利用feeding RNAi技术筛选线虫体内低氧应答剪接相关因子的研究
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摘要
高等哺乳动物的基因表达调控主要包括转录前、转录中和转录后三个水平, mRNA前体的剪接加工就是转录后水平调控基因表达的一种重要机制。可变剪接是指从一个mRNA前体中通过不同的剪接方式(选择不同的剪接位点组合)产生不同的mRNA剪接异构体的过程。Pre-mRNA剪接的变异和相关因子的突变是造成人类疾病的一大原因。
低氧应答是指机体在低于正常氧气浓度下产生的应对低氧损伤的一系列应答机制,以确保机体适应低氧环境并对更严重的缺氧压力产生保护作用。研究证明,秀丽线虫的低氧诱导因子HIF-1的恒定性调控通路和人类的相应通路之间具有高度保守性,低氧也可以引发细胞损伤和死亡,启动一系列细胞应答以减轻缺氧造成的损伤。有报道指出,线虫13%的基因具有可变剪接形式,人类的多数剪接因子在线虫中都能够找到同源基因。另有报道发现细胞低氧应答通路中的转录因子——低氧诱导因子HIF-1α的一种可变剪接可对HIF-1α起负性调控作用。但可变剪接机制作为一种重要的基因表达调控手段,在低氧应答中的作用及可能的调节机制到底如何,目前仍然所知甚少。
RNAi是指外源的dsRNA可特异地使机体内与之序列互补的内源基因失活的一种基因沉默机制。Feeding RNAi筛选技术就是利用RNA干涉原理,通过给线虫喂食表达外源的dsRNA的RNAi菌种达到沉默靶标基因的目的。
本研究利用物理缺氧小室,在O2浓度为0.2%的条件下,检测到了缺氧在细胞水平和行为学水平对线虫的损伤。并且在前人的基础上不断优化本实验室线虫物理缺氧模型,最终采用浸泡法,成功构建了稳定的线虫物理缺氧耐受性检测体系。通过绘制不同时间点线虫缺氧死亡率曲线,确定了野生型线虫N2低氧条件下的半数死亡期为11h。在此基础上,利用feeding RNAi技术,以线虫体内350个文库中含有的RBP编码蛋白为筛选对象,成功筛选出了6个参与低氧的RBP编码基因;同时,以已知参与低氧的具有可变剪接形式的基因为筛选对象,成功筛选出了4个可能的低氧应答剪接因子,并通过基因序列检测和功能分析初步揭示了这些阳性基因参与低氧应答的可能机制及与已知靶基因的潜在联系,为缺氧损伤的预防治疗及调控机制提供了重要线索,有助于从基因转录表达水平和可变剪接调控水平了解缺血缺氧相关的多种疾病(如心脑血管疾病)的分子机制、临床治疗及新药开发。
The gene expression of advanced mammalian includes three levels: pre-transcription, transcription and post-transcription. The splicing of pre-mRNA is an important mechanism to regulate gene expression in post-transcriptional levels. Alternative splicing is defined as a process that pre-mRNA can be spliced in different ways (choose a different combination of splice sites) to product different isomers of mRNA splicing.
Hypoxia is a mechanism of responsive regulation when the O2 concentration is below normoxia, it is purposed to protect body from lacking of O2. Studies have shown that it is highly conserved between the constant regulation pathway of hypoxia inducible factor-1, HIF-1(HIF-1) in C.elegans and the corresponding human pathway. Hypoxia can lead to cell injury and death and start a series of cell responses in order to reduce the damage caused by hypoxia. It is also reported that 13% genes in C.elegans have alternative splicing forms, the majority of human splicing factor can find homologous gene in C.elegans. It has been reported that an alternative splicing of the transcription factor of the hypoxic response pathway (hypoxia inducible factor-1α, HIF-1α) was found playing a negative role in regulation of HIF-1α. However, it is still known very little about what role the alternative splicing mechanism as an important means of gene expression and the possible regulatory mechanism in hypoxic response pathway.
RNAi refers to a gene-silencing mechanism that exogenous dsRNA may combine specially with the complementary sequence in endogenous gene so that inactivate it. Feeding RNAi screening technology is to use the RNA interference principle, feeding worms the RNAi strains which express the dsRNA and contain a specific gene fragment, in order to silence the target gene and clear the gene specific functions.
This study used hypoxic chamber, set the O2 concentration at 0.2%, detected the hypoxic phenotype of C.elegans from cell level and behavior research. Based on the formers’study, we optimized our experiment condition, and finally through socking worms in 1ⅹM9 buffer, we successfully built a physical hypoxic tolerance detection system. By protracting time mortality curves of physical hypoxic in C.elegans, confirmed 11 hours is the half hypoxic death percent of C.elegans. Based on this, using feeding RNAi technology, aimed at 350 RBP genes and 20 potential hypoxic related splicing factors contained in RNAi library of C.elegans, we screened 6 hypoxia-related encoding RBP genes and 4 the potential hypoxic related splicing factors from C.elegans by feeding RNAi technology. After sequencing and detecting the positive genes, we began to reveal the mechanism what these positive genes may be involved in hypoxia response and the potential relationships with the known target genes through analyzing gene function. It has provided an important clue to the prevention and treatment for hypoxic injury and understanding the regulatory mechanism. It will be helpful to clinical treatment and new drug development, and it also will be helpful to understand the mechanisms of a variety of hypoxia-ischemia-related diseases (such as cardio-cerebral vascular diseases) in the gene expression from transcription and alternative splicing regulatory level.
