Gadd45a蛋白与长链非编码RNA相互作用的分子机制研究
发布时间：2018-11-29 09:54
【摘要】：非编码RNA (non-coding RNA, ncRNA)是一类蛋白编码功能极低或者不具有蛋白编码潜能的转录本,长度超过200bp的ncRNA为长链非编码RNA(long non-coding RNA, lncRNA).目前研究发现lncRNA主要通过与蛋白(多数为RNA结合蛋白)、DNA分子或者RNA分子相互作用,在基因转录及转录后调控、翻译及翻译后调控等多个层次发挥重要调节作用,维持细胞的生物学过程。越来越多的研究发现,长链非编码RNA与多种疾病,尤其是肿瘤相关。Gadd45a (growth arrest and DNA damage,生长阻滞和DNA损伤基因)是Gadd蛋白家族成员之一。Gadd45a蛋白是一种应激蛋白,多种应激情况均可诱导Gadd45a蛋白的表达。Gadd45a蛋白主要通过与其他蛋白相互作用,参与细胞周期调控、DNA损伤修复及细胞凋亡等。Gadd45a蛋白在多种肿瘤中低表达,通过抑制细胞生长、侵袭转移、肿瘤血管形成等功能,抑制肿瘤发生发展。近年研究发现Gadd45a蛋白也可以与RNA结合,但是究竟与哪些RNA,尤其是lncRNA结合,与RNA结合后参与什么生物学过程研究尚少。本研究在结肠癌细胞系中利用RNA-蛋白免疫共沉淀(RNA immunoprecipitation,RIP)测序方法,探讨与Gadd45a蛋白结合的RNA,特别是lncRNA。测序结果中包括72条长链非编码RNA,我们从中挑选了lncRNA Growth arrest-specific 5(GAS5)作为继续研究的对象。首先用RIP-PCR及RNA pull down实验验证Gadd45a蛋白可以与lncRNA GAS5结合。LncRNA GAS5并不能影响Gadd45a蛋白在细胞中的含量及分布,但是进一步运用过表达和敲降的研究策略,通过免疫共沉淀联合银染实验,得出lncRNA GAS5可以介导Gadd45a蛋白与HSP90蛋白的结合。Gadd45a蛋白与HSP90蛋白结合后,可以竞争性抑制该蛋白与CDC37蛋白的结合。同样,lncRNAGAS5也可通过Gadd45a蛋白抑制HSP90蛋白与CDC37蛋白的结合。HSP90蛋白在99例结肠癌组织及81例正常组织中的表达检测结果显示该蛋白在肿瘤组织中明显高表达,并且还可以作为结肠癌一个独立的预后相关因子。综上所述,本研究通过RIP测出方法发现并验证了一系列可以与Gadd45a蛋白相互作用的mRNA及非编码RNA,尤其是长链非编码RNA.。Gadd45a蛋白可以与lncRNA GAS5相互作用,并且在lncRNA GAS5的协助下与HSP90蛋白结合,从而竞争性抑制该蛋白与其辅助伴侣分子CDC37蛋白的结合,发挥类似HSP90蛋白抑制剂的功能,抑制肿瘤细胞的生长。
[Abstract]:Non-coding RNA (non-coding RNA, ncRNA) is a class of transcripts with very low protein coding function or no protein coding potential, and ncRNA with a length longer than 200bp is a long-chain non-coding RNA (long non-coding RNA, lncRNA). At present, it has been found that lncRNA plays an important role in gene transcription and posttranscriptional regulation, translation and posttranslational regulation by interacting with protein (mostly RNA binding protein), DNA molecule or RNA molecule). Maintain the biological process of the cell. A growing number of studies have found that long chain noncoding RNA is a member of the Gadd protein family and is associated with many diseases, especially tumor-related growth arrest and DNA damage genes. Gadd45a protein is a stress protein. The expression of Gadd45a protein can be induced by various stress conditions. Gadd45a protein is mainly involved in cell cycle regulation, DNA damage repair and cell apoptosis by interacting with other proteins. Gadd45a protein is expressed in many kinds of tumors by inhibiting cell growth. Invasion and metastasis, tumor angiogenesis and other functions, inhibit tumor development. In recent years, it has been found that Gadd45a protein can bind to RNA, but there are few studies on which RNA, especially lncRNA, and what biological processes are involved after binding to RNA. In this study, RNA- protein immunoprecipitation (RNA immunoprecipitation,RIP) sequencing was used in colon cancer cell lines to investigate RNA, binding to Gadd45a protein, especially lncRNA.. The sequencing results included 72 long chain noncoding RNA, from which lncRNA Growth arrest-specific 5 (GAS5) was selected for further study. Firstly, RIP-PCR and RNA pull down experiments were used to verify that Gadd45a protein could bind to lncRNA GAS5. LncRNA GAS5 did not affect the content and distribution of Gadd45a protein in cells, but it further applied the strategy of over-expression and knockdown. By immunoprecipitation combined with silver staining, it was concluded that lncRNA GAS5 could mediate the binding of Gadd45a protein to HSP90 protein, and that Gadd45a protein could competitively inhibit the binding of Gadd45a protein to CDC37 protein after binding with HSP90 protein. Similarly, lncRNAGAS5 could inhibit the binding of HSP90 protein to CDC37 protein through Gadd45a protein. The expression of HSP90 protein in 99 cases of colon cancer and 81 cases of normal tissues showed that the expression of HSP90 protein was significantly higher in tumor tissues. It