槐果碱和血根碱通过靶向miR-21和miR-16调控肿瘤细胞增殖、凋亡的机制研究

发布时间：2018-02-14 02:51

本文关键词： miR-16 miR-21 槐果碱血根碱增殖、凋亡　出处：《西安电子科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：癌症是威胁人类生命的一种严重病症。癌症治愈的几率远小于其他疾病,几乎是不治之症。癌症治疗一直是医学领域最为活跃的研究课题之一。如果癌症能够提早发现和治疗,治愈的几率也将大幅提高。研究发现,在肿瘤细胞中miR-21特异性高表达,miR-16低表达显示这两种microRNA可作为癌症治疗的潜在靶标。此外,现有研究发现,一些天然药物能够改变microRNA的表达,从而影响肿瘤细胞的生长、增殖或分化过程。本文主要筛选能够调控miR-21和miR-16表达的天然药物以及研究它们对肿瘤细胞增殖和凋亡的影响。因此,本文首选通过萤火虫荧光素酶基因的3’UTR插入与miR-21或者miR-16完全互补的序列,构建成响应miR-21或miR-16表达的miR-21荧光报告基因和miR-16荧光报告基因,通过检测荧光变化来间接反映miR-21和mi R-16的表达量。荧光素酶实验发现槐果碱上调miR-21的表达,血根碱下调miR-16的表达。首先,对miR-21和槐果碱进行细胞和动物机制的研究。具体的实验有实时定量PCR、免疫印迹实验(Western blot)、细胞增殖检测实验(CCK-8)等。通过实时定量PCR证明槐果碱上调miR-21的表达;通过免疫印迹法证明槐果碱下调mi R-21的靶基因PTEN的表达;通过信号通路实验,发现槐果碱通过NF-KB和P38MAPK信号通路上调miR-21的表达;细胞增殖检测实验发现anti-miR-21 RNA和槐果碱能够共同抑制A549细胞的增殖。槐果碱裸鼠肿瘤治疗研究,发现槐果碱对肿瘤生长有抑制作用。其次,miR-16和血根碱进行了细胞和动物机制的研究。具体的实验有实时定量PCR、免疫印迹实验、caspase-3活性检测实验和裸鼠肿瘤治疗实验。实时定量PCR实验发现了血根碱下调miR-16的表达;免疫印迹实验发现了血根碱下调Bcl-2蛋白;通过caspase-3活性检测实验,表明了miR-16和血根碱协同激活caspase-3的表达;裸鼠成瘤实验发现血根碱能够抑制裸鼠肿瘤生长,即血根碱具有抗癌作用。总之,通过研究发现槐果碱通过靶向miR-21对肿瘤细胞增殖有抑制作用,血根碱通过靶向miR-16促进细胞凋亡,因而槐果碱和血根碱可以作为潜在的药物用于肿瘤的治疗。
[Abstract]:Cancer is a serious disease that threatens human life. Cancer is much less likely to be cured than other diseases and almost incurable. Cancer treatment has been one of the most active research topics in medicine. The likelihood of cure will also be significantly increased. Studies have found that the high expression of miR-21 specifically in cancer cells suggests that the two types of microRNA may be potential targets for cancer treatment. Some natural drugs can alter the expression of microRNA, thus affecting the growth of tumor cells. Proliferation or differentiation. In this paper, we select natural drugs that can regulate the expression of miR-21 and miR-16 and study their effects on the proliferation and apoptosis of tumor cells. In this paper, we first constructed the miR-21 fluorescent reporter gene and the miR-16 fluorescent reporter gene in response to the expression of miR-21 or miR-16 by inserting 3UTR of the luciferase gene of the firefly worm into a sequence that is completely complementary to miR-21 or miR-16. Luciferase assay showed that Sophorine upregulated the expression of miR-21 and blood root alkaloids down-regulated the expression of miR-16. The cellular and animal mechanisms of miR-21 and sophorline were studied. The specific experiments were real-time quantitative PCRs, Western blotl assay and cell proliferation assay (CCK-8). It was proved by real-time quantitative PCR that Sophorine upregulated the expression of miR-21. The results showed that Sophorine down-regulated the expression of PTEN in target gene of miR-21 by Western blot, and up-regulated the expression of miR-21 through NF-KB and p38 MAPK signaling pathway through signal pathway experiment. Cell proliferation assay showed that anti-miR-21 RNA and sophorline could inhibit the proliferation of A549 cells. It was found that Sophorine had inhibitory effect on tumor growth. Secondly, the cellular and animal mechanisms of miR-16 and root alkaloid were studied. The specific experiments included real-time quantitative PCR, Western blot assay, caspase-3 activity detection and tumor therapy in nude mice. Real-time quantitative PCR assay showed that root alkaloids down-regulated the expression of miR-16; Western blotting showed that root alkaloids down-regulated Bcl-2 protein; caspase-3 activity test showed that miR-16 and root alkaloids co-activated caspase-3 expression; in nude mice tumorigenesis, root alkaloids inhibited tumor growth in nude mice. In conclusion, it was found that Sophorine inhibits the proliferation of tumor cells through targeted miR-21, and root alkaloids promote cell apoptosis through targeted miR-16. Therefore, Sophorine and root alkaloids can be used as potential drugs for the treatment of tumors.
【学位授予单位】：西安电子科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730.5

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