發(fā)布時(shí)間：2018-03-19 03:16

  本文選題：核旁斑點(diǎn)結(jié)構(gòu)蛋白1　切入點(diǎn)：DNA損傷應(yīng)激反應(yīng)　出處：《浙江大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：核旁斑點(diǎn)結(jié)構(gòu)蛋白1(paraspeckle protein1, PSPC1)是第一個(gè)被發(fā)現(xiàn)的核旁斑點(diǎn)(paraspeckle)的結(jié)構(gòu)蛋白,但是到目前為止,PSPC1的功能尚不清楚。在我們的前期蛋白組學(xué)研究過程中發(fā)現(xiàn),化療藥物順鉑(cisplatin)染毒HeLa細(xì)胞后PSPC1蛋白水平增加。這一現(xiàn)象提示,PSPC1可能參與順鉑誘導(dǎo)的DNA損傷應(yīng)激反應(yīng)過程(DNA damage response, DDR)。因此,在本研究中,我們研究探討了PSPC1在DDR中發(fā)揮的生物學(xué)功能。首先,我們明確了在HeLa細(xì)胞中,不但順鉑可以誘導(dǎo)PSPC1蛋白水平增加,而且D NA損傷劑(DNA damaging agent)甲磺酸甲酯(Methyl methanesulfonate, MMS)也可以誘導(dǎo)PSPC1蛋白水平增加。如果通過小RNA干擾(siRNA)抑制HeLa細(xì)胞內(nèi)PSPC1蛋白表達(dá),不僅細(xì)胞生長被抑制,而且細(xì)胞凋亡、細(xì)胞的活性氧(reactive oxygen species, ROS)水平、細(xì)胞的DNA損傷程度都有顯著增加,這一結(jié)果表明PSPC1很可能參與了細(xì)胞的DDR。但是進(jìn)一步研究發(fā)現(xiàn)PSPC1不與DDR中的重要蛋白yH2AX共定位,也不與同源重組修復(fù)(homologous recombination repair, HR)中的核心蛋白p53結(jié)合蛋白1(p53binding protein1,53BP1)或RAD51共定位。此外,在PSPC1缺失細(xì)胞中DNA修復(fù)的動(dòng)力學(xué)過程沒有發(fā)生明顯變化。以上結(jié)果表明PSPC1可能并不直接參與上述3個(gè)蛋白介導(dǎo)的DNA修復(fù)過程。但有趣的是,PSPC1蛋白表達(dá)被抑制后,正常的細(xì)胞周期被破壞,細(xì)胞更多的會(huì)被阻滯在G2/M期。如果在順鉑誘導(dǎo)的處于G1/S期阻滯的HeLa細(xì)胞中抑制PSPC1表達(dá),將會(huì)誘導(dǎo)細(xì)胞逃逸G1/S周期檢驗(yàn)點(diǎn),導(dǎo)致細(xì)胞進(jìn)入G2/M期；而在MMS誘導(dǎo)的G2/M期阻滯的細(xì)胞中,降低PSPC1表達(dá)水平則會(huì)增強(qiáng)G2/M期阻滯,兩種情況最終都導(dǎo)致更多的細(xì)胞死亡；而高表達(dá)PSPC1后,更多的細(xì)胞被阻滯在S期,進(jìn)入G2/M期的細(xì)胞數(shù)量減少。免疫共沉淀結(jié)果顯示,PSPC1可以與ATR相互作用蛋白(ATR-interaction protein, ATRIP)相互作用,提示PSPC1很可能通過與ATRIP相互作用參與細(xì)胞周期調(diào)控過程�？傊�,本研究揭示了PSPC1在DDR中可能的生物學(xué)功能,即通過調(diào)節(jié)G1/S周期檢驗(yàn)點(diǎn)影響DDR過程。在細(xì)胞DNA損傷時(shí),PSPC1蛋白水平增加從而導(dǎo)致細(xì)胞阻滯在S期,利于DNA修復(fù)的進(jìn)行；而當(dāng)PSPC1表達(dá)缺失時(shí),細(xì)胞無法阻滯在S期而進(jìn)入G2/M期,DNA無法被修復(fù)而導(dǎo)致?lián)p傷程度增高,最終導(dǎo)致更多的細(xì)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R114

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Hope E.Hamrick;Penelope J.Duerksen—Hughes;;ATM and ATR:Sensing DNA damage[J];World Journal of Gastroenterology;2004年02期

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本文編號(hào)：1632582