抑制上皮-间充质转化对肾癌细胞转移和肾癌放疗的影响

Impact of epithelial-to-mesenchymal transition inhibition on renal cancer metastasis and renal cancer radiotherapy

目的 观察抑制上皮-间充质转化（EMT）与肾癌细胞转移的相关性及其在肾癌放疗中的作用.方法 实验设对照组、脂质体转染组（Lipofectamine组）、微小RNA（miRNA,miR）-200类似物转染组（miR-200组）和无义序列组（Negative-Mimics转染组）.用miR-200类似物转染肾癌细胞,实时定量聚合酶链反应（Real-time PCR）检测EMT相关分子E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin） mRNA变化确定miR-200对EMT的抑制效果;Transwell迁移实验、划痕实验检测各组肾癌细胞迁移能力;用2Gy剂量X线照射各组细胞,微核实验、免疫荧光检测、p53结合蛋白1焦点数（53BP1 foci）实验检测肾癌细胞损伤程度,克隆存活实验检测细胞存活能力.结果 Real-time PCR结果证明E-cadherin、N-cadherin和Vimentin在对照组中的表达量为1.01 ±0.01、1.00±0.02、1.01 ±0.02;与对照组比较,这3个基因在Lipofectamine组和Negative-Mimics组的表达量为0.92±0.04、1.07±0.01、0.99 ±0.03;0.94±0.03、0.99±0.02、0.97 ±0.03,差异无统计学意义（P＞0.05）;在miR-200组的表达量为3.16 ±0.07、0.75±0.07、0.22±0.03,差异有统计学意义（P＜0.05）,证明miR-200能显著抑制肾癌细胞EMT过程.对照组、Lipofectamine组、Negative-Mimics转染组和miR-200类似物转染组Transwell迁移实验和划痕实验结果分别为（203 ±4）、（191 ±5）、（198±2）、（201 ±5）个细胞和（21.9±1.9）、（22.5±2.6）、（21.8±3.5）、（20.7±1.8）个细胞,组间比较差异均无统计学意义（P＞0.05）;X线处理后,对照组微核率和克隆存活率为（9.3±0.4）个、（1.02±0.03）％,53BP1 foci为（18.4±1.1）个;与对照组比较,Lipofectamine组和Negative-Mimics转染组微核和克隆存活率为（9.4±0.3）个、（1.09±0.05）％和（9.1±0.2）个、（0.94±0.02）％,53BP1 foci为（18.5±0.3）个和（17.9±0.4）个,差异无统计学意义（P＞0.05）;miR-200类似物转染组微核率和克隆形成率为（15.2±0.2）个和（0.39 ±0.03）％,53BP1foci为（24.9±1.1）个,差异有统计学意义（P＜0.05）.结论 EMT抑制对肾癌细胞转移能力没有影响,但是能够增强放射治疗对肾癌细胞的杀伤作用.

Objective To investigate if the inhibition of epithelial-to-mesenchymal transition （EMT） is related to renal cancer metastasis and the impact on renal cancer radiotherapy.Methods The mRNA expression level of E-cadherin,N-cadherin and Vimentin was tested through real-time quantitative polymerase chain reaction （Real-time PCR） to evaluate the inhibition of microRNA （miRNA,miR）-200 mimics transfection on EMT.Scratch assays and Transwell migration assays were performed to study the migration ability of cells in each group.When cells were treated with 2Gy X-rays,the micronucleus assay and p53 binding protein 1 （53BP1） assay and clonogenic survival assay were performed in each group to evaluate the cellular damage.Results The Real-time PCR results showed that the expression level of E-cadherin,N-cadherin and Vimentin in the control group was 1.01 ± 0.01,1.00 ± 0.02,1.01 ±0.02;0.92±0.04,1.07±0.01,0.99 ±0.03;and 0.94 ±0.03,0.99±0.02,0.97 ±0.03 in the cells transfected with Lipofectamine and Negative-Mimics respectively （P 〉 0.05）;3.16 ± 0.07,0.75 ± 0.07,0.22 ± 0.03 in the cells transfected with miR-200 mimics （P 〈 0.05）,suggesting miR-200 mimics transfection could remarkably inhibit the EMT （P 〈 0.05）.The results of transwell migration assay and scratch assay were （203 ± 4）,（191 ± 5）,（198 ± 2）,（201 ± 5） and （21.9 ± 1.9）,（22.5 ±2.6）,（21.8 ±3.5）,（20.7 ± 1.8） cells in the control,Lipofectamine,Negative-Mimics and miR-200 groups respectively （P 〉 0.05）.When exposed to X-rays,in the control group,the frequency of micronucleus,the clonogenic survival fraction and the number of 53BP1 foci were （9.3 ± 0.4）,（1.02 ± 0.03）%,and （18.4 ± 1.1）,and those in the group transfected with Lipofectamine and Negative-Mimics were （9.4±0.3）,（1.09±0.05）%,（18.5 ±0.3） and （9.1 ±0.2）,（0.94±0.02）%,（17.9 ±0.4） respectively （P 〉 0.05）.In the miR-200 mimics transfected group,the frequency of micronucleus,the clonogenic survival fraction and the number of 53BP1 foci were （15.2 ± 0.2）,（0.39 ± 0.03） % and （24.9 ± 1.1,P 〈 0.05）.Conclusion Inhibition of EMT didn＇ t impact metastasis of the renal cancer cells matastasis,but can enhance the killing effects of radiotherapy on renal cancer cells.