發(fā)布時(shí)間：2018-09-03 07:34

【摘要】：研究背景端粒是位于真核染色體末端,由短的DNA重復(fù)序列TTAGGG和相關(guān)蛋白質(zhì)組成的復(fù)合物。其主要作用是保護(hù)染色體,既可防止染色體被核酸酶降解,同時(shí)又能阻止相鄰染色體間的融合。另外,端粒的長度反應(yīng)細(xì)胞的可分裂次數(shù),因此,被稱為細(xì)胞的“生命鐘”。正常情況下,端粒DNA會(huì)隨著細(xì)胞的每次分裂而縮短50-200個(gè)堿基對(duì),當(dāng)其縮短到一定長度時(shí),染色體則無法完成正常復(fù)制,細(xì)胞的有絲分裂也會(huì)隨之停滯,轉(zhuǎn)而進(jìn)入衰老階段。端粒酶是一種RNA依賴的DNA聚合酶,由催化組分端粒酶逆轉(zhuǎn)錄酶(hTERT)、端粒酶模板RNA (hTER)以及端粒酶相關(guān)蛋白質(zhì)組成,其主要作用是延長端粒的長度。在正常的成熟體細(xì)胞中,端粒酶幾乎沒有活性,但是,在病理狀態(tài)下,如腫瘤細(xì)胞中,其活性異常增高,使端粒長度得以延長,從而保證了腫瘤細(xì)胞的無限分裂增殖。由此可見,端粒酶活性的異常升高所導(dǎo)致的端粒長度的維持,與腫瘤的發(fā)生發(fā)展密切相關(guān)。硼替佐米是已經(jīng)應(yīng)用于臨床的用來治療多發(fā)性骨髓瘤的一種藥物,作為26S蛋白酶體的抑制劑,其已知的主要作用機(jī)制是通過抑制NF-κB信號(hào)途徑,進(jìn)而激活caspase3,同時(shí)抑制抗凋亡基因bcl-2的表達(dá),來誘導(dǎo)腫瘤細(xì)胞的凋亡。雖然硼替佐米在多發(fā)性骨髓瘤的治療中已經(jīng)取得了良好療效,但仍有部分耐藥病例出現(xiàn),因此,我們亟需尋找硼替佐米新型的作用機(jī)制以及靶點(diǎn)分子,以使其獲得更加合理的臨床應(yīng)用。腫瘤抑制因子aridla是染色質(zhì)重構(gòu)復(fù)合體SWI/SNF的重要組成成分,可結(jié)合于DNA的特定區(qū)域,此復(fù)合體中的另一成員BRG1或BRM可水解ATP產(chǎn)生能量,利用這些能量,SWI/SNF重構(gòu)復(fù)合體可使染色質(zhì)中核小體的組蛋白核心發(fā)生移位甚至脫落,從而轉(zhuǎn)移核小體的位置,改變?nèi)旧|(zhì)的構(gòu)象,使下游基因的轉(zhuǎn)錄起始部位暴露或隱藏,由此來調(diào)控下游基因的轉(zhuǎn)錄。目前,眾多的基因測(cè)序結(jié)果顯示,在卵巢癌、胃癌、腎癌、胰腺癌等絕大多數(shù)腫瘤中都存在ARID1A基因的突變或缺失,這提示aridla可能作為一種抑癌基因來行使作用。但是,具體的抑癌機(jī)制目前研究尚少。研究目的1.探討硼替佐米誘導(dǎo)腫瘤細(xì)胞凋亡的過程中,對(duì)端粒酶活性和端粒功能的影響,以發(fā)掘其新的作用機(jī)制,并對(duì)腫瘤細(xì)胞產(chǎn)生硼替佐米耐藥過程中hTERT所發(fā)揮作用進(jìn)行研究。2.探討胃癌細(xì)胞中aridla對(duì)hTERT表達(dá)的調(diào)控及其相關(guān)機(jī)制,為aridla的抑癌作用提供證據(jù)支持。研究方法和結(jié)果1.硼替佐米降低端粒酶活性和端粒穩(wěn)定性誘導(dǎo)腫瘤細(xì)胞凋亡(1)硼替佐米抑制腫瘤細(xì)胞端粒酶活性,同時(shí)降低其端粒穩(wěn)定性。硼替佐米處理白血病細(xì)胞HEL和胃癌細(xì)胞BGC-823前后,分別用PCR-ELISA試劑盒檢測(cè)端粒酶活性,qRT-PCR檢測(cè)hTERT、hTER及端粒結(jié)合蛋白TRF1、TRF2、 POT1、RAP1、TPP1及TIN2的mRNA表達(dá),Western Blotting檢測(cè)TRF1、TRF2、POT1的蛋白表達(dá),流式-熒光原位雜交(Flow-FISH)和qPCR檢測(cè)端粒長度,免疫-熒光原位雜交(Immuno-FISH)檢測(cè)端粒DNA的損傷。結(jié)果表明,硼替佐米可下調(diào)hTERT、hTER的mRNA表達(dá)水平,降低端粒酶活性,使端粒結(jié)合蛋白的表達(dá)廣泛失調(diào),縮短端粒長度并導(dǎo)致端粒功能異常。(2)hTERT高表達(dá)可減弱由硼替佐米引起的端粒功能異常。硼替佐米處理穩(wěn)定高表達(dá)hTERT基因的細(xì)胞株HEL-hTERT、BGC-823-hTERT后,Flow-FISH和qPCR檢測(cè)細(xì)胞端粒長度,Immuno-FISH檢測(cè)端粒DNA損傷,qRT-PCR檢測(cè)端粒結(jié)合蛋白TRF1、TRF2、POT1、RAP1、TPP1及TIN2的mRNA表達(dá),Western Blotting檢測(cè)TRF1、TRF2、POT1的蛋白表達(dá)。