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目的:构建靶向人角蛋白18(cytokeratin 18,CK18)基因的shRNA真核表达载体,稳定转染人乳腺癌MCF-7细胞株,观察其对MCF-7细胞增殖的影响。方法:设计两条靶向CK18基因的RNA干扰序列,命名为CK18-shRNA1、CK18-shRNA2,同时设计阴性对照,将合成的寡核苷酸链与Psilencer3.1-H1/Hygro质粒连接形成重组质粒,并经脂质体介导稳定转染MCF-7细胞,G418筛选后扩增获得稳定转染细胞株,分别采用RT-PCR和Western blotting法检测细胞株中CK18mRNA和蛋白的表达,并进一步通过MTT法检测重组表达载体稳定转染对细胞增殖的影响。结果:重组表达载体(Psilenc-er3.1-CK18-shRNA1、Psilencer3.1-CK18-shRNA2和Psilencer3.1-NC-shRNA)经PCR及DNA测序分析证明序列插入正确,经500μg/ml G418筛选出稳定转染Psilencer3.1-CK18-shRNA的MCF-7细胞,Psilencer3.1-CK18-shRNA2转染可有效抑制MCF-7细胞中CK18 mRNA和蛋白的表达,而Psilencer3.1-CK18-shRNA1转染不影响MCF-7细胞中CK18 mRNA和蛋白的表达水平。与阴性对照Psilencer3.1-NC-shRNA组相比,Psilencer3.1-CK18-shRNA2转染可有效抑制MCF-7细胞的增殖[(0.40±0.01)vs(0.55±0.06),P<0.05]。结论:靶向CK18的shRNA表达载体稳定转染细胞后可抑制人乳腺癌MCF-7细胞的增殖。
OBJECTIVE: To construct shRNA eukaryotic expression vector targeting cytokeratin 18 (CK18) gene and stably transfected it into human breast cancer cell line MCF-7 to observe its effect on the proliferation of MCF-7 cells. METHODS: Two RNAi sequences targeting CK18 gene were designed and named as CK18-shRNA1 and CK18-shRNA2. At the same time, a negative control was designed and the synthesized oligonucleotide chain was ligated with Psilencer3.1-H1 / Hygro plasmid to form a recombinant plasmid , And transfected into MCF-7 cells by lipofectamine. The stable transfected cell lines were screened by G418, and the expression of CK18 mRNA and protein were detected by RT-PCR and Western blotting respectively. The effect of stable transfection of recombinant expression vector on cell proliferation was detected by MTT assay. Results: The recombinant plasmids were inserted into Psilenc-er3.1-CK18-shRNA1, Psilencer3.1-CK18-shRNA2 and Psilencer3.1-NC-shRNA by PCR and DNA sequencing. Psilencer3.1-CK18-shRNA2 transfected MCF-7 cells stably transfected with Psilencer3.1-CK18-shRNA can effectively inhibit the expression of CK18 mRNA and protein in MCF-7 cells, while Psilencer3.1-CK18-shRNA1 transfection Does not affect the expression of CK18 mRNA and protein in MCF-7 cells. Compared with the negative control Psilencer3.1-NC-shRNA group, Psilencer3.1-CK18-shRNA2 transfection could effectively inhibit the proliferation of MCF-7 cells [(0.40 ± 0.01) vs (0.55 ± 0.06), P <0.05]. Conclusion: The shRNA targeting CK18 can inhibit the proliferation of human breast cancer MCF-7 cells after stably transfected the cells.