【作者】 顾红军；

【导师】 李强；

【作者基本信息】 第二军医大学 ， 内科学， 2009， 博士

【摘要】 肺癌是肿瘤相关的首要致死原因,肺腺癌约占原发性肺癌的30%。许多病人在诊断时病情已到晚期,主要的治疗手段对提高生存率已到一个平台期。鉴于化疗效果差,毒性和费用高,因此迫切需要深入探讨肺腺癌发生发展的分子生物学机制,以寻找新的治疗途径,改善其预后。组蛋白乙酰化状态由组蛋白乙酰化酶(Histone acetyltransferases,HATs)和组蛋白去乙酰化酶(Histone deacetylases,HDACs)调节。HATs和HDACs之间的动态平衡控制着染色质的结构和基因的表达,它们的功能紊乱是肿瘤发生发展的重要分子机制。HATs和HDACs是两个通过控制组蛋白乙酰化/去乙酰化状态调节转录作用相反的酶。在组蛋白N端赖氨酸的ε-氨基团乙酰化中和了赖氨酸的阳性电荷,减少了组蛋白与DNA的结合,增加了转录因子与DNA的结合;而去乙酰化导致了染色体的紧密性,抑制了基因的表达。HDACs属于组蛋白去乙酰化酶超家族,共有Ⅰ-Ⅳ四型。Ⅰ型HDACs包括HDAC1、2、3、8。Ⅱ型HDACs包括HDAC4、5、6、7、9、10。Ⅲ型HDACs包括SIRT2(Sir related proteins)1、2、3、4、5、6、7。Ⅳ型HDACs是HDAC11。不同的肿瘤类型HDACs的表达谱不同,肿瘤细胞中HDAC1高表达可明显增加肿瘤细胞的增殖能力,并且HDAC1高表达可影响细胞外基质而使肿瘤细胞移行和侵袭力明显加强。研究发现胃癌和前列腺癌细胞中HDAC1高表达。在胃癌、结直肠癌、宫颈的非典型增生以及子宫内膜间质肉瘤HDAC2过表达。Glaser KB等在HeLa细胞株中用siRNA干扰Ⅰ型和Ⅱ型HDACs(HDAC1,2,3,4,7)后发现,HDAC1和HDAC3是肿瘤细胞存活和增殖所必需的,用siRNA干扰HDAC1和HDAC3可引起组蛋白超乙酰化,抑制HeLa细胞增殖和诱导凋亡。siRNA单独干扰人骨肉瘤U2OS细胞、乳腺癌MCF7细胞HDAC1可引起细胞阻滞在G1或G2/M期细胞静止而不能进行有丝分裂从而导致细胞生长抑制,并增加细胞凋亡,而单独干扰HDAC2则无此效果。这些研究表明HDAC1在肿瘤细胞增殖中起至关重要的作用。HDAC1在肺癌中的表达,以及对肺癌细胞株的发生和发展是否起重要作用,尚无相关报道。HDACi(HDACs inhibitor,HDACi)已被广泛应用于体内外研究肿瘤发展等相关基因去抑制的效果。HDACi对基因表达的调控不是通过直接修饰DNA序列,而是通过协助DNA紧密地结合在组蛋白氨基端上阻止肿瘤抑制基因的转录和表达来发挥作用的。许多HDACi如曲古菌素A(trichostatin A,TSA)、丁酸酯等通过诱导细胞生长抑制、分化和凋亡而起到抗肿瘤作用。HDACi还可激活死亡受体通路和诱导凋亡。这些研究已证明HDACi的抗增殖和抗肿瘤作用。小分子HDACi对不同的HDACs几乎没有选择性。HDACi的效果更多依赖于肿瘤细胞的类别而不是所选抑制剂的类型。TSA对多个非小细胞肺癌细胞株增殖均有抑制作用,而且对HDACⅠ类和Ⅱ类均有抑制作用,在非小细胞肺癌细胞株中尚无明确抑制HDACs靶位的报道。TSA作为一种高特异性、稳定而有效的组蛋白去乙酰化酶抑制剂而备受关注,是目前国际上公认的研究酰基化对细胞影响的可靠模式。本课题通过检测肺腺癌和癌旁组织中的HDAC1 mRNA和蛋白表达差异,初步探讨HDAC1基因和肺腺癌的关系。在体外用RNA干扰技术沉默肺癌细胞株HDAC1,观察细胞增殖、凋亡和周期的变化,同时与TSA干预比较,探讨HDAC1在肺腺癌增殖中的作用,明确HDACi抑制肿瘤细胞增殖的主要靶点,对药物的筛选和新的药物合成,有着极为重要的意义。进一步观察细胞Bax,Bcl-2,cyclin B1和p21表达初步探讨HDAC1与细胞增殖、凋亡和周期的关系。一、人肺腺癌组织中HDAC1 mRNA和蛋白的表达目的:检测组蛋白去乙酰化酶家族成员HDAC1在肺腺癌组织和癌旁组织中的表达情况,初步探讨HDAC1与肺腺癌的关系。材料和方法:收集手术切除的肺腺癌组织及相应的癌旁组织共6例,HE染色明确病理类型。采用Real-time PCR和Western blotting技术检测6例肺腺癌组织和癌旁组织中HDAC1 mRNA和蛋白的表达情况。免疫组化明确HDAC1的表达位置。结果:Real-time PCR和Western blotting结果表明发现肺腺癌患者HDAC1在癌组织中表达显著高于癌旁组织高。免疫组化发现HDAC1在细胞核中高表达。结论:肺腺癌HDAC1基因较癌旁组织表达高,其与腺癌的发生可能相关。二、HDAC1 siRNA和TSA对肺腺癌细胞体外增殖及凋亡的影响目的:探讨HDAC1 siRNA对肺腺癌细胞株增殖、细胞周期及凋亡的影响,与HDACs多靶点的抑制剂TSA的作用进行比较,明确HDAC1在肺腺癌增殖中的作用。材料和方法:采用HDAC1 siRNA瞬间转染腺癌细胞株A549和NCI-H1299。采用Real-time PCR和Western blotting检测HDAC1 siRNA瞬间转染的效率;采用MTT法测定细胞瞬间转染HDAC1 siRNA后的增殖情况;应用流式细胞术检测HDAC1siRNA瞬间转染细胞后细胞周期及凋亡的影响,并与TSA干预作用相对比。结果:肺腺癌细胞株A549和NCI-H1299在HDAC1 siRNA转染后,HDAC1在mRNA水平和蛋白水平均出现明显的下降。HDAC1 siRNA转染腺癌细胞株增加了AC-H4的表达。在A549细胞转染HDAC1 siRNA48h后,与TSA(0.2μM)作用48h导致存活率下降相近(69%＆63%)。在NCI-H1299细胞中HDAC1 siRNA转染48h后,与TSA(0.4μM)作用48h导致存活率下降相近(83%＆76%)。HDAC1 siRNA沉默基因后和一定浓度下对多个靶点HDACs有乙酰化抑制作用的TSA干预,得到相似的抑制肿瘤增殖效应。在A549细胞HDAC1 siRNA转染后发现,早期凋亡增加与转染前相比(13.45%vs 5.22%),和TSA干预相似。在NCI-H1299细胞株中予以一定浓度的TSA未出现早期凋亡增加,HDAC1 siRNA转染后发现凋亡增加(11.76%vs 8.09%)。HDAC1 siRNA转染后腺癌细胞株均出现G2期阻滞。