本文選題：膀胱癌 + 藤黃酸��； 參考：《華中科技大學》2015年博士論文

【摘要】：[背景]藤黃酸(GA)作為新近發(fā)現(xiàn)的天然抗腫瘤藥物,在膀胱癌中顯示了強有效的抗癌作用,極具臨床應用前景,闡明其作用機制已成為其臨床應用的研究重點。長鏈非編碼RNA (LncRNA)過去被視為基因組轉(zhuǎn)錄的“噪音”,近年研究發(fā)現(xiàn)LncRNA參與生物調(diào)節(jié)的各個環(huán)節(jié),其表達異常與多種疾病的發(fā)生、發(fā)展密切相關(guān)。LncRNA GAS5在多種腫瘤中低表達,被認為是重要的抑癌基因,但GAS5與膀胱癌發(fā)生、發(fā)展之間的關(guān)系仍不明確,作用機制也有待深入研究。[目的]1.檢測長鏈非編碼RNA GAS5在膀胱癌中的表達情況,分析其與膀胱癌臨床分級、分期之間的關(guān)系；2.探討GAS5在膀胱癌中的作用并闡述其作用機制；3.進一步完善新型化療藥GA抑制膀胱癌的作用機制。[方法]1.收集至我院行膀胱癌根治性切除術(shù)的病例43例,同時獲取癌組織與周圍正常組織,提取RNA行RT-QPCR實驗檢測長鏈非編碼RNA GAS5的表達水平,并分析GAS5的表達與臨床分級、分期之間的關(guān)系；2.分別合成GAS5 siRNA與shRNA沉默GAS5,構(gòu)建GAS5過表達載體(GV144-GAS5)過表達GAS5,分別轉(zhuǎn)染膀胱癌細胞株T24、EJ；3.GAS5在膀胱癌細胞中的作用：MTT檢測細胞生存率,流式細胞術(shù)檢測細胞凋亡率,Western blot檢測蛋白表達水平；4.雙熒光素酶報告基因檢測GAS5對EZH2啟動子活性的影響；5.RIP實驗檢測GAS5與轉(zhuǎn)錄因子E2F4的結(jié)合作用；6. CHIP實驗檢測E2F4與EZH2啟動子的結(jié)合作用；7.構(gòu)建EZH2過表達載體與shRNA載體,轉(zhuǎn)染T24、EJ細胞,RT-QPCR實驗檢測EZH2對miR-101的調(diào)節(jié)作用；8.雙熒光素酶報告基因檢測EZH2對miR-101的啟動子活性的影響；9.不同濃度(0、1.5、2.0、2.5 μM)GA分別作用于T24、EJ細胞,RT-QPCR檢測GA對GAS5的調(diào)節(jié)作用：10.沉默GAS5前后,分別用MTT、流式、Western blot檢測GA對膀胱癌細胞的作用：11. balb-c裸鼠構(gòu)建膀胱癌異位移植模型,檢測GA通過GAS5體外抑制膀胱癌生長。[結(jié)果]1.43例膀胱癌標本中33例GAS5表達下調(diào)(76.74%)；非基層浸潤性膀胱癌(Ta+Tis+T1)中下調(diào)率為59.09%,浸潤性癌中下調(diào)率為95.24%；無淋巴結(jié)轉(zhuǎn)移的下調(diào)率為78.57%,有淋巴結(jié)轉(zhuǎn)移的下調(diào)率為75.86%；無遠處轉(zhuǎn)移的下調(diào)率為77.5%,有遠處轉(zhuǎn)移的下調(diào)率為66.67%。2.過表達GAS5后膀胱癌細胞增率下降,凋亡率上升,caspase-3蛋白活活化增加：沉默GAS5后膀胱癌細胞增值率上升,凋亡率下降,caspase-3蛋白活化減少。3. Western blot顯示GAS5能夠抑制EZH2蛋白的表達,RT-QPCR顯示GAS5能夠抑制：EZH2 mRNA的表達,雙熒光素報告基因檢測提示GAS5能夠抑制EZH2啟動子的活性。4.RIP實驗提示GAS5能與轉(zhuǎn)錄因子E2F4特異性的結(jié)合,CHIP實驗提示E2F4能夠特異性的結(jié)合到EZH2啟動子區(qū)域。5. RT-QPCR提示EZH2能夠抑制miR-101的表達,雙熒光素報告基因檢測提示EZH2能夠抑制miR-101-2啟動子的活性。6.GA能夠正性調(diào)節(jié)GAS5的表達,且呈劑量正相關(guān)性；GA能夠顯著誘導膀胱癌細胞的凋亡,沉默GAS5后GA誘導膀胱癌細胞凋亡的能力顯著下降。7.動物實驗顯示GA、GAS5均能夠顯著抑制膀胱癌的生長；沉默GAS5后,GA抑制膀胱癌生長的能力顯著下降。[結(jié)論]1.GAS5在膀胱癌組織中顯著低表達,且肌層浸潤性膀胱癌中低表達率明顯高于非基層浸潤性膀胱癌,其表達水平與淋巴結(jié)轉(zhuǎn)移、遠處轉(zhuǎn)移無明顯關(guān)系;2.膀胱癌中,EZH2可以從轉(zhuǎn)錄水平負性調(diào)節(jié)miR-101的表達；3.GAS5可以通過轉(zhuǎn)錄因子E2F4抑制EZH2啟動子活性,抑制EZH2 mRNA、蛋白的表達,進而促進miR-101的表達,誘導膀胱癌細胞凋亡;4.GA通過GAS5阻斷EZH2-miR-101通路促進體內(nèi)外膀胱癌細胞凋亡。
[Abstract]:[background] GA, as a newly discovered natural antineoplastic drug, has shown strong effective anticancer effect in bladder cancer and has a great potential for clinical application. It has become a key point in its clinical application. Long chain noncoding RNA (LncRNA) has been regarded as a "noise" of genome transcription in the past. In recent years, the study of LncRNA has been found. Each link with biological regulation, its expression abnormality and the occurrence of a variety of diseases, development closely related to the low expression of.LncRNA GAS5 in a variety of tumors, is considered to be an important tumor suppressor gene, but the relationship between the development of GAS5 and the development of bladder cancer is still unclear, and the mechanism of action needs to be further studied. [Objective]1. detection of long chain non coding RNA GAS5 is in the process of detection. The relationship between the expression of bladder cancer and the clinical classification and staging of bladder cancer; 2. to explore the role of GAS5 in bladder cancer and to elaborate its mechanism of action; 3. to further improve the mechanism of the inhibitory effect of new chemotherapeutic drugs GA on bladder cancer. [methods]1. collect 43 cases of radical resection of bladder cancer in our hospital and obtain cancer at the same time. The expression level of long chain non coding RNA GAS5 was detected by RNA RT-QPCR test, and the relationship between the expression of GAS5 and the clinical classification and stages was analyzed. 