宫颈癌Siha细胞miR21基因表达与顺铂敏感相关性研究

发布时间：2018-06-06 22:04

  本文选题：宫颈肿瘤 + miR　； 参考：《中华肿瘤防治杂志》2015年14期

【摘要】：目的探讨miR21基因表达改变对宫颈癌Siha细胞顺铂敏感性的影响。方法采用脂质体介导法分别以micrOFFTMmiR21inhibitor、micrONTMmiR21mimic、miR21阴性对照试剂转染Siha细胞,实验分为miR21下调组、miR21上调组、阴性对照组及空白对照组。实时荧光定量PCR检测转染后各组细胞中miR21基因的表达水平;CCK-8比色法检测各组细胞对顺铂的半抑制率浓度(50%inhibitory concentration,IC50值);AnnexinⅤ/PI法检测顺铂处理后各组细胞的凋亡率;实时荧光定量PCR和蛋白质印迹法,分别从mRNA和蛋白水平检测顺铂对各组细胞中PTEN、PDCD4基因表达的影响。结果实时荧光定量PCR检测miR21基因的表达结果显示,miR21下调组的表达明显低于阴性对照组,miR21上调组的表达明显高于阴性对照组,F=255.525,P0.001。CCK-8比色法检测结果显示,顺铂对miR21下调组的IC50值为(2.44±0.69)μg/mL,阴性对照组为(3.96±0.07)μg/mL;miR21上调组为(6.93±0.07)μg/mL,空白对照组为(4.05±0.03)μg/mL,F=870.118,P0.001。AnnexinⅤ/PI检测结果显示,5μg/mL顺铂诱导miR21下调组的凋亡率为(64.36±2.47)%,阴性对照组为(4.2±0.17)%;miR21上调组为(0.06±0.03)%,空白对照组为(4.14±0.25)%,χ2=10.385,P=0.016。实时荧光定量PCR检测PTEN和PDCD4结果显示,miR21下调组PTEN的mRNA表达相较于阴性对照组显著增加,miR21上调组PTEN的mRNA表达相较于阴性对照组降低,F=174.057,P0.001;miR21下调组PDCD4的mRNA表达相较于阴性对照组显著增加,miR21上调组PDCD4的mRNA表达相较于阴性对照组降低,F=491.283,P0.001。蛋白质印迹法检测结果显示,miR21下调组PTEN蛋白表达量为8.845±0.062,阴性对照组7.695±0.056;miR21上调组为6.908±0.058,空白对照组为1,F=16.036,P0.01。miR21下调组PDCD4蛋白表达量为9.936±0.036,阴性对照组为8.728±0.019;miR21上调组为7.838±0.066,空白对照组为1,F=55.323,P0.001。结论下调miR21基因可能增加宫颈癌Siha细胞对顺铂的敏感性,上调miR21基因后可能减弱宫颈癌Siha细胞对顺铂的敏感性。
[Abstract]:Objective to investigate the effect of miR21 gene expression on cisplatin sensitivity in Siha cells. Methods Siha cells were transfected with micrOFFTMmiR21inhibitory or micrONTMmiR21mimicmiR21 negative control reagent by liposome-mediated method. The cells were divided into three groups: miR21 up-regulated group, negative control group and blank control group. The expression level of miR21 gene in the transfected cells was detected by real-time fluorescence quantitative PCR and the apoptosis rate of the cells treated with cisplatin was detected by CCK-8 colorimetric assay and the IC50 value of inhibitory concentration of Cisplatin was determined by Annexin 鈪，

本文编号：1988241