p55PIK调控AFP表达的信号通路机制研究

发布时间：2018-05-03 17:29

  本文选题：AFP + p55PIK　； 参考：《湖北工业大学》2017年硕士论文

【摘要】：目的:p55PIK是PI3K的一个调节亚基,在调节细胞周期以及炎症进程中都发挥着重要作用,并且p55PIK在肝癌临床肿瘤样品中大量表达,前期实验发现向肝癌细胞中添加p55PIK特异性抑制剂P15多肽,可以起到抑制肝癌细胞生长,以及下调肝癌诊断指标AFPmRNA表达水平的效应,所以本课题的研究目的是阐明p55PIK调控AFP表达的分子机制。方法:以肝癌细胞系HepG2为研究对象,超表达和沉默p55PIK,然后利用实时定量PCR以及westernblot的方法检测细胞中AFP的mRNA含量以及蛋白含量,明确肝癌细胞中p55PIK对AFP的调节效果;用p55PIK特异性抑制剂P15处理肝癌细胞,检测NF-κB的活性,证实在肝癌细胞中p55PIK可以调节NF-κB的活性;以肝癌细胞系HepG2为研究对象,分别加入NF-κB特异性抑制剂PDTC以及p55PIK特异性抑制剂P15后,利用实时定量PCR以及westernblot的方法检测细胞中AFP的mRNA含量以及蛋白含量,证实肝癌细胞中NF-κB对AFP的调节效果;通过同时上调p55PIK以及抑制NF-κB的方法,观察抑制NF-κB是否可以抵消上调p55PIK导致的AFP上调,证实P55PIK通过NF-kB信号路径解调节AFP表达;利用荧光素酶报告系统,确定AFP基因启动子上受NF-κB调控的片段;利用生物信息学的手段,分析并预测AFP基因启动子上受NF-κB调控的片段中可能与NF-κB结合的位点;利用点突变技术,观察上述DNA位点对p55PIK/NF-κB对AFP转录活性的影响,明确哪些位点在p55PIK/NF-κB调控AFP转录活性中发挥重要作用。结果:观察到AFP的mRNA水平与加入的P15量呈浓度依赖性,随着加入的P15量增加AFP的mRNA表达量逐渐减少,并且沉默p55PIK后AFP的蛋白表达量显著降低,超表达p55PIK后AFP的表达量显著增加;向HepG2细胞中加入NF-κB抑制剂(PDTC)后,会起到下调AFP的mRNA及蛋白表达水平的效应;分别向HepG2细胞中加入P15和PDTC后,检测NF-κB通路关键蛋白的表达情况,加入P15后p65表达量没有显著变化,p65(ser536)磷酸化水平显著降低,IKBα表达量显著升高,IKBα(ser36)磷酸化水平显著降低,加入PDTC后p65表达量显著降低,p65(ser536)磷酸化水平显著降低,IKBα表达量显著升高,IKBα(ser36)磷酸化水平显著降低,观察(control,p55PIK,p55PIK+PDTC)实验组,PDTC可以抵消由于过表达p55PIK而引起的AFP表达量升高;构建得到AFP基因上游调控区不同片段的荧光素酶报告基因载体(-5229/-16)-AFP,(-3375/-16)-AFP,(-1865/-16)-AFP和(-225/-16)-AFP,酶切以及测序鉴定分子量序列正确,检测分别加入P15和PDTC对不同长度的AFP启动子转录活性的影响,结果显示(-5184/+29)-AFP和(-3330/+29)-AFP变化最为显著,即NF-κB的调控位点主要在(-5184,-1820)片段上;对报告基因活性变化最大的片段(-5184,-1820)进行预测,位点(-4717/-4726),(-4372/4381),(-3171/3180)可能是NF-κB与AFP启动子结合并调控该片段的转录活性的位点;以全长AFP基因启动子报告基因载体(-5229/-16)-AFP为模板,对上述位点进行点突变,并比较添加P15和PDTC前后报告基因载体突变对AFP基因转录活性的影响程度,结果显示(-4717/-4726),(-4372/4381)突变后P15和PDTC不再能够调节AFP的转录活性,即位点(-4717/-4726),(-4372/4381)是在p55PIK/NF-κB调控AFP转录活性的关键位点。结论:在肝癌细胞中,p55PIK介导的信号通路参与了肝癌生物标志蛋白AFP的表达;实验结果还表明p55PIK对AFP表达的调控是通过NF-κB信号通路实现的;在AFP启动子上游的NF-κB的作用位点(-4717/-4726),(-4372/4381)在p55PIK/NF-κB调节AFP转录活性中发挥重要作用。
[Abstract]:Objective: p55PIK is a regulatory subunit of PI3K, which plays an important role in regulating cell cycle and inflammatory processes. And p55PIK is expressed in a large number of cancer samples in liver cancer. The early experiments have found that the addition of p55PIK specific inhibitor, P15 polypeptide, can inhibit the growth of hepatoma cells and reduce the diagnosis of liver cancer. The purpose of this study is to elucidate the molecular mechanism of p55PIK regulation of the expression of AFP. Methods: using the hepatoma cell line HepG2 as the research object, the purpose of this study is to overexpress and silence p55PIK, and then use the real-time quantitative PCR and Westernblot to detect the mRNA content and protein content of AFP in the cell, and to clear the liver of the AFP. The effect of p55PIK on the regulation of AFP in cancer cells, the activity of NF- kappa B was detected by p55PIK specific inhibitor P15, and it was proved that p55PIK could regulate the activity of NF- kappa B in the hepatoma cells. The quantitative PCR and Westernblot methods were used to detect the mRNA content and protein content of AFP in the cells, and to verify the regulation effect of NF- kappa B on AFP in the hepatoma cells. P expression, using the luciferase reporter system to determine the