核旁斑点结构蛋白1（PSPC1）在DNA损伤应激反应中的生物学功能研究

发布时间：2018-03-19 03:16

本文选题：核旁斑点结构蛋白1　切入点：DNA损伤应激反应　出处：《浙江大学》2014年博士论文　论文类型：学位论文

【摘要】：核旁斑点结构蛋白1(paraspeckle protein1, PSPC1)是第一个被发现的核旁斑点(paraspeckle)的结构蛋白,但是到目前为止,PSPC1的功能尚不清楚。在我们的前期蛋白组学研究过程中发现,化疗药物顺铂(cisplatin)染毒HeLa细胞后PSPC1蛋白水平增加。这一现象提示,PSPC1可能参与顺铂诱导的DNA损伤应激反应过程(DNA damage response, DDR)。因此,在本研究中,我们研究探讨了PSPC1在DDR中发挥的生物学功能。首先,我们明确了在HeLa细胞中,不但顺铂可以诱导PSPC1蛋白水平增加,而且D NA损伤剂(DNA damaging agent)甲磺酸甲酯(Methyl methanesulfonate, MMS)也可以诱导PSPC1蛋白水平增加。如果通过小RNA干扰(siRNA)抑制HeLa细胞内PSPC1蛋白表达,不仅细胞生长被抑制,而且细胞凋亡、细胞的活性氧(reactive oxygen species, ROS)水平、细胞的DNA损伤程度都有显著增加,这一结果表明PSPC1很可能参与了细胞的DDR。但是进一步研究发现PSPC1不与DDR中的重要蛋白yH2AX共定位,也不与同源重组修复(homologous recombination repair, HR)中的核心蛋白p53结合蛋白1(p53binding protein1,53BP1)或RAD51共定位。此外,在PSPC1缺失细胞中DNA修复的动力学过程没有发生明显变化。以上结果表明PSPC1可能并不直接参与上述3个蛋白介导的DNA修复过程。但有趣的是,PSPC1蛋白表达被抑制后,正常的细胞周期被破坏,细胞更多的会被阻滞在G2/M期。如果在顺铂诱导的处于G1/S期阻滞的HeLa细胞中抑制PSPC1表达,将会诱导细胞逃逸G1/S周期检验点,导致细胞进入G2/M期；而在MMS诱导的G2/M期阻滞的细胞中,降低PSPC1表达水平则会增强G2/M期阻滞,两种情况最终都导致更多的细胞死亡；而高表达PSPC1后,更多的细胞被阻滞在S期,进入G2/M期的细胞数量减少。免疫共沉淀结果显示,PSPC1可以与ATR相互作用蛋白(ATR-interaction protein, ATRIP)相互作用,提示PSPC1很可能通过与ATRIP相互作用参与细胞周期调控过程。总之,本研究揭示了PSPC1在DDR中可能的生物学功能,即通过调节G1/S周期检验点影响DDR过程。在细胞DNA损伤时,PSPC1蛋白水平增加从而导致细胞阻滞在S期,利于DNA修复的进行；而当PSPC1表达缺失时,细胞无法阻滞在S期而进入G2/M期,DNA无法被修复而导致损伤程度增高,最终导致更多的细胞走向死亡。
[Abstract]:Paraphoric speckle protein 1 (PSPC1) is the first structural protein of paraspeckle protein 1 (PSPC1), but the function of PSPC1 is not clear. The increase of PSPC1 protein level in HeLa cells induced by cisplatin suggests that PSPC1 may be involved in the stress response of DNA damage induced by cisplatin. We studied the biological function of PSPC1 in DDR. Firstly, we confirmed that not only cisplatin can induce the increase of PSPC1 protein level in HeLa cells, DNA damaging damaging methyl methanesulfonate (MMS) can also induce an increase in the level of PSPC1 protein. If the expression of PSPC1 protein in HeLa cells is inhibited by small RNA interference, not only cell growth is inhibited, but also apoptosis is induced. The level of reactive oxygen speciesin cells and the degree of DNA damage in cells were significantly increased, which suggested that PSPC1 might be involved in DDR of cells. However, further studies showed that PSPC1 did not co-locate with yH2AX, an important protein in DDR. Nor were they co-located with the core protein p53 binding protein 1 p53 binding protein 1 (53 BP1) or RAD51 in homologous recombination repair. in addition, There was no significant change in the dynamic process of DNA repair in PSPC1 deletion cells. These results suggest that PSPC1 may not be directly involved in the DNA repair process mediated by the above three proteins, but it is interesting to note that the expression of PSPC1 protein is inhibited. The normal cell cycle was destroyed and more cells were blocked in G2 / M phase. If the PSPC1 expression was inhibited in the Cisplatin induced G1 / S phase block HeLa cells, it would induce the cells to escape the G1 / S cycle test point and induce the cells to enter the G2 / M phase. In G2 / M phase block cells induced by MMS, the decrease of PSPC1 expression increased G2 / M phase arrest, both of which eventually led to more cell death, and more cells were blocked in S phase after high PSPC1 expression. The results of immunoprecipitation showed that PSPC1 could interact with ATR interacting protein ATR-interaction protein (ATRIPP), suggesting that PSPC1 might be involved in cell cycle regulation by interacting with ATRIP. This study revealed the possible biological function of PSPC1 in DDR, that is, by regulating the G 1 / S cycle test point, the DDR process was affected. During the DNA injury, the protein level of pPSPC1 increased, resulting in cell arrest in S phase, which was conducive to the repair of DNA. When the expression of PSPC1 was absent, the cells could not be blocked in S phase and could not be repaired into the G _ 2 / M phase, which led to the increase of the degree of damage, and eventually to the death of more cells.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

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