Gadd45a蛋白與長鏈非編碼RNA相互作用的分子機制研究
發(fā)布時間：2018-11-29 09:54
【摘要】：非編碼RNA (non-coding RNA, ncRNA)是一類蛋白編碼功能極低或者不具有蛋白編碼潛能的轉(zhuǎn)錄本,長度超過200bp的ncRNA為長鏈非編碼RNA(long non-coding RNA, lncRNA).目前研究發(fā)現(xiàn)lncRNA主要通過與蛋白(多數(shù)為RNA結(jié)合蛋白)、DNA分子或者RNA分子相互作用,在基因轉(zhuǎn)錄及轉(zhuǎn)錄后調(diào)控、翻譯及翻譯后調(diào)控等多個層次發(fā)揮重要調(diào)節(jié)作用,維持細胞的生物學(xué)過程。越來越多的研究發(fā)現(xiàn),長鏈非編碼RNA與多種疾病,尤其是腫瘤相關(guān)。Gadd45a (growth arrest and DNA damage,生長阻滯和DNA損傷基因)是Gadd蛋白家族成員之一。Gadd45a蛋白是一種應(yīng)激蛋白,多種應(yīng)激情況均可誘導(dǎo)Gadd45a蛋白的表達。Gadd45a蛋白主要通過與其他蛋白相互作用,參與細胞周期調(diào)控、DNA損傷修復(fù)及細胞凋亡等。Gadd45a蛋白在多種腫瘤中低表達,通過抑制細胞生長、侵襲轉(zhuǎn)移、腫瘤血管形成等功能,抑制腫瘤發(fā)生發(fā)展。近年研究發(fā)現(xiàn)Gadd45a蛋白也可以與RNA結(jié)合,但是究竟與哪些RNA,尤其是lncRNA結(jié)合,與RNA結(jié)合后參與什么生物學(xué)過程研究尚少。本研究在結(jié)腸癌細胞系中利用RNA-蛋白免疫共沉淀(RNA immunoprecipitation,RIP)測序方法,探討與Gadd45a蛋白結(jié)合的RNA,特別是lncRNA。測序結(jié)果中包括72條長鏈非編碼RNA,我們從中挑選了lncRNA Growth arrest-specific 5(GAS5)作為繼續(xù)研究的對象。首先用RIP-PCR及RNA pull down實驗驗證Gadd45a蛋白可以與lncRNA GAS5結(jié)合。LncRNA GAS5并不能影響Gadd45a蛋白在細胞中的含量及分布,但是進一步運用過表達和敲降的研究策略,通過免疫共沉淀聯(lián)合銀染實驗,得出lncRNA GAS5可以介導(dǎo)Gadd45a蛋白與HSP90蛋白的結(jié)合。Gadd45a蛋白與HSP90蛋白結(jié)合后,可以競爭性抑制該蛋白與CDC37蛋白的結(jié)合。同樣,lncRNAGAS5也可通過Gadd45a蛋白抑制HSP90蛋白與CDC37蛋白的結(jié)合。HSP90蛋白在99例結(jié)腸癌組織及81例正常組織中的表達檢測結(jié)果顯示該蛋白在腫瘤組織中明顯高表達,并且還可以作為結(jié)腸癌一個獨立的預(yù)后相關(guān)因子。綜上所述,本研究通過RIP測出方法發(fā)現(xiàn)并驗證了一系列可以與Gadd45a蛋白相互作用的mRNA及非編碼RNA,尤其是長鏈非編碼RNA.。Gadd45a蛋白可以與lncRNA GAS5相互作用,并且在lncRNA GAS5的協(xié)助下與HSP90蛋白結(jié)合,從而競爭性抑制該蛋白與其輔助伴侶分子CDC37蛋白的結(jié)合,發(fā)揮類似HSP90蛋白抑制劑的功能,抑制腫瘤細胞的生長。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.2
，
本文編號：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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.2
，
本文編號：2364678
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2364678.html
最近更新
教材專著
