抑癌基因PTEN在线粒体上的定位促进A431细胞的凋亡

The mitochondrial localization of tumor suppressor PTEN promotes apoptosis in A431 cells

摘要:

为研究抑癌基因PTEN在线粒体上的定位对细胞凋亡的影响,构建N端融合有细胞色素C氧化酶亚基七(CoxⅦ)的PTEN腺病毒重组体(Mito-PTEN)。将CoxⅦ-PTEN片段克隆至腺病毒穿梭载体pAdTrack-CMV中,PmeⅠ线性化穿梭质粒,并与腺病毒基因组载体pAdeasy-1共转化大肠杆菌菌株BJ5183,鉴定重组体,并将阳性重组体转化至大肠杆菌菌株DH5α中进行扩增;经PacⅠ酶切后的重组体通过脂质体方法转染用于腺病毒包装的细胞HEK-293A,包装重组腺病毒,收集病毒液,反复扩增,进行病毒滴度的测定;通过绿色荧光蛋白(GFP)标签检测转染及感染效率;用Mito-PTEN腺病毒感染人表皮鳞状癌细胞A431,以流式细胞分析仪检测细胞凋亡情况。成功构建了Mito-PTEN的腺病毒重组体,并进行了该重组体的病毒包装,滴度为10⁷pfu/mL,通过免疫印迹检测目的蛋白,并发现Mito-PTEN可以促进A431细胞的凋亡。Mito-PTEN的成功构建以及它对A431细胞凋亡的促进作用为研究PTEN在线粒体上的功能以及可能的在肿瘤治疗方面的应用提供了理论依据。

Abstract:

To study the function of mitochondrial PTEN in mediation of cellular apoptosis, the adenoviral recombinant of Mito-PTEN, which contains CoxⅦ (subunit Ⅶ of Cytochrome C Oxidase) gene in N-terminus, were generated. Using CoxVⅡ-PTEN-EYFPN1 as a template, CoxⅦ-PTEN was cloned into the shuttle vector pAdTrack-CMV with the restriction endonuclease sites XhoⅠand XbaⅠ. The shuttle plasmid was linearize with PmeⅠand co-transformed with adenoviral backbone vector pAdeasy-1 into E.coli BJ5183. Following selection and identification, the positive recombinant plasmids were transformed into E.coli DH5α for propagation. To package the adenoviruses, recombinant plasmid candidate was linearize using PacⅠ and transfected into HEK-293A cells with Lipofectamine 2000. Through freeze-thaw-vortex cycles, recombinant viral particles were collected and harvested, and utilized to infect 293A cells for further amplification. The method of TCID50 was employed to determine virus titers. With green fluorescent protein (GFP) as marker, the efficiency of transfection and infection was monitored by fluorescence microscopy, and the apoptosis of A431 cells after infection of Mito-PTEN-Ad was analyzed by flow cytometry. Adenoviral recombinant of Mito-PTEN was packaged successfully with the TCID₅₀ as 10⁷pfu/mL and the expressed protein was detected by western blot. In addition, it has been demonstrated that Mito-PTEN promoted apoptosis of A431 cells. Take together, the successful generation of adenoviral recombinant of Mito-PTEN, which could induce apoptosis in A431 cells, sets up a basis for further functional studies of mitochondrial PTEN and provides us a potential tool for cancer treatment in future.