结论:HDAC1 siRNA转染对肺腺癌细胞株增殖有抑制作用,诱导肺腺癌细胞株阻滞在G2期,促细胞凋亡。三、HDAC1 siRNA和TSA对肺腺癌细胞的作用机制研究目的:探讨HDAC1沉默引起对肺腺癌细胞株增殖、凋亡变化的机制。方法:采用脂质体法HDAC1 siRNA瞬时转染和TSA干预人肺腺癌A549和H1299细胞株。采用Real-time PCR检测HDAC1 siRNA转染对Bcl-2、Bax、p21和细胞周期cyclinB1 mRNA水平的影响,并与TSA干预比较。采用Western blotting检测HDAC1 siRNA转染前后Bcl-2、Bax、p21和细胞周期蛋白cyclinB1的蛋白水平变化。结果:Real-time PCR检测HDAC1 siRNA转染后和TSA干预后A549细胞内Bcl-2和CyclinB1 mRNA与对照组相比分别降低37%和57%。与对照组相比Bax和p21mRNA水平分别增高4.82和3.38倍。HDAC1 siRNA转染NCI-H1299细胞株CyclinB1mRNA与对照组相比减低49%,Bcl-2 mRNA无明显差异,Bax和p21 mRNA与对照组相比分别增高3.88和3.91倍。Western-blotting测定蛋白表达水平与mRNA结果一致。在A549细胞中HDAC1 siRNA转染后,p53表达增高。结论:HDAC1沉默能抑制原癌基因Bcl-2,增加抑癌基因Bax、p21的表达,而诱导细胞凋亡;并能通过减低Cyclin B1的表达,导致细胞停滞在G2期。通过上述研究,本课题得出以下结论:1、肺腺癌组织中HDAC1基因及蛋白的表达水平均显著高于癌旁组织,其与腺癌的发生可能相关。2、HDAC1 siRNA转染肺腺癌细胞株的增殖有抑制作用,并能诱导肺腺癌细胞阻滞在G2期,促进肺腺癌细胞凋亡。3、HDAC1 siRNA转染能抑制肺腺癌细胞内原癌基因Bcl-2和细胞周期调控蛋白cyclinB1表达,增加抑癌基因Bax、p21的表达,从而使肿瘤细胞停滞于G2期,诱导细胞凋亡。全文总结:HDAC1在肺腺癌的发生、发展中具有一定的促进作用。通过干扰HDAC1的表达可对肺腺癌细胞的增殖、凋亡和细胞周期产生显著的影响。本实验结果为HDAC1成为肺腺癌靶向治疗的新靶点奠定了实验基础。HDAC家族其它成员的作用需要在后续研究中进一步明确。更多还原

【Abstract】 Lung cancer is known to be the leading cause of cancer death for both man and woman in the word.Lung adenocarcinoma is 30%of all the patients with lung cancer. Most of the lung adenocarcinoma patients are in the late stage of the disease when first diagnosed and the the survival rate stays on the platform.New therapeutic approaches in treatment of lung adenocarcinoma are anxiously needed considering the poor cure rate and high cost and toxicity of chemotherapy treatment used currently.Detailed exploration of the molecular biology of lung adenocarcinoma is also required for understanding this fatal malignant disease profoundly.The condition of acetylation are regulated by histone acetyltransferases(HATs)and histone deacetylases(HDACs).The dynamic balance between histone acetyltransferases and histone deacetylases controls the structure of chromatin and expression of genes.The chaos of HATs and HDACs is one of the important molecular mechanism of carcinogenesis.HATs and HDAC are two enzymes with opposing effects that regulate the transcription of genes by controlling the acetylation status of histones.The acetylation atε-amino group of specific lysines in the N-terminus of histones neutralizes the positive charge on lysine.This is thought to reduce the affinity of histone complexes to DNA chains,thus enhancing the access of transcriptional factors to DNA.On the other hand,deacetylation results in a more condensed chromatin.HDACs which belong to histone deacetylases superfamily have been classified into four distinct families:TypeⅠ,Ⅱ,ⅢandⅣ.The members of TypeⅠHDACs include HDAC1,HDAC2,HDAC3 and HDAC8.Overexpression of HDAC1 in human cells can not only increase the proliferation