SKP2基因抑制剂的高通量筛选与功能研究

发布时间：2018-03-04 20:53

本文选题：SKP2前列腺癌　切入点：报告基因试验　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:异常的基因表达是肿瘤的一个重要标志,通常驱动着细胞增殖,分化和恶性肿瘤细胞的转移。细胞周期失衡是肿瘤发生的重要因素。前列腺癌是公认的男性发病率最高的非上皮源性恶性肿瘤之一,也是因癌症导致死亡的重要因素。多项研究指出,与细胞周期调控密切相关的SKP2基因的异常表达在前列腺癌的发展中起到了重要的作用。在多种人类肿瘤中SKP2均呈现过表达,其异位表达也会促进肿瘤的发生。这些都提示SKP2的表达异常在恶性肿瘤的发展中具有重要的意义,而抑制SKP2基因的过表达显然是恶性肿瘤治疗中颇具前景的战略。转录治疗是基因治疗的热点内容,通过直接干预转录过程对肿瘤细胞异常基因表达进行纠正。随着越来越多的治疗癌症的靶向基因得以确定,以及新药研发技术的不断提高,转录治疗也得以迅速发展。如何在大量不同种类的化合物中筛选出有效的抗肿瘤药物,是药物研发的重要环节。报告基因试验的推广使得药物的高通量筛选(HTS)变得更便捷、经济和高效。然而,受到不完整的顺式作用原件的调控以及表观遗传环境差异的影响,随机插入的报告基因常常因位于侧面的凝聚染色质而表观沉默,影响了传统的报告基因试验的可靠性。考虑到这些问题,Yan等研发出一种基于内部核糖体进入位点原位报告基因试验,报告基因可以被设计并选择性地插入一段基因序列最接近内源基因编码区下游的位置,并在原有的转录调节机制下与内源基因共同表达。这种新型的报告基因试验避免了传统方法的不足,其有效性有待进一步论证。目的:考虑到前列腺癌的高发病率、死亡率以及治疗效果的不确切,以及SKP2基因异常表达在前列腺癌中的重要作用,我们期待能够应用Yan等研发的基于内部核糖体进入位点原位报告基因试验,通过高通量筛选,找到能够在转录水平抑制SKP2基因过表达的化合物,并通过后续实验验证其有效性并尝试探寻其机制,以期为恶性肿瘤的研究和治疗提供新的思路。方法:利用前列腺癌细胞DU145细胞系,通过Yan等研发的基于内部核糖体进入位点原位报告基因试验技术,构建共表达SKP2-FLuc DU145细胞。然后,利用上述重组细胞通过luciferase试验进行HTS药物筛选,我们共筛选了来自NCI化合物库、She实验组和Chembridge化合物库超过8000种化合物。利用前列腺癌细胞(DU145、PC3、22RV1和C4-2B细胞系),通过real-time PCR试验、Western blot试验和MTT试验等将筛选出的化合物做进一步验证。结果:通过上述新型的报告基因试验技术,我们成功通过HTS和luciferase试验初步筛选出10个化合物。将这些化合物进一步做real-time PCR试验,得到在转录水平抑制SKP2的化合物48X(NCI librarys)。通过多个前列腺癌细胞系Western blot试验,我们进一步证实该化合物对前列腺癌细胞SKP2具有抑制作用,并且该效应与剂量和作用时间呈正相关,并且在加药后8小时即可有效抑制SKP2。MTT结果证实,4组前列腺癌细胞系细胞在经过48X化合物处理后,出现细胞增殖能力降低的功能性改变。结论:本研究进一步证实了Yan等开发的基于内部核糖体进入位点原位报告基因试验技术的可行性和有效性。本研究所筛选出的48X(NCI librarys)化合物对前列腺癌细胞SKP2基因具有在转录水平上的抑制作用,并且对前列腺癌细胞具有功能性效应(细胞增殖能力下降)。
[Abstract]:Background: abnormal gene expression is an important sign of the tumor, usually drives cell proliferation, differentiation and metastasis of malignant tumor cells. Cell cycle imbalance is an important factor in tumor. Prostate cancer is known as the male incidence of non epithelial malignant tumor with the highest, is also an important factor leading to death due to cancer. A number of studies have indicated that the abnormal expression of SKP2 gene is closely related with the regulation of the cell cycle in the development of prostate cancer plays an important role in a variety of human tumors. SKP2 showed the expression of the ectopic expression may promote the occurrence of tumors. These results suggest that abnormal expression of SKP2 plays an important role in the development of malignant tumor, inhibit the overexpression of the SKP2 gene is obviously the treatment of malignant tumor, promising strategy. Antiretroviral therapy is a hot topic in gene therapy, through direct intervention The process of transcription to correct abnormal gene expression of tumor cells with cancer treatment. More and more target genes to be determined, and the research and development of new drugs and technology continue to improve, antiretroviral therapy is also developing rapidly. How to select the effective anti-tumor drugs in a large number of different types of compounds, is an important link in drug development. Promotion report gene test allows high throughput drug screening (HTS) has become more convenient, economical and efficient. However, affected by the incomplete cis acting original epigenetic regulation and environmental differences, often because of the random insertion of reporter gene is located on the side of the condensed chromatin and epigenetic silencing, affects the reliability of reporter gene the traditional test. Considering these problems, Yan developed an internal ribosome entry site based on in situ test report gene, the reporter gene can be designed and Selectively inserted a gene sequence of endogenous gene encoding region closest to the downstream position of the mechanism and regulation of endogenous gene transcription in the original expression. This new type of reporter gene assay to avoid the shortcomings of traditional methods, its effectiveness should be further argued. Objective: Considering prostate cancer high incidence and mortality the treatment effect is not exact, an important role in prostate cancer and abnormal expression of SKP2 gene, we look forward to the application of Yan based on the development of the internal ribosome entry site reporter gene by in situ test, high-throughput screening, found to inhibit SKP2 gene expression of the compounds at the transcriptional level, and through subsequent experiments to verify its effectiveness and try to explore its mechanism, to provide new ideas for research and treatment for malignant tumors. Methods: the prostate cancer cell line DU145, via Y An based on the development of the internal ribosome entry site reporter gene in situ test technique, construct the co expression of SKP2-FLuc DU145 cells. Then, the HTS drug screening by luciferase test using the recombinant cells, we were selected from the NCI compound library, experimental group She and Chembridge compound library of more than 8000 kinds of compounds. The prostate cancer cells (DU145. PC3,22RV1 and C4-2B cell lines by real-time PCR), Western test, blot test and MTT test will be screened compounds for further verification. Results: by the new report by test technology, we successfully passed the HTS and luciferase test results showed that 10 compounds. These compounds will further make the real-time PCR test, get inhibition of SKP2 at the transcriptional level of compound 48X (NCI librarys). Through a number of prostate cancer cell line Western blot test, we enter a Further confirmed that the compounds have inhibitory effects on prostate cancer SKP2 cells, and the positive effect and dose and time related, and confirmed in 8 hours after dosing can effectively inhibit the SKP2.MTT results, the prostate cancer cell line 48X cells in the 4 groups after treatment of functional compounds, reduce the cell proliferation. Conclusion: This study further confirmed that the Yan based on the development of the internal ribosome entry site reporter gene assay technology feasibility and effectiveness. This study selected 48X (NCI librarys) compounds have inhibitory effect on the transcription level of SKP2 gene in prostate cancer cells, and has the functional effects on prostate cancer cells (the cells decreased the proliferation ability).

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3

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