can also be used as an independent prognostic factor for colon cancer. In conclusion, a series of mRNA and non-coding RNA, that can interact with Gadd45a protein, especially long chain non-coding RNA..Gadd45a protein, can interact with lncRNA GAS5 by RIP method. With the help of lncRNA GAS5, the protein binds to HSP90 protein, which competitively inhibits the binding of the protein to its cocompanion molecule, CDC37 protein, and plays a role similar to HSP90 protein inhibitor and inhibits the growth of tumor cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2
，
本文编号：2364678
[Abstract]:Non-coding RNA (non-coding RNA, ncRNA) is a class of transcripts with very low protein coding function or no protein coding potential, and ncRNA with a length longer than 200bp is a long-chain non-coding RNA (long non-coding RNA, lncRNA). At present, it has been found that lncRNA plays an important role in gene transcription and posttranscriptional regulation, translation and posttranslational regulation by interacting with protein (mostly RNA binding protein), DNA molecule or RNA molecule). Maintain the biological process of the cell. A growing number of studies have found that long chain noncoding RNA is a member of the Gadd protein family and is associated with many diseases, especially tumor-related growth arrest and DNA damage genes. Gadd45a protein is a stress protein. The expression of Gadd45a protein can be induced by various stress conditions. Gadd45a protein is mainly involved in cell cycle regulation, DNA damage repair and cell apoptosis by interacting with other proteins. Gadd45a protein is expressed in many kinds of tumors by inhibiting cell growth. Invasion and metastasis, tumor angiogenesis and other functions, inhibit tumor development. In recent years, it has been found that Gadd45a protein can bind to RNA, but there are few studies on which RNA, especially lncRNA, and what biological processes are involved after binding to RNA. In this study, RNA- protein immunoprecipitation (RNA immunoprecipitation,RIP) sequencing was used in colon cancer cell lines to investigate RNA, binding to Gadd45a protein, especially lncRNA.. The sequencing results included 72 long chain noncoding RNA, from which lncRNA Growth arrest-specific 5 (GAS5) was selected for further study. Firstly, RIP-PCR and RNA pull down experiments were used to verify that Gadd45a protein could bind to lncRNA GAS5. LncRNA GAS5 did not affect the content and distribution of Gadd45a protein in cells, but it further applied the strategy of over-expression and knockdown. By immunoprecipitation combined with silver staining, it was concluded that lncRNA GAS5 could mediate the binding of Gadd45a protein to HSP90 protein, and that Gadd45a protein could competitively inhibit the binding of Gadd45a protein to CDC37 protein after binding with HSP90 protein. Similarly, lncRNAGAS5 could inhibit the binding of HSP90 protein to CDC37 protein through Gadd45a protein. The expression of HSP90 protein in 99 cases of colon cancer and 81 cases of normal tissues showed that the expression of HSP90 protein was significantly higher in tumor tissues. It can also be used as an independent prognostic factor for colon cancer. In conclusion, a series of mRNA and non-coding RNA, that can interact with Gadd45a protein, especially long chain non-coding RNA..Gadd45a protein, can interact with lncRNA GAS5 by RIP method. With the help of lncRNA GAS5, the protein binds to HSP90 protein, which competitively inhibits the binding of the protein to its cocompanion molecule, CDC37 protein, and plays a role similar to HSP90 protein inhibitor and inhibits the growth of tumor cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2
，
本文编号：2364678
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