of cancer cells but also enhance the migration or invasion abivility of cells through extracellular matrix.Overexpression of HDAC1 mRNA and protein has been observed in human gastric and prostate tumors.Gastric carcinomas,colorectal carcinomas,cervical dysplasias and endometrial stromal sarcomas all overexpress HDAC2 as compared to their normal counterparts.It has been reported that specific siRNAs targeting HDAC3 and HDAC1 produced a concentration-dependent inhibition of histone hyperacetylation in HeLa cells and can impede the cells proliferation as well as inducing apoptosis.Senese et al showed that using RNA interference-mediated HDAC1 protein knockdown can cause cell cycle arrest at either G1 phase or G2/M phase, resulting in the loss of mitotic cells,cell growth inhibition,and an increase in the percentage of apoptotic cells.On the contrary,HDAC2 knockdown had no effect on cell proliferation unless HDAC1 was knocked down concurrently.These data demonstrated that TypeⅠHDACs,such as HDAC1,are important in the regulation of proliferation and survival in cancer cells.HDAC inhibitor(HDACi) is a new kind of anticancer agents which can cause cell cycle arrest,differentiation,and/or apoptosis of tumours by blocking deacetylation function of HDACs.The modificative effects of HDACi seem to be dependent upon the type of tumour cell rather than the specific HDAC inhibitors used.For NSCLC cells in culture,these compounds lead to growth arrest,cells differentiation,or apoptosis. Although the involvement of HDACs in the development of cancer has been proven,the detailed mechanisms underlying the regulation of NSCLS cell proliferation,apoptosis and cell cycle by individual HDACs are still unclear.The inhibitory effects of different members of HDACi are nonspecific for different HDAC isoforms.Also,the regulation of these enzymes during cancer growth is largely unknown.It significance of new drug screening and synthesis to define target of HDACi is considerable.This study is about to detect the expression of HDACs mRNA and protein in lung adenocarcinoma and latero-cancer tissues to exploring the effects of HDACs in lung adenocarcinoma.The role of HDAC1 in tumor cell proliferation and apoptosis is investigated using RNA interference technology in two different human lung adenocarcinoma cell lines(A549 and NCI-H1299).The involvments of Bax,Bcl-2,cyclin B1 and p21 in the effects of HDAC1 on lung adenocarcinoma are also determined in our study.1.The expression of HDAC1 mRNA and protein in human lung adenocarcinomaObjective:To detect the expression of HDAC1 mRNA and protein in human lung adenocarcinoma and paracancerous tissues and explore its role in lung adenocarcinoma.Methods and materials:Six