Experiment research in the regulation of Maspin gene promoter methylation targeted by short hairpin RNAs on HN13 cells migration and invasion ability
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摘要: 目的 观察shRNA诱导Maspin基因启动子甲基化对口腔鳞状细胞癌HN13细胞株体外侵袭和迁移能力的影响。 方法 针对Maspin基因的启动子区域甲基化位点设计特异性shRNA序列sequence 1、2、3、4及非特异性序列sequence 0,构建重组腺病毒,以空载病毒为对照组,转染至HN13细胞株,观察转染率、细胞生长曲线及甲基化程度。同时,采用实时RT-PCR和WB实验检测各组Maspin的mRNA与蛋白表达水平;采用Transwell小室实验检测各组HN13细胞的迁移和侵袭数目。 结果 Maspin-shRNA重组腺病毒转染率达到95%以上,转染后sequence 1、2、4细胞生长速率有所升高,与对照组相比差异明显,且sequence 1、2、4的甲基化程度也明显高于对照组(P<0.05)。转染后培养7 d,与对照组相比,sequence 0细胞Maspin的mRNA与蛋白表达水平有所升高,sequence 1、2、4有所降低,组间差异均有统计学意义(P<0.05);与对照组相比,sequence 0细胞迁移和侵袭数目有所降低,sequence 1、2、4有所升高,其组间差异均有统计学意义(P<0.05)。 结论 Maspin基因启动子甲基化可促进体外口腔鳞状细胞癌HN13细胞的侵袭和迁移。
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关键词:
- shRNA重组腺病毒 /
- Maspin /
- 启动子甲基化 /
- HN13细胞
Abstract: Objective To observe the effect of Maspin gene promoter methylation targeted by short hairpin RNAs on HN13 cells migration and invasion ability. Methods Both specific (sequence 1,sequence 2,sequence 3,sequence 4) and nonspecific (sequence 0) shRNA sequences were designed in view of the Maspin gene promoter region methylation site,which were soon used to conduct recombinant adenovirus and transfected to HN13 cells,using no-load adenovirus as the controls.The transfection rate,cell growth curve and the degree of methylation were observed;At the same time,the mRNA and protein expression level were detected by RT-PCR and WB assay,the HN13 cells migration and invasion ability were detected by Transwell chamber experiment. Results The transfection efficiency of Maspin-shRNA recombinant adenovirus was above 95%.After the transfection,the cell growth rate of sequence 1,2,4 increased,which was obviously different to the controls,and their methylation levels were higher than the controls too (P<0.05).After 7 days'culture,the Maspin mRNA and protein expression level of sequence 0 were both higher than the controls,and the Maspin mRNA and protein expression level of sequence 1,2,4 were both lower than the controls,all the difference had statistical significance (P<0.05).The number of cell migration and invasion of sequence 0 were both lower than the controls,and the number of cell migration and invasion of sequence 1,2,4 were both higher than the controls,all the difference had statistical significance (P<0.05). Conclusion Maspin gene promoter methylation could promote the migration and invasion ability of HN13 cells.-
Key words:
- Recombinant adenovirus mediated shRNA /
- Maspin /
- Gene promoter methylation /
- HN13 cells
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