联合基因Survivin、CDK1的shRNAs靶向干扰对鼻咽癌CNE-2细胞增值的影响

发布时间：2018-05-07 19:01

本文选题：短发夹样RNA + Survivin基因　；参考：《基因组学与应用生物学》2014年02期

【摘要】：为了构建具有干扰效应的靶向Survivin和CDK1基因及两者串联的短发夹样RNA(short hairpin RNA,shRNA)表达载体,并研究其靶向干扰对鼻咽癌细胞CNE-2生物学行为的改变。本研究通过构建pU6-SurvivinshRNA、pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA重组载体,用脂质体Lipofectamine 2000法将其转染入CNE-2细胞株中,应用RT-PCR方法初步筛选具有干扰效应的重组载体,逆转录实时荧光定量PCR(RT-qPCR)检测Survivin、CDK1 mRNA的基因表达水平,Western blot检测Survivin和CDK1蛋白表达以及噻唑蓝溴化四唑(methyl thiazolyl tetrazolium,MTT)检测癌细胞增殖活性的变化。结果表明:成功构建具有干扰效应的shRNAs表达载体;联合或单基因Survivin、CDK1的shRNAs表达载体干扰CNE-2细胞后,其对应的mRNA和目的蛋白表达均降低,CNE-2癌细胞增殖活性降低,联合基因shRNAs表达载体具有一定程度的干扰增强作用。
[Abstract]:In order to construct the expression vector of targeted Survivin and CDK1 genes and short hairpin RNA-like shRNAs with interference effect, and to study the effect of targeting interference on the biological behavior of nasopharyngeal carcinoma (NPC) cell line CNE-2. In this study, pU6-survivin RNA-pU6-CDK1shRNA and pU6-SurvivinshRNA-CDK1shRNA recombinant vector were constructed and transfected into CNE-2 cell line by liposome Lipofectamine 2000 method. The interfering recombinant vector was screened by RT-PCR method. The expression level of survivin CDK1 mRNA gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of Survivin and CDK1 protein was detected by Western blot, and the proliferative activity of cancer cells was detected by methyl thiazolyl tetrazoliumium (MTT). The results showed that the shRNAs expression vector with interference effect was successfully constructed, and the expression of mRNA and target protein decreased after shRNAs expression vector combined with or single gene survivin CDK1 interfered with CNE-2 cells. The co-gene shRNAs expression vector has a certain degree of interference enhancement.
【作者单位】：海南大学农学院;南方医科大学珠江医院医学检验中心;海南省人民医院医学检验中心;
【基金】：国家自然科学基金项目(81060223)资助
【分类号】：R739.63

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