沉默Bmi-1基因表达稳定细胞株的建立及其生物学特性的研究

Establishment and Biochemical Characteristics Study of Cell Line with Stable Silencing of Bmi-1 Gene

B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-cell-specific moloney murine leukemia virus insertionsite 1,Bmi-1)基因是多梳基因家族成员,参与细胞增殖调控.研究发现Bmi-1基因可能参与肿瘤的形成,可能成为肿瘤潜在的治疗靶点.用RNA干扰(RNA interference,RNAi)沉默Bmi-1基因表达观察其对乳腺癌细胞株MCF-7侵袭和转移等生物学特性的影响,以探讨Bmi-1在乳腺癌发生发展中的作用.PT67细胞包装质粒后产生的逆转录病毒感染MCF-7细胞,嘌呤霉素筛选建立稳定细胞株,稳定抑制Bmi-1的细胞株命名为MCF-7/Bmi-1si.通过RT-PCR和Western blot分别从mRNA和蛋白水平检测Bmi-1的表达量;平板克隆形成实验检测细胞克隆形成能力;Transwell侵袭小室模型检测细胞体外侵袭和转移能力.MCF-7/Bmi-1si组与MCF-7和MCF-7/GFPsi组相比,Bmi-1 mRNA和蛋白表达量明显减少,克隆形成数及形成率也明显减少(P<0.05).侵袭和转移实验表明:与MCF-7和MCF-7/GFPsi组相比,MCF-7/Bmi-1si组细胞在Transwell侵袭小室中24 h穿膜细胞数明显减少(P<0.05).结果表明沉默Bmi-1基因表达稳定细胞株构建成功,Bmi-1基因表达的沉默能显著降低MCF-7细胞的体外增殖及侵袭转移能力.

B-cell-specific moloney murine leukemia virus insertion site 1 （Bmi-1） gene is a component of the Polycomb group, which is involved in regulation of cell proliferation. Study reveal that Bmi-1 may participate in tumorigenesis and may be used as a potential therapeutic target for the treatment of cancer. RNA interfer- ence （RNAi） was introduced to silence Bmi-1 gene aiming to study the effect on biochemical characteristics of MCF-7 such as invasion and metastasis and the role of Bmi-1 on breast cancer tumorigenesis. Retroviruses were produced using the retroviral vector and PT67 packaging cell line. Retroviruses were used to infect MCF-7 cell, then puromycin-resistant cells were pooled. A stable cell line with a persistent silencing of Brui- t was established, which was named as MCF-7/Bmi-lsi. The expression of Bmi-1 was appraised with RT- PCR and Western blot assays, respectively. The colony-forming unit assay was used to measure the ability ofcell proliferation, and Transwell chamber model was employed to test the ability of cell invasion and metas- tasis in vitro. Compared with MCF-7 and MCF-7/GFPsi, the mRNA and protein expression of Bmi-1 in MCF- 7/Bmi-1 were significantly less （P〈0.05）. The number and rate of colony formation in MCF-7/Bmi-lsi were also largely decreased （P〈0.05）. The results of invasion and metastasis assays indicated： the cells in MCF-7/ Bmi-lsi moved from the upper chamber into the lower one for 24 h were less than those in the control groups MCF-7 and MCF-7/GFPsi （P〈0.05）. The stable cell line with a persistent silencing of Bmi-1 gene was successfully established. Results reveal that the stable cell line with a persistent silencing of Bmi-1 gene was successfully established. Silencing of Bmi-1 gene can inhibit the ability of proliferation, invasion and metas- tasis in MCF-7 cells in vitro.