低氧应答是指机体在低于正常氧气浓度下产生的应对低氧损伤的一系列应答机制,以确保机体适应低氧环境并对更严重的缺氧压力产生保护作用。研究证明,秀丽线虫的低氧诱导因子HIF-1的恒定性调控通路和人类的相应通路之间具有高度保守性,低氧也可以引发细胞损伤和死亡,启动一系列细胞应答以减轻缺氧造成的损伤。有报道指出,线虫13%的基因具有可变剪接形式,人类的多数剪接因子在线虫中都能够找到同源基因。另有报道发现细胞低氧应答通路中的转录因子——低氧诱导因子HIF-1α的一种可变剪接可对HIF-1α起负性调控作用。但可变剪接机制作为一种重要的基因表达调控手段,在低氧应答中的作用及可能的调节机制到底如何,目前仍然所知甚少。
RNAi是指外源的dsRNA可特异地使机体内与之序列互补的内源基因失活的一种基因沉默机制。Feeding RNAi筛选技术就是利用RNA干涉原理,通过给线虫喂食表达外源的dsRNA的RNAi菌种达到沉默靶标基因的目的。
本研究利用物理缺氧小室,在O2浓度为0.2%的条件下,检测到了缺氧在细胞水平和行为学水平对线虫的损伤。并且在前人的基础上不断优化本实验室线虫物理缺氧模型,最终采用浸泡法,成功构建了稳定的线虫物理缺氧耐受性检测体系。通过绘制不同时间点线虫缺氧死亡率曲线,确定了野生型线虫N2低氧条件下的半数死亡期为11h。在此基础上,利用feeding RNAi技术,以线虫体内350个文库中含有的RBP编码蛋白为筛选对象,成功筛选出了6个参与低氧的RBP编码基因;同时,以已知参与低氧的具有可变剪接形式的基因为筛选对象,成功筛选出了4个可能的低氧应答剪接因子,并通过基因序列检测和功能分析初步揭示了这些阳性基因参与低氧应答的可能机制及与已知靶基因的潜在联系,为缺氧损伤的预防治疗及调控机制提供了重要线索,有助于从基因转录表达水平和可变剪接调控水平了解缺血缺氧相关的多种疾病(如心脑血管疾病)的分子机制、临床治疗及新药开发。
The gene expression of advanced mammalian includes three levels: pre-transcription, transcription and post-transcription. The splicing of pre-mRNA is an important mechanism to regulate gene expression in post-transcriptional levels. Alternative splicing is defined as a process that pre-mRNA can be spliced in different ways (choose a different combination of splice sites) to product different isomers of mRNA splicing.
Hypoxia is a mechanism of responsive regulation when the O2 concentration is below normoxia, it is purposed to protect body from lacking of O2. Studies have shown that it is highly conserved between the constant regulation pathway of hypoxia inducible factor-1, HIF-1(HIF-1) in C.elegans and the corresponding human pathway. Hypoxia can lead to cell injury and death and start a series of cell responses in order to reduce the damage caused by hypoxia. It is also reported that 13% genes in C.elegans have alternative splicing forms, the majority of human splicing factor can find homologous gene in C.elegans. It has been reported that an alternative splicing of the transcription factor of the hypoxic response pathway (hypoxia inducible factor-1α, HIF-1α) was found playing a negative role in regulation of HIF-1α. However, it is still known very little about what role the alternative splicing mechanism as an important means of gene expression and the possible regulatory mechanism in hypoxic response pathway.
RNAi refers to a gene-silencing mechanism that exogenous dsRNA may combine specially with the complementary sequence in endogenous gene so that inactivate it. Feeding RNAi screening technology is to use the RNA interference principle, feeding worms the RNAi strains which express the dsRNA and contain a specific gene fragment, in order to silence the target gene and clear the gene specific functions.
This study used hypoxic chamber, set the O2 concentration at 0.2%, detected the hypoxic phenotype of C.elegans from cell level and behavior research. Based on the formers’study, we optimized our experiment condition, and finally through socking worms in 1ⅹM9 buffer, we successfully built a physical hypoxic tolerance detection system. By protracting time mortality curves of physical hypoxic in C.elegans, confirmed 11 hours is the half hypoxic death percent of C.elegans. Based on this, using feeding RNAi technology, aimed at 350 RBP genes and 20 potential hypoxic related splicing factors contained in RNAi library of C.elegans, we screened 6 hypoxia-related encoding RBP genes and 4 the potential hypoxic related splicing factors from C.elegans by feeding RNAi technology. After sequencing and detecting the positive genes, we began to reveal the mechanism what these positive genes may be involved in hypoxia response and the potential relationships with the known target genes through analyzing gene function. It has provided an important clue to the prevention and treatment for hypoxic injury and understanding the regulatory mechanism. It will be helpful to clinical treatment and new drug development, and it also will be helpful to understand the mechanisms of a variety of hypoxia-ischemia-related diseases (such as cardio-cerebral vascular diseases) in the gene expression from transcription and alternative splicing regulatory level.
引文
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