cases of lung adenocarcinoms tissue samples and matched latero-cancer tissues were collected and detected HE staining to confirm their adenocacinoma characteristics.The mRNA and protein levels of HDAC1 of the samples were detected by Real-time PCR and Western blotting analysis,respectively.The location of HDAC1 expression is determined by streptavidin peroxidase immunohistochemistry techniques.Results:The results of Realtime-PCR showed that the average level of HDAC1 mRNA expressoion in adenocarcinoma tissues are significant higher than that in matched latero-cancer tissues(2.30 vs 1,P=0.03),which was cofirmed by Western blotting analysis. Immunohistochemistry showed HDAC1 protein is localized in cell nucleus.Conclusions:The expression of HDAC1 mRNA and protein levels were both significant higher in human lung adenocarcinoma tissues than in latero-cance tissues,indicating its possible involvement in lung adenocarcinoma carcinogenesis.2.Effects of RNAi knockdown of HDAC1 and trichostatin A(TSA) treatment on the growth of lung adenocarcinoma cell linesObjective:To evaluate the role of RNAi knockdown of HDAC1 in tumor cell proliferation by comparing with TSA treatment.Methods and materials.The role of HDAC1 in tumor cell proliferation was investigated using specific RNA interference targeting HDAC1 and TSA treatment in A549 and NCI-H1299 human lung adenocarcinoma cell lines.Cells were classified into three groups:the control group,negative siRNA group,and HDAC1 siRNA group. Realtime-PCR and Western blotting analysis were performed to detect the mRNA and protein expression of HDAC1 and Ac-H4,its acetylated product,in each group.The effects on cells proliferation,cell cycle progression and apoptosis were determined by flow cytometry,annexin-V staining and MTT assay,respectively.Results:The expression of HDAC1 mRNA and protein in lung adenocarcinoma cell lines were significantly suppressed after specific HDAC1 siRNA transfection.Western blotting analysis revealed an increased acetylation level of a subset of histones H4 after suppression of HDAC1.The growth of two cell lines with HDAC1 knockdown were both significantly inhibited when compared with negative siRNA controls.The survival rates of both A549 and NCI-H1299 cells after HDAC1 siRNA transfection significantly decreased (69%and 83%,respectively),while after TSA treatment the survival rates of these two cells decreased to a similar extent(63%and 76%,respectively).Either RNAi knockdown or TSA treatment resulted in increased early apoptotic rate of A549(13.45%vs 5.22%) and NCI-H1299(11.76%vs 8.09%).G2/M cyclin arrest was also observed in two cell lines with suppressed HDAC1 expression when compared with the controls.Conclusions:Transfection