癌变·畸变·突变2024，Vol.36Issue(5) ：395-398.DOI:10.3969/j.issn.1004-616x.2024.05.010

采用MS2 pulldown和质谱分析法检测BT549细胞中的LncRNA SNHG15结合蛋白

Identification of LncRNA SNHG15 binding proteins in BT549 cells using MS2 pulldown and mass spectrometry

陈少英 彭佩 周艳春 顾巍
癌变·畸变·突变2024，Vol.36Issue(5) ：395-398.DOI:10.3969/j.issn.1004-616x.2024.05.010
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  • 万方数据

采用MS2 pulldown和质谱分析法检测BT549细胞中的LncRNA SNHG15结合蛋白

Identification of LncRNA SNHG15 binding proteins in BT549 cells using MS2 pulldown and mass spectrometry

陈少英 1彭佩 1周艳春 1顾巍1
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作者信息

  • 1. 汕头大学医学院病理生理学教研室,广东 汕头 515041
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摘要

目的:鉴定乳腺癌BT549细胞中与长链非编码RNA SNHG15结合的蛋白.方法:用慢病毒感染乳腺癌BT549细胞,构建稳定过表达SNHG15的BT549-SNHG15-MS2细胞株.通过MS2 pulldown和质谱分析法,检测BT549细胞中与SNHG15结合的蛋白.随后采用RNA免疫共沉淀(RIP)和逆转录-定量PCR(RT-qPCR)法,以HEAD框肽5(DEAD box helicases 5,DDX5)为模板验证共沉淀蛋白和SNHG15的结合.结果:经MS2 pulldown和质谱分析筛选出386个潜在的SNHG15结合蛋白.RNA免疫沉淀结合RT-qPCR检测结果证实DDX5与SNHG15的结合.结论:本研究介绍了一种有效分析RNA-蛋白质相互作用的生物学方法,同时为深入研究SNHG15的作用机制提供了新的实验证据.

Abstract

OBJECTIVE:To identify proteins which interacted with long noncoding RNA SNHG15 in breast cancer cells.METHODS:BT549 cells stably overexpressing SNHG15 were established by infection with lentivirus expressing MS2bs-tagged SHNG15.MS2 pulldown and Mass Spectrometry were used to identify proteins associated with SNHG15.Using DDX5(DEAD box helicase 5)as an example,interactions of SHNG15 with identified proteins were further verified by RNA immunoprecipitation(RIP)and RT-qPCR.RESULTS:In total,386 proteins were identified which interacted with SNHG15.DDX5 was truly associated with SNHG15.CONCLUSION:These data indicated an effective method to purify RNA-associated proteins and provided new information for the further study of the function of SNHG15.

关键词

长链非编码RNA/MS2/pulldown/小核仁RNA宿主基因15/RNA免疫沉淀/RNA结合蛋白

Key words

long noncoding RNA/MS2 pulldown/SNHG15/RNA immunoprecipitation/RNA binding protein

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出版年

2024
癌变·畸变·突变
中国环境诱变剂学会

癌变·畸变·突变

CSTPCD
影响因子：0.35
ISSN：1004-616X
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