2. the GAS5 siRNA and shRNA were synthesized and GAS5, and GAS5 overexpression vector (GV144-GAS5) was constructed, respectively, and transfected to bladder cancer cell line respectively. Function in bladder cancer cells: MTT detection of cell survival, flow cytometry to detect cell apoptosis rate, Western blot detection of protein expression level; 4. double luciferase reporter gene detection of GAS5 on the activity of EZH2 promoter; 5.RIP test to detect the binding of GAS5 to transcription factor E2F4; 6. CHIP test for E2F4 and EZH2 initiation 7. construction of EZH2 overexpression vector and shRNA carrier, transfection of T24, EJ cells, RT-QPCR test to detect the regulation of EZH2 to miR-101, and 8. double luciferase reporter gene to detect the effect of EZH2 on the promoter activity of miR-101; 9. different concentrations (0,1.5,2.0,2.5 um M) GA 5 regulating effect: before and after 10. silent GAS5, MTT, flow, and Western blot were used to detect the effect of GA on bladder cancer cells: 11. Balb-c nude mice were constructed for ectopic transplantation of bladder cancer, and GA was used to detect the growth of bladder cancer in vitro through GAS5. [results 33 cases of]1.43 cases with bladder cancer were downregulated (76.74%), and non grass-roots invasive bladder cancer (T). A+Tis+T1) the middle and lower rates were 59.09%, the downregulation rate of invasive cancer was 95.24%, the downregulation rate of lymph node metastasis was 78.57%, the downregulation rate of lymph node metastasis was 75.86%, and the downregulation rate of distant metastasis was 77.5%. The down regulation rate of distant metastasis was 66.67%.2. over expression of GAS5, the increase of bladder cancer cell rate, the increase of apoptosis rate, caspase-3 protein Activation increased: after silence GAS5, the increment rate of bladder cancer cells increased, the apoptosis rate decreased, the activation of caspase-3 protein decreased by.3. Western blot, and GAS5 could inhibit the expression of EZH2 protein. RT-QPCR showed that GAS5 could inhibit the expression of EZH2 mRNA, and the dual fluorescein reporter gene detection showed that GAS5 could inhibit the activity of promoter. The results suggest that GAS5 can bind to the specificity of the transcription factor E2F4. The CHIP experiment suggests that E2F4 can specifically bind to.5. RT-QPCR in the EZH2 promoter region, suggesting that EZH2 can inhibit the expression of miR-101. The dual fluorescein reporter gene detection suggests that EZH2 can inhibit the active.6.GA of miR-101-2 promoter to regulate the expression, and the dose is in a dose. Positive correlation, GA can significantly induce apoptosis of bladder cancer cells, after silence GAS5, the ability of GA to induce apoptosis of bladder cancer cells decreased significantly..7. animal experiments showed that GA and GAS5 could significantly inhibit the growth of bladder cancer. After silent GAS5, the ability of GA to inhibit the growth of bladder cancer decreased significantly. [conclusion]1.GAS5 is significantly lower in bladder cancer tissue. The low expression rate of the musculocutaneous invasive bladder cancer was significantly higher than that of non base invasive bladder cancer. The expression level was not associated with lymph node metastasis and distant metastasis; in 2. bladder cancer, EZH2 could regulate the expression of miR-101 from the transcriptional level; 3.GAS5 could inhibit the EZH2 promoter activity through the transcription factor E2F4 and inhibit EZH2 mRNA, eggs. The expression of white cells further promoted the expression of miR-101 and induced apoptosis of bladder cancer cells. 4.GA blocked the EZH2-miR-101 pathway through GAS5 and promoted the apoptosis of bladder cancer cells in vivo and in vitro.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.14

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