【摘要】：目的研究泛素化酶CRL4蛋白復(fù)合體家族成員CRL4-WD40重復(fù)序列結(jié)構(gòu)域蛋白70(WDR70)在卵巢癌細(xì)胞中的DNA修復(fù)功能,以及卵巢癌組織中該泛素化酶基因的突變規(guī)律。方法利用免疫熒光方法,檢測(cè)CRL4骨架蛋白DDB1及WDR70基因特異性沉默的卵巢癌細(xì)胞與其對(duì)應(yīng)的對(duì)照組細(xì)胞在化療藥物或放射線照射誘導(dǎo)產(chǎn)生DNA雙鏈斷裂后,組蛋白H2AX(γH2AX)及單鏈DNA結(jié)合蛋白32(RPA32)磷酸化灶點(diǎn)顯示的差異;BrdU標(biāo)記和染色實(shí)驗(yàn)檢測(cè)WDR70基因?qū)NA復(fù)制是否存在影響,同時(shí)利用免疫組化染色檢測(cè)卵巢癌組織臨床病理標(biāo)本及正常卵巢組織標(biāo)本中的WDR70和組蛋白H2B單泛素化(uH2B)染色差異,以闡明CRL4的DNA損傷應(yīng)答特征,RT-PCR測(cè)定卵巢癌組織中WDR70的基因表達(dá)水平,并采用DNA測(cè)序確定WDR70突變位點(diǎn)。結(jié)果免疫熒光染色結(jié)果顯示,CRL4-WDR70的不同蛋白亞基(DDB1、WDR70)在細(xì)胞周期檢驗(yàn)點(diǎn)激活和uH2B介導(dǎo)的DNA末端回切過(guò)程中起著不同的作用:DDB1參與以上兩個(gè)機(jī)制的調(diào)控,而WDR70只促進(jìn)末端回切、RPA32在DNA斷裂點(diǎn)的招募和同源重組修復(fù)。BrdU標(biāo)記和染色結(jié)果顯示W(wǎng)DR70基因?qū)NA復(fù)制并不存在影響。免疫組化結(jié)果顯示,卵巢癌組織臨床病理標(biāo)本及正常卵巢組織標(biāo)本中的WDR70和uH2B表達(dá)存在差異。RT-PCR結(jié)果顯示W(wǎng)DR70基因的全長(zhǎng)、5′和3′轉(zhuǎn)錄本水平在50%的卵巢癌組織中水平減低,出現(xiàn)多處外顯子突變位點(diǎn)。結(jié)論 CRL4在DNA修復(fù)過(guò)程中具有促進(jìn)H2B單泛素化、促進(jìn)DNA末端回切和激活細(xì)胞周期檢驗(yàn)點(diǎn)等多種重要功能,是維持基因組穩(wěn)定性、遏阻卵巢癌發(fā)生的重要抗癌機(jī)制。
[Abstract]:Objective to study the DNA repair function of CRL4-WD40 repeat domain protein 70 (WDR70), a member of the Ubiquitin CRL4 protein complex family, in ovarian cancer cells and the mutation rule of the ubiquitin gene in ovarian cancer cells. Methods CRL4 skeleton protein DDB1 and WDR70 gene specific silencing ovarian cancer cells and their corresponding control cells were detected by immunofluorescence method after DNA double strand breaks were induced by chemotherapeutic drugs or irradiation. The difference of phosphorylation sites between histone H2AX (緯 H2AX) and single-stranded DNA binding protein 32 (RPA32) was observed. The effect of WDR70 gene on DNA replication was detected by BrdU labeling and staining. Immunohistochemical staining was used to detect the difference between WDR70 and histone H2B monoubiquitin (uH2B) staining in clinicopathological specimens of ovarian cancer and normal ovarian tissues. In order to elucidate the characteristics of DNA damage response of CRL4, RT-PCR was used to detect the expression of WDR70 gene in ovarian cancer tissues, and DNA sequencing was used to determine the WDR70 mutation site. Results the results of immunofluorescence staining showed that different protein subunits (DDB1,WDR70) of CRL4-WDR70 played different roles in the activation of cell cycle detection points and DNA terminal recutting mediated by uH2B. DDB1 was involved in the regulation of these two mechanisms. However, WDR70 only promoted terminal reverse cutting, RPA32 recruitment at DNA break point and homologous recombination repair. The results of BrdU labeling and staining showed that WDR70 gene had no effect on DNA replication. Immunohistochemical results showed that the expression of WDR70 and uH2B were different between clinicopathological specimens of ovarian cancer and normal ovarian tissues. RT-PCR results showed the full length of WDR70 gene. The levels of 5 'and 3' transcripts were decreased in 50% of ovarian cancer tissues, and several exon mutation sites were found. Conclusion CRL4 has many important functions in the process of DNA repair, such as promoting monoubiquitization of H2B, promoting DNA terminal reversion and activating cell cycle test points. It is an important anticancer mechanism to maintain genomic stability and inhibit the occurrence of ovarian cancer.
【作者單位】： 四川大學(xué)華西第二醫(yī)院婦產(chǎn)科;出生缺陷與相關(guān)婦兒疾病教育部重點(diǎn)實(shí)驗(yàn)室(四川大學(xué));
【基金】：四川省科技廳應(yīng)用基礎(chǔ)計(jì)劃項(xiàng)目(No.2017FZ0034) 四川省科技廳軟科學(xué)計(jì)劃項(xiàng)目(No.2017ZR0169)資助
【分類號(hào)】：R737.31

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