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

发布时间：2018-07-09 22:26

本文选题：shRNA + Survivin基因　；参考：《海南大学》2014年硕士论文

【摘要】：目的：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,并研究其对鼻咽癌细胞CNE-2干扰后,癌细胞的生物学行为(增殖、凋亡)的改变。方法：根据Genbank记录的Survivin及CDK1序列,遵循shRNA设计原则并合成相应的寡核苷酸链SurvivinshRNA、 CDK1shRNA’构建pU6-SurvivinshRNA、 pU6-CDK1shRNA重组载体,用脂质体Lipofectamine2000方法将其转染入CNE-2细胞株中,RT-PCR初步筛选具有干扰效应的pU6-SurvivinshRNA、 pU6-CDK1shRNA;将上述具有干扰效应的SurvivinshRNA和CDK1shRNA串联进行pU6-SurvivinshRNA-CDK1shRNA表达载体的构建,经酶切及测序鉴定后,以脂质体Lipofectamine2000转染CNE-2细胞株后；收集转染单基因及联合基因重组质粒的CNE-2细胞,进行逆转录实时荧光定量PCR(RT-qPCR)检测癌细胞Survivin和CDK1的mRNA表达水平,Western blot检测Survivin和CDK1蛋白表达的变化,噻唑蓝溴化四唑(]methyl thiazolyl tetrazolium,MTT)法检测癌细胞增殖活性的变化,流式细胞仪检测癌细胞凋亡的变化。结果：(1)经酶切及测序结果分析：pU6-SurvivinshRNA、pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA重组载体均成功构建；(2)RT-qPCR的结果显示：pU6-SurvivinshRNA和pU6-SurvivinshRNA-CDK1shRNA各干扰组均下调Survivin mRNA的表达,分别为52.7%和82.4%,pU6-CDK1shRNA干扰组的Survivin mRNA的表达没有下调；pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1ShRNA各干扰组均下调CDK1mRNA的表达,分别为56.7%和85.0%, pU6-SurvivinshRNA干扰组的CDK1mRNA的表达没有下调；(3) pU6-SurvivinshRNA和pU6-SurvivinshRNA-CDK1shRNA干扰组均下调Survivin蛋白的表达,pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA干扰组均下调CDK1蛋白的表达。(4)MTT法检测癌细胞增殖活性的结果显示：pU6-Survivin,hRNA、 PU6-CDKlshRNA和pU6-SurvivinshRNA-CDK1shRNA干扰组均能抑制细胞的生长,抑制率分别为25.60%、15.62%和32.54%；(5)流式细胞仪检测癌细胞凋亡的结果显示：pU6-SurvivinshRNA、 pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA干扰组均促进癌细胞凋亡,凋亡率分别为10.27%,8.00%和14.87%。结论：(1)pU6-SurvivinshRNA、 pU6-CDK1shRNA和pU6-SurvivinshRNA-CDK1shRNA重组载体均成功构建；(2) pU6-SurvivinShRNA、pU6-CDK1ShRNA和pU6-SurvivinshRNA-CDK1shRNA重组载体干扰鼻咽癌细胞,均可以抑制癌细胞的增殖,促进癌细胞的凋亡；(3)Survivin基因和CDK1基因有望成为鼻咽癌肿瘤基因治疗的靶基因,多基因联合干扰可以提高效应。
[Abstract]:Aim: to construct the expression vector of survivin and CDK1 gene targeting survivin and CDK1 gene and short hairpin (short hairpin RNA (shRNA) in tandem, and to study the changes of biological behavior (proliferation and apoptosis) of nasopharyngeal carcinoma cell line CNE-2 after the interference of survivin and CDK1 gene. Methods: according to the sequence of survivin and CDK1 recorded in Genbank, following the shRNA design principle and synthesizing the corresponding oligonucleotide chain survivshRNAs, CDK1shRNAs' constructed pU6-survivin RNA, pU6-CDK1shRNA recombinant vector. Lipofectamine2000 was transfected into CNE-2 cell line by RT-PCR to screen interference pU6-survivin shRNAs (pU6-CDK1shRNAs), the interfering survivshRNA and CDK1shRNA were connected in tandem to construct pU6-survivshRNA-CDK1shRNA expression vector. CNE-2 cells transfected with liposome Lipofectamine2000 were collected and transfected into CNE-2 cells, and the expression levels of survivin and CDK1 were detected by reverse transcription-real-time fluorescence quantitative PCR (RT-qPCR). The expression of survivin and CDK1 protein was detected by Western blot. Methyl thiazolyl tetrazoliumium tetrazolium tetrazol@@ Results: (1) the recombinant vectors of pU6-survivshRNA-CDK1shRNA and pU6-survivvinshRNA-CDK1shRNA were successfully constructed by restriction endonuclease digestion and sequencing, (2) the results of RT-qPCR showed that the expression of survivin mRNA was down-regulated in the two interference groups (52.7% and 82.4pU6-CDK1shRNA respectively). Both pU6-CDK1shRNA and pU6-survivvinshRNA-CDK1ShRNA down-regulated the expression of CDK1 mRNA in 56.7% and 85.0%, respectively. The expression of CDK1 mRNA was not down-regulated in pU6-survivin RNA interference group. (3) both pU6-survivvinshRNA-CDK1shRNA interference group and pU6-survivvinshRNA-CDK1shRNA interference group down-regulated the expression of survivin protein. (5) the apoptosis of cancer cells was detected by flow cytometry. The results showed that the cell apoptosis was promoted by cell apoptosis in the groups of cell apoptosis induced by the interference of cell apoptosis, the percentage of apoptosis was 10.277.00% and the percentage of apoptosis was 14.87% (P < 0.05), respectively, in the interference groups of pU6-CDK1shRNA and pU6-survivin shRNA-CDK1shRNA. Conclusion: (1) the recombinant vectors of pU6-survivvinshRNA, pU6-CDK1shRNA and pU6-survivvinshRNA-CDK1shRNA were successfully constructed, (2) pU6-survivin shRNA-CDK1shRNA and pU6-survivvinshRNA-CDK1shRNA interference vector could inhibit the proliferation of cancer cells and promote the apoptosis of cancer cells. (3) survivin gene and CDK1 gene are expected to be the target genes for gene therapy of nasopharyngeal carcinoma.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.63

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