新型化疗药藤黄酸通过长链非编码RNA GAS5诱导膀胱癌细胞凋亡的研究

发布时间：2018-06-21 23:13

本文选题：膀胱癌 + 藤黄酸　；参考：《华中科技大学》2015年博士论文

【摘要】：[背景]藤黄酸(GA)作为新近发现的天然抗肿瘤药物,在膀胱癌中显示了强有效的抗癌作用,极具临床应用前景,阐明其作用机制已成为其临床应用的研究重点。长链非编码RNA (LncRNA)过去被视为基因组转录的“噪音”,近年研究发现LncRNA参与生物调节的各个环节,其表达异常与多种疾病的发生、发展密切相关。LncRNA GAS5在多种肿瘤中低表达,被认为是重要的抑癌基因,但GAS5与膀胱癌发生、发展之间的关系仍不明确,作用机制也有待深入研究。[目的]1.检测长链非编码RNA GAS5在膀胱癌中的表达情况,分析其与膀胱癌临床分级、分期之间的关系；2.探讨GAS5在膀胱癌中的作用并阐述其作用机制；3.进一步完善新型化疗药GA抑制膀胱癌的作用机制。[方法]1.收集至我院行膀胱癌根治性切除术的病例43例,同时获取癌组织与周围正常组织,提取RNA行RT-QPCR实验检测长链非编码RNA GAS5的表达水平,并分析GAS5的表达与临床分级、分期之间的关系；2.分别合成GAS5 siRNA与shRNA沉默GAS5,构建GAS5过表达载体(GV144-GAS5)过表达GAS5,分别转染膀胱癌细胞株T24、EJ；3.GAS5在膀胱癌细胞中的作用：MTT检测细胞生存率,流式细胞术检测细胞凋亡率,Western blot检测蛋白表达水平；4.双荧光素酶报告基因检测GAS5对EZH2启动子活性的影响；5.RIP实验检测GAS5与转录因子E2F4的结合作用；6. CHIP实验检测E2F4与EZH2启动子的结合作用；7.构建EZH2过表达载体与shRNA载体,转染T24、EJ细胞,RT-QPCR实验检测EZH2对miR-101的调节作用；8.双荧光素酶报告基因检测EZH2对miR-101的启动子活性的影响；9.不同浓度(0、1.5、2.0、2.5 μM)GA分别作用于T24、EJ细胞,RT-QPCR检测GA对GAS5的调节作用：10.沉默GAS5前后,分别用MTT、流式、Western blot检测GA对膀胱癌细胞的作用：11. balb-c裸鼠构建膀胱癌异位移植模型,检测GA通过GAS5体外抑制膀胱癌生长。[结果]1.43例膀胱癌标本中33例GAS5表达下调(76.74%)；非基层浸润性膀胱癌(Ta+Tis+T1)中下调率为59.09%,浸润性癌中下调率为95.24%；无淋巴结转移的下调率为78.57%,有淋巴结转移的下调率为75.86%；无远处转移的下调率为77.5%,有远处转移的下调率为66.67%。2.过表达GAS5后膀胱癌细胞增率下降,凋亡率上升,caspase-3蛋白活活化增加：沉默GAS5后膀胱癌细胞增值率上升,凋亡率下降,caspase-3蛋白活化减少。3. Western blot显示GAS5能够抑制EZH2蛋白的表达,RT-QPCR显示GAS5能够抑制：EZH2 mRNA的表达,双荧光素报告基因检测提示GAS5能够抑制EZH2启动子的活性。4.RIP实验提示GAS5能与转录因子E2F4特异性的结合,CHIP实验提示E2F4能够特异性的结合到EZH2启动子区域。5. RT-QPCR提示EZH2能够抑制miR-101的表达,双荧光素报告基因检测提示EZH2能够抑制miR-101-2启动子的活性。6.GA能够正性调节GAS5的表达,且呈剂量正相关性；GA能够显著诱导膀胱癌细胞的凋亡,沉默GAS5后GA诱导膀胱癌细胞凋亡的能力显著下降。7.动物实验显示GA、GAS5均能够显著抑制膀胱癌的生长；沉默GAS5后,GA抑制膀胱癌生长的能力显著下降。[结论]1.GAS5在膀胱癌组织中显著低表达,且肌层浸润性膀胱癌中低表达率明显高于非基层浸润性膀胱癌,其表达水平与淋巴结转移、远处转移无明显关系;2.膀胱癌中,EZH2可以从转录水平负性调节miR-101的表达；3.GAS5可以通过转录因子E2F4抑制EZH2启动子活性,抑制EZH2 mRNA、蛋白的表达,进而促进miR-101的表达,诱导膀胱癌细胞凋亡;4.GA通过GAS5阻断EZH2-miR-101通路促进体内外膀胱癌细胞凋亡。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14

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