| 张淑君,单梦圆,翟羽佳,李闯,季丙元.PIWIL1基因沉默对人乳腺癌MCF-7细胞增殖的影响[J].济宁医学院学报,2019,42(2):117-121 |
| PIWIL1基因沉默对人乳腺癌MCF-7细胞增殖的影响 |
| Effects of PIWIL1 gene silencing on proliferation of human breast cancer MCF-7 cells |
| 投稿时间：2019-01-29 |
| DOI：10.3969/j.issn.1000-9760.2019.02.010 |
| 中文关键词: shRNA;PIWIL1;乳腺癌;MCF-7细胞株 |
| 英文关键词: shRNA;PIWIL1;Breat cancer;MCF-7 cell Lines |
| 基金项目:济宁医学院大学生科研项目（JYXS2017KJ013） |
| 作者 | 单位 | E-mail | | 张淑君 | 济宁医学院精神卫生学院, 济宁 272013 | | | 单梦圆 | 济宁医学院精神卫生学院, 济宁 272013 | | | 翟羽佳 | 济宁医学院精神卫生学院, 济宁 272013 | | | 李闯 | 济宁医学院精神卫生学院, 济宁 272013 | | | 季丙元 | 济宁医学院精神卫生学院, 济宁 272013 | jby2006@126.com |
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| 中文摘要: |
| 目的 PIWIL1基因沉默对人乳腺癌MCF-7细胞增殖活性的影响，为乳腺癌的基因诊断和治疗提供理论依据。方法 针对PIWIL1 mRNA序列设计合成siRNA并筛选最佳siRNA，构建pGCsilencerTMH1/GFP/Neo/PIWIL1 RNAi和pGCsilencerTMH1/GFP/Neo/Non RNAi重组质粒；将重组质粒导入乳腺癌MCF-7细胞株，G418筛选获得稳定转染的细胞株；实验分为脂质体对照组、阴性质粒转染对照组（阴性对照组）及PIWIL1-shRNA重组质粒转染组（PIWIL1-shRNA组），实时荧光定量PCR法检测PIWIL1 mRNA表达水平的变化，Western blot检测细胞中PIWIL1蛋白表达，CCK-8检测干扰质粒对MCF-7细胞增殖活性的影响。结果 荧光显微镜检测显示稳定表达重组质粒pGCsilencerTMH1/GFP/Neo/PIWIL1 RNAi的乳腺癌MCF-7细胞株筛选成功，其PIWIL1 mRNA抑制率（74%）明显高于阴性对照组（5.4%），PIWIL1-shRNA组细胞PIWIL1蛋白表达水平显著低于脂质体对照组和阴性对照组细胞，MCF-7细胞增殖活性显著低于阴性对照组和脂质体对照组差异具有统计学意义（P<0.05）。结论 重组质粒pGCsilencerTMH1/GFP/Neo/PIWIL1 RNAi可抑制乳腺癌细胞中PIWIL1基因的表达，进而降低细胞活性。 |
| 英文摘要: |
| Objective To investigate the effects of silencing PIWIL1 gene on the viability of human breat cancer MCF-7 cells,and to provide evidence in diagnosis and treatment for breat cancer.Methods Specific siRNA for PIWIL1 was designed and synthesized,and the optimal siRNA was selected.Then,the two recombinant plasmids pGCsilencerTMH1/GFP/Neo/PIWIL1 RNAi and pGCsilencer TMH1/GFP/Neo/Non RNAi were constructed.The two plasmids were transfected into MCF-7 cells,and the positive cell clones were obtained by G418 selection.The breat cancer cel1 lines MCF-7 were divided into three groups:liposome-treated control group,negative plasmid-transfected control group and PIWIL1-shRNA transfected group.The mRNA and protein levels of PIWIL1 was determined by real-time quantitative PCR and Western blot,respectively.The proliferation of cells was also observed by CCK-8 viability assay.Results The MCF-7 cell lines stably expressing recombinant plasmid pGCsilencer TMH1/GFP/Neo/PIWIL1 RNAi was successfully selected.The results of real-time quantitative PCR indicated that the inhibitory rates of mRNA in pGCsilencer TMH1/GFP/Neo/PIWIL1 transfected group (74%) was higher than that in negative plasmid transfected control group (5.4%).And the difference was statistically significant (P<0.05).The PIWIL1 protein levels was also signicantly decreased in RNAi cells.The CCK-8 assay results showed that the cell proliferation in PIWIL1-shRNA transfected group was lower than that in negative plasmid transfected control group and liposome-treated control group.Conclusion The recombinant plasmid pGCsilencer TMH1/GFP/Neo/PIWIL1 can suppress the expression of PIWIL1 in breat cancer cells resulting in the reduction of cell viability. |
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