of specific HDAC1 siRNA effectively inhibits the proliferation of lung adenocarcinoma cells.The suppression of HDAC1 expression resulted in both G2/M cell cycle arrest and increased cells apotosis.These data suggest that inhibition of HDAC1 may provide a valuable approach for lung adenocarcinoma treatment. It is worthwhile evaluating HDAC1 as a potential candidate for anticancer therapy in lung adenocarcinoma.3.Effect of RNAi knockdown of HDAC1 on the expresison of Bax,Bcl-2,p21 and cyclin B1 gene in lung adenocarcinoma cell linesObjective:To evaluate the role of transient transfection of HDAC1 siRNA inducing gene concerning about proliferation and apotosis in lung adenocarcinoma cell line.Methods and materials:Transient transfection of A549 and NCI-H1299 cell lines with specific HDAC1 siRNA were performed.The level of mRNA and protein expression on Bcl-2,Bax,p21 and cyclinB1 were detected by Realtime-PCR and Western blotting analysis.Results:The levels of Bcl-2 and CyclinB1 mRNA in A549 cells were significantly decreased after HDAC1 siRNA transfection compared to negative control(37%vs 57%).On the contrary,Bax and p21 expression in A549 cells were significantly increased(4.82 and 3.38 folds, respectively).In NCI-H1299 cells the mRNA expression of CyclinB1 were significantly decreased (49%) after HDAC1 siRNA transfection,while Bax and p21 mRNA levels were increased(3.88 and 3.91 folds,respectively).The results of western blotting analysis were coincident with those of Realtime-PCR analysis.Conclusions:Specific HDAC1 siRNA transfection can inhibit the expression of Bcl-2 and cyclin B1 gene while increase the expression of Bax and p21.Based on the above experiments,the final conclusions were as followings:1.The expression of HDAC1 mRNA and protein levels were higher in human lung adenocarcinoma tissues than that in latero-cance tissues,indicating possible involvement of HDAC1 in carcinogenesis of lung adenocarcinoma.2.Transfection of specific HDAC1 siRNA can effectively inhibit the proliferation of lung adenocarcinoma cell lines.RNAi knockdown of HDAC1 in cells resulted in G2/M cycle arrest and increased cells apoptosis,indicating the potential of HDAC1 as a candidate for anticancer therapy in lung adenocarcinoma.3.RNAi knockdown of HDAC1 can inhibit the expression of Bcl-2 and cyclin B1 gene while increasing the expression of Bax and p21 gene.In summary,we found in this study that RNA interference for HDAC1 induces cell cycle arrest and apoptosis in lung adenocarcinoma.We suppose that HDAC1 may play an important role in carcinogenesis of lung adenocarcinoma and may be a valuable new target for cancer treatment.The role of other member of HDACs should be further studied.更多还原