.19 and 60.66±11.22,respectively,and the number of clones of the 769-P cell in the Calycosin group was decreased,the difference was statistically significant(t=5.67,P<0.01).The relative migration rates of 769-P cell in the control group and the Calycosin group were(43.13±3.82)%and(14.27±3.25)%,respectively,after the 769-P cell were treated with Calycosin,the cell migration ability was weakened(t=5.71,P<0.05).The relative expression levels of miRNA-1246 in 769-P cell of the control group and the Calycosin group was 1.03±0.12 and 6.99±1.84,respectively,and the relative expression levels of CXCR4 mRNA was 7.17±2.96 and 0.98±0.06,respectively,showed that Calycosin can up-regulate the expression of miRNA-1246 in 769-P cell(t=3.24,P<0.01),and down-regulate the expression of CXCR4 mRNA(t=4.18,P<0.01).Compared with the control group,the Calycosin could down-regulate the expression of CXCR4 protein and ERK pathway protein in 769-P cell.Conclusion Calycosin can inhibit the proliferation and migration of renal cancer 769-P cell,and its mechanism may be related to up-regulating the expression of miRNA-1246 and blocking the CXCR4/ERK pathway.
作者 黄耿 张晓玲 桂定文 王小英 罗清 Huang Geng;Zhang Xiaoling;Gui Dingwen;Wang Xiaoying;Luo Qing(Department of Urology,Huangshi Central Hospital,Huangshi 435000,China)
出处 《国际外科学杂志》 2024年第6期366-371,I0004,共7页 International Journal of Surgery
基金 湖北省卫生健康科研基金(WJ2019H158)。
关键词 肾肿瘤 微RNAS 细胞增殖 细胞迁移分析 毛蕊异黄酮 MiRNA-1246 趋化因子受体4 Kidney neoplasms MicroRNAs Cell proliferation Cell migration assays Calycosin MiRNA-1246 Chemokine receptor 4
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