端粒与端粒酶在硼替佐米和ARID1A抑制肿瘤进展中的功能学研究

发布时间：2018-09-03 07:34

【摘要】：研究背景端粒是位于真核染色体末端,由短的DNA重复序列TTAGGG和相关蛋白质组成的复合物。其主要作用是保护染色体,既可防止染色体被核酸酶降解,同时又能阻止相邻染色体间的融合。另外,端粒的长度反应细胞的可分裂次数,因此,被称为细胞的“生命钟”。正常情况下,端粒DNA会随着细胞的每次分裂而缩短50-200个碱基对,当其缩短到一定长度时,染色体则无法完成正常复制,细胞的有丝分裂也会随之停滞,转而进入衰老阶段。端粒酶是一种RNA依赖的DNA聚合酶,由催化组分端粒酶逆转录酶(hTERT)、端粒酶模板RNA (hTER)以及端粒酶相关蛋白质组成,其主要作用是延长端粒的长度。在正常的成熟体细胞中,端粒酶几乎没有活性,但是,在病理状态下,如肿瘤细胞中,其活性异常增高,使端粒长度得以延长,从而保证了肿瘤细胞的无限分裂增殖。由此可见,端粒酶活性的异常升高所导致的端粒长度的维持,与肿瘤的发生发展密切相关。硼替佐米是已经应用于临床的用来治疗多发性骨髓瘤的一种药物,作为26S蛋白酶体的抑制剂,其已知的主要作用机制是通过抑制NF-κB信号途径,进而激活caspase3,同时抑制抗凋亡基因bcl-2的表达,来诱导肿瘤细胞的凋亡。虽然硼替佐米在多发性骨髓瘤的治疗中已经取得了良好疗效,但仍有部分耐药病例出现,因此,我们亟需寻找硼替佐米新型的作用机制以及靶点分子,以使其获得更加合理的临床应用。肿瘤抑制因子aridla是染色质重构复合体SWI/SNF的重要组成成分,可结合于DNA的特定区域,此复合体中的另一成员BRG1或BRM可水解ATP产生能量,利用这些能量,SWI/SNF重构复合体可使染色质中核小体的组蛋白核心发生移位甚至脱落,从而转移核小体的位置,改变染色质的构象,使下游基因的转录起始部位暴露或隐藏,由此来调控下游基因的转录。目前,众多的基因测序结果显示,在卵巢癌、胃癌、肾癌、胰腺癌等绝大多数肿瘤中都存在ARID1A基因的突变或缺失,这提示aridla可能作为一种抑癌基因来行使作用。但是,具体的抑癌机制目前研究尚少。研究目的1.探讨硼替佐米诱导肿瘤细胞凋亡的过程中,对端粒酶活性和端粒功能的影响,以发掘其新的作用机制,并对肿瘤细胞产生硼替佐米耐药过程中hTERT所发挥作用进行研究。2.探讨胃癌细胞中aridla对hTERT表达的调控及其相关机制,为aridla的抑癌作用提供证据支持。研究方法和结果1.硼替佐米降低端粒酶活性和端粒稳定性诱导肿瘤细胞凋亡(1)硼替佐米抑制肿瘤细胞端粒酶活性,同时降低其端粒稳定性。硼替佐米处理白血病细胞HEL和胃癌细胞BGC-823前后,分别用PCR-ELISA试剂盒检测端粒酶活性,qRT-PCR检测hTERT、hTER及端粒结合蛋白TRF1、TRF2、 POT1、RAP1、TPP1及TIN2的mRNA表达,Western Blotting检测TRF1、TRF2、POT1的蛋白表达,流式-荧光原位杂交(Flow-FISH)和qPCR检测端粒长度,免疫-荧光原位杂交(Immuno-FISH)检测端粒DNA的损伤。结果表明,硼替佐米可下调hTERT、hTER的mRNA表达水平,降低端粒酶活性,使端粒结合蛋白的表达广泛失调,缩短端粒长度并导致端粒功能异常。(2)hTERT高表达可减弱由硼替佐米引起的端粒功能异常。硼替佐米处理稳定高表达hTERT基因的细胞株HEL-hTERT、BGC-823-hTERT后,Flow-FISH和qPCR检测细胞端粒长度,Immuno-FISH检测端粒DNA损伤,qRT-PCR检测端粒结合蛋白TRF1、TRF2、POT1、RAP1、TPP1及TIN2的mRNA表达,Western Blotting检测TRF1、TRF2、POT1的蛋白表达。结果表明,硼替佐米处理后HEL-hTERT和BGC-823-hTERT细胞中端粒长度无明显改变,端粒DNA损伤和端粒结合蛋白的表达失调程度明显弱于相同浓度硼替佐米处理的HEL-control和BGC-823-control细胞。(3)hTERT高表达可抑制由硼替佐米所诱导的肿瘤细胞凋亡。硼替佐米处理稳定高表达hTERT基因的细胞株HEL-hTERT、BGC-823-hTERT与对照细胞株HEL-control、BGC-823-control后,台盼蓝染色计数确定细胞的存活率,Annexin-V染色后流式细胞术检测细胞的凋亡率。结果表明,硼替佐米处理后,HEL-hTERT和BGC-823-hTERT细胞的存活率明显高于HEL-control和BGC-823-control细胞,而HEL-hTERT和BGC-823-hTERT细胞的凋亡比例明显低于HEL-control和BGC-823-control细胞。(4)hTERT通过减弱DNA损伤和上调bcl-2表达抑制肿瘤细胞凋亡。硼替佐米处理稳定高表达hTERT基因的细胞株HEL-hTERT、BGC-823-hTERT与对照细胞株HEL-control、BGC-823-control后,免疫荧光检测DNA损伤,即53BP1的凝集,Western Blotting检测bcl-2的表达。结果表明,HEL-hTERT与BGC-823-hTERT细胞中的DNA损伤明显少于HEL-control、BGC-823-control细胞,HEL-hTERT细胞内bcl-2表达明显高于HEL-control细胞,且在硼替佐米处理后HEL-hTERT细胞内仍存留较多量的bcl-2蛋白。2.染色质重构分子ARID1A负性调控hTERT抑制胃癌进展(1)arid1a负性调控hTERT表达。胃癌细胞系AGS和SGC中转染arid1a高表达或对照质粒PcDNA 6.0后,采用qRT-PCR和western blotting分别检测hTERT的mRNA及蛋白表达水平。结果表明,aridla高表达后,hTERT的mRNA及蛋白表达均下降。(2) arid1a抑制hTERT启动子活性。AGS和SGC细胞中转染aridla高表达或对照质粒PcDNA 6.0后,再转染hTERT启动子报告基因质粒,然后采用双荧光素酶报告基因系统检测hTERT启动子活性。结果表明,arid1a高表达后,hTERT启动子活性明显降低。(3)arid1a抑制c-myc在hTERT启动子区的结合,但不改变c-myc的表达量。AGS和SGC细胞中转染arid1a高表达或对照质粒PcDNA 6.0后,qRT-PCR检测c-myc的mRNA表达,结果显示arid1a未影响c-myc的mRNA水平。在转染了arid1a高表达或对照质粒PcDNA6.0的AGS和SGC细胞中,转染c-myc结合位点突变的hTERT启动子质粒,用双荧光素酶报告基因系统检测突变的hTERT启动子活性,结果显示,其活性未受arid1a高表达的影响。研究结论1.硼替佐米可以通过降低HEL和BGC-823细胞的端粒酶活性和端粒稳定性诱导细胞凋亡,而hTERT可减弱硼替佐米对端粒功能的影响,介导肿瘤细胞对硼替佐米的耐药。2. Aridla通过阻碍c-myc在hTERT启动子区的结合来抑制hTERT的表达,从而抑制胃癌进展。
