缺氧对miR-194-1启动子活性及对前列腺癌细胞迁移侵袭的影响

发布时间：2018-07-06 18:56

本文选题：miR-194-1 + HIF-1α　；参考：《中山大学》2017年硕士论文

【摘要】：本研究以雄激素非依赖型前列腺癌细胞系DU-145为研究背景,探讨缺氧对miR-194-1启动子活性的影响,对DU-145细胞迁移和侵袭能力的影响,以及缺氧诱导因子HIF-1α与mi R-194-1的关系。揭示HIF-1α及miR-194在前列腺癌的发生发展中的作用和意义,为DU-145发病机理研究、病理诊断及预后判断等提供新的依据,同时将HIF-1α或miRNA作为靶基因的基因治疗可能成为日后治疗前列腺癌的一种有效途径。首先基于表观遗传学数据库预测了miRNA-194-1上游启动子序列和缺氧反应元件(HRE)序列,构建含启动子序列的荧光素酶报告载体,通过截短表达报告载体和瞬时转染检测萤火虫荧光素酶强度的方法确定其启动子序列。为了验证缺氧对miR-194-1转录的影响,我们构建了含有HRE缺失突变的miR-194-1启动子载体,并检测启动子的荧光活性。然后用氯化钴模拟缺氧,研究缺氧处理对前列腺上皮细胞和前列腺癌细胞的HIF-1α与miR-194-1表达的影响。构建HIF-1α过表达载体以及合成HIF-1αsiRNA序列并转染细胞,通过RT-qPCR和Western Blot实验检测HIF-1α和miR-194-1的表达水平,进一步验证HIF-1α与miR-194-1的关系,之后在细胞水平通过划痕实验和transwell实验探讨缺氧对前列腺癌细胞DU-145迁移和侵袭的影响。结果:(1)成功构建pGL3-Basic-194-1和缺失HRE序列的pGL3-Basic-194-1-△HRE荧光素酶载体,转染后pGL3-Basic-194-1的启动子荧光活性显著升高,这表明启动子序列是正确的;pGL-3-Basic-194-1-△HRE比空载体的荧光活性还要低,且在常氧和缺氧条件下,荧光活性都没发生变化,表明缺失HRE序列后,启动子活性显著降低,且不再受缺氧调控,验证了miR-194-1核心启动子序列中包含HRE序列。(2)miR-194-1在前列腺癌细胞中的表达量低于前列腺上皮细胞系,提示了其作为日后前列腺癌诊断治疗的可能性。(3)成功构建HIF-1α过表达载体和合成siRNA序列,转染细胞后,检测到HIF-1α过表达会引起miR-194-1表达的下调;抑制HIF-1α会导致miR-194-1表达的上调。(4)HIF-1α过表达或缺氧会使DU-145细胞的迁移和侵袭能力增强;siRNA抑制HIF-1α表达,则细胞迁移和侵袭能力下降。结论:实验成功构建了miR-194-1的启动子序列,且验证了缺氧会调控miR-194-1启动子的转录调控。并且缺氧或HIF-1α过表达会促进DU-145细胞的迁移和侵袭;转染siRNA会抑制DU-145的迁移和侵袭。
[Abstract]:In this study, androgen independent prostate cancer cell line DU-145 was used to investigate the effects of hypoxia on the activity of miR-194-1 promoter, migration and invasion of DU-145 cells, and the relationship between hypoxia inducible factor HIF-1 伪 and miR-194-1. To reveal the role and significance of HIF-1 伪 and miR-194 in the occurrence and development of prostate cancer, and to provide a new basis for the study of pathogenesis, pathological diagnosis and prognosis of DU-145. At the same time, gene therapy with HIF-1 伪 or miRNA as target gene may be an effective way to treat prostate cancer in the future. Firstly, the upstream promoter sequence and hypoxia response element (HRE) sequence of miRNA-194-1 were predicted based on epigenetics database, and luciferase report vector containing promoter sequence was constructed. The promoter sequence was determined by truncated expression report vector and transient transfection to detect the luciferase intensity of firefly. In order to verify the effect of hypoxia on the transcription of miR-194-1, we constructed the promoter vector containing HRE deletion mutation and detected the fluorescence activity of the promoter. Then the effects of hypoxia on the expression of HIF-1 伪 and miR-194-1 in prostate epithelial cells and prostate cancer cells were studied. HIF-1 伪 overexpression vector was constructed and HIF-1 伪 siRNA sequence was synthesized and transfected into cells. The expression levels of HIF-1 伪 and miR-194-1 were detected by RT-qPCR and Western Blot to further verify the relationship between HIF-1 伪 and miR-194-1. Then the effects of hypoxia on the migration and invasion of prostate cancer cell DU-145 were investigated at cell level by scratch test and transwell assay. Results: (1) pGL3-Basic-194-1 and pGL3-Basic-194-1-HRE luciferase vector were constructed successfully. The fluorescence activity of pGL3-Basic-194-1 promoter was significantly increased after transfection, which indicated that the promoter sequence was correct and the fluorescence activity of pGL3-Basic-194-1-HRE was lower than that of empty vector. The fluorescence activity did not change in normoxic and hypoxic conditions, indicating that the promoter activity decreased significantly after HRE deletion, and was no longer regulated by hypoxia. It was verified that the core promoter sequence of miR-194-1 contained HRE sequence. (2) the expression of miR-194-1 in prostate cancer cells was lower than that in prostate epithelial cells. The results suggested that HIF-1 伪 overexpression vector and siRNA sequence were successfully constructed. After transfection, the overexpression of HIF-1 伪 resulted in down-regulation of miR-194-1 expression. Inhibition of HIF-1 伪 resulted in upregulation of miR-194-1 expression. (4) overexpression of HIF-1 伪 or hypoxia increased migration and invasion ability of DU-145 cells. SiRNA inhibited HIF-1 伪 expression, then cell migration and invasion decreased. Conclusion: the promoter sequence of miR-194-1 was successfully constructed, and it was verified that hypoxia could regulate the transcription of miR-194-1 promoter. Hypoxia or overexpression of HIF-1 伪 promoted migration and invasion of DU-145 cells, and transfection of siRNA inhibited migration and invasion of DU-145 cells.
【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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