AFP gene promoter regulated by NF- kappa B, and using bioinformatics to analyze and predict the loci that may be associated with NF- kappa B in the NF- kappa B regulated segments of the AFP gene promoter, and use point mutation technique to observe the reflection of p55PIK/NF- kappa B to the transcriptional activity of p55PIK/NF- kappa B. Make sure which sites play an important role in the p55PIK/NF- kappa B regulation of AFP transcriptional activity. Results: the mRNA level of AFP was observed in a concentration dependent manner with the P15 amount added, and the mRNA expression of AFP decreased gradually with the increase of P15 amount added, and the amount of protein expression of AFP after p55PIK decreased significantly. After adding the NF- kappa B inhibitor (PDTC) to the HepG2 cells, the mRNA and protein expression level of AFP were downregulated. After adding P15 and PDTC to the HepG2 cells, the expression of the key protein in the NF- kappa B pathway was detected. The level of phosphorylation of IKB alpha (ser36) decreased significantly. After adding PDTC, the expression of p65 decreased significantly, the level of phosphorylation of p65 (ser536) decreased significantly, the expression of IKB a significantly increased, and the level of IKB alpha (ser36) phosphorylation was significantly reduced, and the experimental group (control, p55PIK, p55PIK+PDTC) was observed. The results showed that AFP gene vector (-5229/-16) -AFP, (-3375/-16) -AFP, (-1865/-16) -AFP and (-225/-16) -AFP, (-1865/-16) -AFP and (-225/-16) -AFP, enzyme digestion and sequencing identification were correct to detect the effect of P15 and PDTC on the transcriptional activity of AFP promoter of different lengths, and the results showed (- 5184/+29) the changes in -AFP and (-3330/+29) -AFP are most significant, that is, the regulation sites of NF- kappa B are mainly on (-5184, -1820) fragments; the fragments (-5184, -1820) that have the largest changes in the activity of the reported gene (-4717/-4726), (-4717/-4726), (-4372/4381), may be the loci of binding and regulating the transcriptional activity of the fragment. Using the full length AFP gene promoter reporter gene carrier (-5229/-16) -AFP as a template, the point mutation was carried out, and the effect of the report gene carrier mutation before and after the addition of P15 and PDTC on the transcriptional activity of the AFP gene was compared. The results showed that (-4717/-4726), P15 and PDTC no longer could regulate the AFP transcriptional activity after (-4372/4381) mutation. (-4717/-4726), (-4372/4381) is a key site in the regulation of AFP transcriptional activity by p55PIK/NF- kappa B. Conclusion: in hepatoma cells, the p55PIK mediated signaling pathway participates in the expression of the hepatocellular carcinoma biomarker AFP; the experimental results also indicate that the regulation of p55PIK on AFP expression is realized through NF- kappa B signaling pathway; The site of action (-4717/-4726), (-4372/4381) plays an important role in regulating p55PIK/NF- transcriptional activity by p55PIK/NF- kappa.

【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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