[Abstract]:Background Telomeres are a complex of short DNA repeats (TTAGGG) and related proteins located at the end of eukaryotic chromosomes. Their main function is to protect chromosomes from being degraded by nucleases while preventing the fusion of adjacent chromosomes. Telomerase is an RNA-dependent DNA polymerase that catalyzes the cell's mitosis. Telomerase reverse transcriptase (hTERT), telomerase template RNA (hTER), and telomerase-related proteins are components of telomerase, whose main function is to lengthen telomere length. Telomerase has little activity in normal mature somatic cells, but in pathological conditions, such as tumor cells, its activity increases abnormally, and telomere length is prolonged. This shows that the maintenance of telomere length caused by abnormal telomerase activity is closely related to the occurrence and development of tumors. Bortezomib is a drug that has been used clinically to treat multiple myeloma, as an inhibitor of 26S proteasome. The mechanism is to induce apoptosis of tumor cells by inhibiting the NF-kappa B signaling pathway, thereby activating caspase 3 and inhibiting the expression of anti-apoptotic gene bcl-2. Although bortezomib has achieved good results in the treatment of multiple myeloma, there are still some cases of drug resistance, so we need to find a new bortezomib-resistant drug. Tumor inhibitor aridla is an important component of the chromatin remodeling complex SWI/SNF, which binds to specific regions of DNA. Another member of this complex, BRG1 or BRM, can hydrolyze ATP to produce energy. Using this energy, SWI/SNF remodeling complex can make staining possible. Histone core of nucleosome in chromatin translocates or even sheds off, thus transferring nucleosome position, changing chromatin conformation, exposing or hiding the transcriptional initiation site of downstream genes, thus regulating the transcription of downstream genes. At present, numerous gene sequencing results show that most of the genes in ovarian cancer, gastric cancer, kidney cancer, pancreatic cancer and so on. There are mutations or deletions of ARID1A gene in tumors, suggesting that aridla may play a role as an anti-oncogene. However, the specific mechanisms of inhibition are still poorly understood. Objective 1. To explore the effects of bortezomib on telomerase activity and telomere function in the process of inducing apoptosis of tumor cells, so as to explore new mechanisms of action. 2. To investigate the regulation of aridla on hTERT expression in gastric cancer cells and its related mechanisms, and to provide evidence for the anti-tumor effect of aridla. Bortezomib inhibited telomerase activity and decreased telomeric stability in tumor cells. Telomerase activity was detected by PCR-ELISA kit before and after treatment with bortezomib in leukemia cells HEL and gastric cancer cells BGC-823, and the mRNA expression of hTERT, hTER and telomere binding protein TRF1, TRF2, POT1, RAP1, TPP1 and TIN2 was detected by qRT-PCR. The expression of TRF1, TRF2, POT1 was detected, telomere length was detected by Flow-FISH and qPCR, and telomere DNA damage was detected by Immuno-FISH. High expression of hTERT could attenuate telomere dysfunction induced by bortezomib. After treatment with stable and high expression of hTERT gene in HEL-hTERT, BGC-823-hTERT, telomere length was detected by Flow-FISH and qPCR, telomere DNA damage was detected by Immuno-FISH, and telomere binding protein TRF1 was detected by qRT-PCR. The expression of