miR-34a和PARP-1在氢醌致癌中蛋白质的乙酰化调控机制及其意义

Our previous study found that the apoptosis could be suppressed by decreased microRNA-34a(miR-34a)expression,the activation of myeloproliferative leukemia virusoncogene(MPL)could be induced by decreased DNA methyltransferase (DNMTs) in cells exposed to hydroquinone.In addition,the reciprocal modulation exist in ribosylation and phosphorylation. This results also showed that miR-34a and poly(ADP-ribose) polymerase-1（PARP-1）may have important function for protein acetylation in hydroquinone carcinogenesis.However,the relation between miRNA and protein acetylation as well as their reciprocal modulation in hydroquinone carcinogenesis are unclear.In this study, PARP-1 and silent information regulatorl（SIRT1）gene deficient TK6 human lymphoblastoid cell strains were established respectively by RNA interference（RNAi）technique,and the high expression of TK6 human lymphoblastoid cell line of PARP-1,SIRT1 and miR-34a genes would be constructed respectively. we applicate methods including report genic system,chromatin immunoprecipitation, acetylation analysis and cell transformation assay to study the modulation relation between miR-34a and PARP-1 as well as their effect on cells malignant phenotype.Meanwhile,we also research the space-time relationship of miR-34a and PARP-1 in biological effect, histone modification and tumor associated protein acetylation were induced by hydroquinone.We would elucidate the reciprocal modulation relationship of miR-34a and PARP-1 as well as their modulating the expression of tumor associated genes and tumour generation in hydroquinone carcinogenesis after this study are completed, and find out early and sensitive biomarker so as to provide information for earlier prevention hydroquinone carcinogenesis.

我们前期研究发现，氢醌可通过减少miR-34a的表达进而抑制细胞凋亡，也可通过降低DNMTs活性导致原癌基因MPL活化；另外，核糖基化与磷酸化间也存在相互调控,且提示miR-34a和PARP-1在氢醌致癌蛋白乙酰化中具有重要作用。然而，miRNA与蛋白乙酰化关系及其在氢醌致癌中的调控机制尚不明确。本项目拟通过建立PARP-1、SIRT1缺陷和高表达及miR-34a高表达细胞株，应用报告基因系统、染色质免疫沉淀、乙酰化分析等技术及细胞转化试验，研究miR-34a与PARP-1间的相互调控关系及其对细胞恶性表型的影响，探讨miR-34a和PARP-1在氢醌致癌中所致生物学效应及肿瘤相关蛋白乙酰化的关系。本研究有助于阐明miR-34a与PARP-1相互调控关系及其在氢醌所致蛋白质乙酰化中具有调控肿瘤相关基因表达和肿瘤形成的作用，并发现氢醌致癌早期敏感生物标志物，为早期预防氢醌致癌奠定理论基础。

苯是工业生产中非常重要的有机溶剂与原料，其毒性作用主要在于其代谢产物氢醌。近年来，表观遗传学已成为后基因组时代基因组功能研究的核心和热点之一。我们前期研究发现，PARP-1在氢醌致癌蛋白乙酰化中具有重要作用。然而，PARP-1与蛋白乙酰化关系及其在氢醌致癌中的调控机制尚不明确。为此我们开展了相关研究：①建立PARP-1、SIRT1缺陷和SIRT1、PARP-1、miR-34a 高表达细胞株以及生物学性状研究；②PARP-1、SIRT1基因对HQ毒作用影响研究；③建立HQ诱导的TK6细胞恶性转化模型并研究恶性转化各阶段细胞肿瘤相关基因的表达；④研究PARP-1在氢醌诱发的细胞恶性转化中作用及蛋白质乙酰化调控机制研究；⑤研究miR-7-5p和Bcl-2在氢醌诱导TK6细胞损伤作用；⑥PARP-1通过聚ADP核糖基化作用调节ZO-2参与氢醌诱导细胞凋亡；⑦SIRT1调控自噬在氢醌致TK6细胞毒性中的作用研究。应用慢病毒介导的RNA干扰技术构建稳定的人淋巴母细胞SIRT1、PARP-1缺陷及SIRT1高表达TK6细胞株和HQ诱导的TK6细胞恶性转化模型，应用报告基因系统、染色质免疫沉淀、乙酰化分析等技术及细胞转化试验，结果发现：SIRT1、PARP-1缺陷均可增加TK6细胞对HQ的敏感性，特异性高表达SIRT1基因可降低TK6细胞对HQ的敏感性，PARP-1对HQ诱导细胞凋亡具有重要作用；HQ染毒TK6细胞过程中，SIRT1可能通过PARP-1、K-ras和凋亡拮抗基因Bcl-2等凋亡相关基因调节P53介导的细胞凋亡；miR-7-5p可通过抑制PARP-1、BRCA1的DNA损伤修复能力使氢醌诱导TK6细胞凋亡增加，Bcl-2高表达可维持线粒体膜电位，避免细胞过多凋亡；SIRT1可通过介导癌基因K-ras的激活、抑癌基因P53的失活和组蛋白H3的去乙酰化而在HQ诱导人淋巴母细胞恶性转化中起重要作用。同时也研究发现HQ处理细胞后，SIRT1降低，PARP-1升高，导致自噬发生；PARP-1在转录和转录后水平调节ZO-2参与HQ诱导的凋亡。研究结果究将有助于阐明PARP-1 、SIRT1在氢醌所致细胞恶性转化中具有调控肿瘤相关基因表达和肿瘤形成的作用，为早期预防氢醌致癌奠定理论基础。