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,P<0.001。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,P<0.001。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,P<0.001;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,P<0.001。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,P<0.01。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,P<0.001。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。
OBJECTIVE To detect the influence of the cervical cancer Siha cell sensitivity to cisplatin,after effectively changes miR21 gene expression.METHODS Using lip2000 to respectively transfect micrOFFTM miR21 inhibitor,micrONTM miR21 mimic,miR21negative control into the cell,and the experiments were divided into miR21down-regulation group,miR21up-regulation group,miR21 negative control group and blank control group.qRT-PCR was used to detect the miR21 expression levels of groups of cells after transfection.CCK-8was used to detect the IC50 values of cisplatin in the cells.Annexin Ⅴ/PI method was used to detect the apoptosis rate of the cell after cisplatin treatment.qRT-PCR and Western blot were respectively used to detect the mRNA and protein expression level of PTEN and PDCD4 after cisplatin treatment.RESULTS qRT-PCR detection results showed that the expression of miR21 gene in miR21down-regulation group was lower than those in miR21 negative control group,the miR21up-regulation group was higher than those in miR21 negative control group（F=255.525,P〈0.001）.CCK-8colorimetry test results showed that IC50 values of cisplatin in miR21down-regulation group（2.44±0.69）μg/mL were significantly lower than those in miR21 negative control group（3.96±0.07）μg/mL;IC50values of cisplatin in miR21up-regulation group（6.93±0.07）μg/mL was significantly higher than those in miR21 negative control group（4.05±0.03）μg/mL（F=870.118,P〈0.001）.In Annexin Ⅴ/PI assay,the 5μg/mL cisplatin induced apoptosis in miR21down-regulation group（64.36±2.47）% was significantly higher than that in miR21 negative control group（4.2±0.17）%;the 5μg/mL cisplatin induced apoptosis in miR21up-regulation group（0.06±0.03）% was significantly lower than that of in miR21 negative control group（4.14±0.25）%（χ2=10.385,P=0.016）.qRT-PCR detection results showed that the expression of PTEN-mRNA in miR21down-regulation group was higher than those in miR21 negative control group,the miR21up-regulation group was lower than those in miR21 negative control group（F=174.057,P〈0.001）.In the same way,the expression of PDCD4-mRNA in miR21down-regulation group was higher than those in miR21 negative control group,the miR21up-regulation group was lower than those in miR21 negative control group（F=491.283,P〈0.001）.Western blot detection results showed that the expression of PTEN protein（8.845±0.062）in miR21down-regulation group was higher than that in miR21 negative control group（7.695±0.056）;The expression of PTEN protein（6.908±0.058）in miR21up-regulation group was lower than that in miR21 negative control group（F=16.036,P〈0.01）.The expression of PDCD4protein（9.936±0.036）in miR21down-regulation group was higher than that in miR21 negative control group（8.728±0.019）;The expression of pdcd4protein（7.838±0.066）in miR21up-regulation group was lower than that in miR21 negative control group（F=55.323,P〈0.001）.CONCLUSION The downregulation of miR21 may increase the sensitivity of cervical cancer Siha cell to cisplatin;The upregulation of miR21 may decrease their sensitivity to cisplatin.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2015年第14期1104-1108,1114,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
广东省科技计划(2011B050300020)