結(jié)果表明,硼替佐米處理后HEL-hTERT和BGC-823-hTERT細(xì)胞中端粒長度無明顯改變,端粒DNA損傷和端粒結(jié)合蛋白的表達(dá)失調(diào)程度明顯弱于相同濃度硼替佐米處理的HEL-control和BGC-823-control細(xì)胞。(3)hTERT高表達(dá)可抑制由硼替佐米所誘導(dǎo)的腫瘤細(xì)胞凋亡。硼替佐米處理穩(wěn)定高表達(dá)hTERT基因的細(xì)胞株HEL-hTERT、BGC-823-hTERT與對(duì)照細(xì)胞株HEL-control、BGC-823-control后,臺(tái)盼藍(lán)染色計(jì)數(shù)確定細(xì)胞的存活率,Annexin-V染色后流式細(xì)胞術(shù)檢測(cè)細(xì)胞的凋亡率。結(jié)果表明,硼替佐米處理后,HEL-hTERT和BGC-823-hTERT細(xì)胞的存活率明顯高于HEL-control和BGC-823-control細(xì)胞,而HEL-hTERT和BGC-823-hTERT細(xì)胞的凋亡比例明顯低于HEL-control和BGC-823-control細(xì)胞。(4)hTERT通過減弱DNA損傷和上調(diào)bcl-2表達(dá)抑制腫瘤細(xì)胞凋亡。硼替佐米處理穩(wěn)定高表達(dá)hTERT基因的細(xì)胞株HEL-hTERT、BGC-823-hTERT與對(duì)照細(xì)胞株HEL-control、BGC-823-control后,免疫熒光檢測(cè)DNA損傷,即53BP1的凝集,Western Blotting檢測(cè)bcl-2的表達(dá)。結(jié)果表明,HEL-hTERT與BGC-823-hTERT細(xì)胞中的DNA損傷明顯少于HEL-control、BGC-823-control細(xì)胞,HEL-hTERT細(xì)胞內(nèi)bcl-2表達(dá)明顯高于HEL-control細(xì)胞,且在硼替佐米處理后HEL-hTERT細(xì)胞內(nèi)仍存留較多量的bcl-2蛋白。2.染色質(zhì)重構(gòu)分子ARID1A負(fù)性調(diào)控hTERT抑制胃癌進(jìn)展(1)arid1a負(fù)性調(diào)控hTERT表達(dá)。胃癌細(xì)胞系A(chǔ)GS和SGC中轉(zhuǎn)染arid1a高表達(dá)或?qū)φ召|(zhì)粒PcDNA 6.0后,采用qRT-PCR和western blotting分別檢測(cè)hTERT的mRNA及蛋白表達(dá)水平。結(jié)果表明,aridla高表達(dá)后,hTERT的mRNA及蛋白表達(dá)均下降。(2) arid1a抑制hTERT啟動(dòng)子活性。AGS和SGC細(xì)胞中轉(zhuǎn)染aridla高表達(dá)或?qū)φ召|(zhì)粒PcDNA 6.0后,再轉(zhuǎn)染hTERT啟動(dòng)子報(bào)告基因質(zhì)粒,然后采用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)hTERT啟動(dòng)子活性。結(jié)果表明,arid1a高表達(dá)后,hTERT啟動(dòng)子活性明顯降低。(3)arid1a抑制c-myc在hTERT啟動(dòng)子區(qū)的結(jié)合,但不改變c-myc的表達(dá)量。AGS和SGC細(xì)胞中轉(zhuǎn)染arid1a高表達(dá)或?qū)φ召|(zhì)粒PcDNA 6.0后,qRT-PCR檢測(cè)c-myc的mRNA表達(dá),結(jié)果顯示arid1a未影響c-myc的mRNA水平。在轉(zhuǎn)染了arid1a高表達(dá)或?qū)φ召|(zhì)粒PcDNA6.0的AGS和SGC細(xì)胞中,轉(zhuǎn)染c-myc結(jié)合位點(diǎn)突變的hTERT啟動(dòng)子質(zhì)粒,用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)突變的hTERT啟動(dòng)子活性,結(jié)果顯示,其活性未受arid1a高表達(dá)的影響。研究結(jié)論1.硼替佐米可以通過降低HEL和BGC-823細(xì)胞的端粒酶活性和端粒穩(wěn)定性誘導(dǎo)細(xì)胞凋亡,而hTERT可減弱硼替佐米對(duì)端粒功能的影響,介導(dǎo)腫瘤細(xì)胞對(duì)硼替佐米的耐藥。2. Aridla通過阻礙c-myc在hTERT啟動(dòng)子區(qū)的結(jié)合來抑制hTERT的表達(dá),從而抑制胃癌進(jìn)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R730.5

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