TRF1, TRF2, POT1, RAP1, TPP1 and TIN2 mRNA was detected by Western Blotting. The results showed that the telomere length of HEL-hTERT and BGC-823-hTERT cells did not change significantly after bortezomib treatment, and telomere DNA damage and telomere-binding protein expression disorder were significantly weaker than those of HEL-contr treated with bortezomib at the same concentration. High expression of hTERT could inhibit the apoptosis of tumor cells induced by bortezomib. After treatment with bortezomib, HEL-hTERT, BGC-823-hTERT and control cell lines HEL-control, BGC-823-control, Trypan blue staining counts were used to determine the cell survival rate, and Annexin-V staining followed by flow cytometry. The results showed that the survival rates of HEL-hTERT and BGC-823-hTERT cells were significantly higher than those of HEL-control and BGC-823-control, while the apoptosis rates of HEL-hTERT and BGC-823-hTERT cells were significantly lower than those of HEL-control and BGC-823-hTERT cells. BGC-823-hTERT and control cell lines HEL-control, BGC-823-hTERT were treated with bortezomib. DNA damage, i.e. 53 BP1 agglutination, was detected by immunofluorescence. The expression of Bcl-2 in HEL-hTERT and BGC-823-hTERT cells was detected by Western Blotting. The expression of Bcl-2 in HEL-control cells was significantly higher than that in HEL-control cells, BGC-823-control cells and HEL-hTERT cells, and there was still a large amount of Bcl-2 protein in HEL-hTERT cells treated with bortezomib. 2. Chromatin remodeling molecule ARID1A negatively regulated the progression of gastric cancer. (1) Ard 1A negatively regulated the expression of hTERT in gastric cancer cell line A. After high expression of arid1a in GS and SGC or control plasmid PcDNA 6.0, the expression of hTERT mRNA and protein were detected by qRT-PCR and Western blotting respectively. The results showed that the expression of hTERT mRNA and protein decreased after high expression of arid1a. (2) Ard1a inhibited the activity of hTERT promoter. The results showed that the activity of hTERT promoter decreased significantly after high expression of arid1a. (3) arid1a inhibited the binding of c-myc to the promoter region of hTERT, but did not change the expression of c-myc. After 1A overexpression or control plasmid PcDNA 6.0, the expression of c-myc mRNA was detected by qRT-PCR. The results showed that arid1a did not affect the level of c-myc mRNA. In AGS and SGC cells transfected with arid1a overexpression or control plasmid PcDNA 6.0, the hTERT promoter plasmid with c-myc binding site mutation was transfected and the mutant hTERT promoter was detected by double luciferase reporter gene system. Promoter activity was not affected by high expression of arid1a. Conclusion 1. Bortezomib can induce apoptosis by decreasing telomerase activity and telomere stability in HEL and BGC-823 cells, while hTERT can attenuate the effect of bortezomib on telomere function and mediate the resistance of tumor cells to bortezomib. 2. Aridla passes through. Inhibition of c-myc binding in hTERT promoter region inhibits the expression of hTERT, thereby inhibiting the progression of gastric cancer.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5

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