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PinX1敲减对乳腺MCF-10A细胞端粒稳定性的调控作用

发表时间:2014-09-15  浏览量:1250  下载量:528
全部作者: 石嵘,周晖,曾文利,陈耀武,马文丽
作者单位: 南方医科大学基因工程研究所;南方医科大学第一临床医学院
摘 要: 目的:研究PinX1敲减对乳腺MCF-10A细胞系端粒稳定性及DNA损伤的影响。方法:在乳腺非致瘤性细胞系MCF-10A细胞中转染 PinX1 shRNA,并于转染后48 h采用嘌呤霉素筛选,建立 PinX1稳定敲减细胞株,采用实时定量real-time PCR,Western blotting及免疫荧光法检测PinX1表达水平,MTT法绘制细胞生长曲线,并进一步对细胞同时进行53BP1抗体免疫荧光染色及端粒的荧光原位杂交(fluorescence in situ hybridization,FISH)染色。结果:real-time PCR,Western blotting及免疫荧光法结果表明,PinX1稳定敲减MCF-10A细胞株PinX1表达水平显著低于对照细胞;MTT法显示PinX1敲减细胞株增殖活性增加;免疫荧光及FISH结果表明,PinX1敲减可引起53BP1聚集形成的凝集点部分与端粒位点重合。结论:PinX1敲减可以通过影响端粒稳定性导致MCF-10A细胞DNA损伤及恶性转化,进而促进细胞的增殖活性。
关 键 词: 分子生物学;端粒稳定性;基因敲减;PinX1;DNA损伤;MCF-10A细胞
Title: Regulation of telomere stability by knocking down of PinX1 in MCF-10A breast cell line
Author: SHI Rong, ZHOU Hui, ZENG Wenli, CHEN Yaowu, MA Wenli
Organization: Institute of Genetic Engineering, Southern Medical University; The First Clinical Medical College, Southern Medical University
Abstract: Objective: To study the effect of PinX1 knocking down on the telomere stability and DNA damage in MCF-10A breast cell line. Methods: PinX1 shRNA was transfected into breast cell line MCF-10A. Puromycin selection was applied 48 h after transfection to acquire a stable PinX1 knocked-down cell line. Real-time PCR, Western blotting and immunofluorescent dyeing were used for detecting the PinX1 expression level, MTT assay was applied for making the cell growth curve. Meanwhile, the cells were checked simultaneously by 53BP1 antibody immunofluorescent dyeing and telomere FISH dyeing. Results: Real-time PCR, Western blotting and immunofluorescent dyeing results showed that PinX1 expression level in the stable PinX1 knock-down MCF-10A cell line was lower than the control cells. MTT assay indicated an enhanced proliferation activity of the PinX1 knocked-down cells. The results of immunofluorescent and FISH dyeing showed that 53BP1 congregated into several foci and co-localized with the telomere spots in the PinX1 knock-down cells. Conclusion: Knock-down of PinX1 could induce DNA damage and malignant transformation in MCF-10A cells by affecting the telomere stability and promote cell proliferation activity as well.
Key words: molecular biology; telomere stability; gene knock-down; PinX1; DNA damage; MCF-10A cell
发表期数: 2014年9月第17期
引用格式: 石嵘,周晖,曾文利,等. PinX1敲减对乳腺MCF-10A细胞端粒稳定性的调控作用[J]. 中国科技论文在线精品论文,2014,7(17):1723-1729.
 
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