本文選題：AFP + p55PIK��； 參考：《湖北工業(yè)大學》2017年碩士論文

【摘要】：目的:p55PIK是PI3K的一個調節(jié)亞基,在調節(jié)細胞周期以及炎癥進程中都發(fā)揮著重要作用,并且p55PIK在肝癌臨床腫瘤樣品中大量表達,前期實驗發(fā)現向肝癌細胞中添加p55PIK特異性抑制劑P15多肽,可以起到抑制肝癌細胞生長,以及下調肝癌診斷指標AFPmRNA表達水平的效應,所以本課題的研究目的是闡明p55PIK調控AFP表達的分子機制。方法:以肝癌細胞系HepG2為研究對象,超表達和沉默p55PIK,然后利用實時定量PCR以及westernblot的方法檢測細胞中AFP的mRNA含量以及蛋白含量,明確肝癌細胞中p55PIK對AFP的調節(jié)效果;用p55PIK特異性抑制劑P15處理肝癌細胞,檢測NF-κB的活性,證實在肝癌細胞中p55PIK可以調節(jié)NF-κB的活性;以肝癌細胞系HepG2為研究對象,分別加入NF-κB特異性抑制劑PDTC以及p55PIK特異性抑制劑P15后,利用實時定量PCR以及westernblot的方法檢測細胞中AFP的mRNA含量以及蛋白含量,證實肝癌細胞中NF-κB對AFP的調節(jié)效果;通過同時上調p55PIK以及抑制NF-κB的方法,觀察抑制NF-κB是否可以抵消上調p55PIK導致的AFP上調,證實P55PIK通過NF-kB信號路徑解調節(jié)AFP表達;利用熒光素酶報告系統(tǒng),確定AFP基因啟動子上受NF-κB調控的片段;利用生物信息學的手段,分析并預測AFP基因啟動子上受NF-κB調控的片段中可能與NF-κB結合的位點;利用點突變技術,觀察上述DNA位點對p55PIK/NF-κB對AFP轉錄活性的影響,明確哪些位點在p55PIK/NF-κB調控AFP轉錄活性中發(fā)揮重要作用。結果:觀察到AFP的mRNA水平與加入的P15量呈濃度依賴性,隨著加入的P15量增加AFP的mRNA表達量逐漸減少,并且沉默p55PIK后AFP的蛋白表達量顯著降低,超表達p55PIK后AFP的表達量顯著增加;向HepG2細胞中加入NF-κB抑制劑(PDTC)后,會起到下調AFP的mRNA及蛋白表達水平的效應;分別向HepG2細胞中加入P15和PDTC后,檢測NF-κB通路關鍵蛋白的表達情況,加入P15后p65表達量沒有顯著變化,p65(ser536)磷酸化水平顯著降低,IKBα表達量顯著升高,IKBα(ser36)磷酸化水平顯著降低,加入PDTC后p65表達量顯著降低,p65(ser536)磷酸化水平顯著降低,IKBα表達量顯著升高,IKBα(ser36)磷酸化水平顯著降低,觀察(control,p55PIK,p55PIK+PDTC)實驗組,PDTC可以抵消由于過表達p55PIK而引起的AFP表達量升高;構建得到AFP基因上游調控區(qū)不同片段的熒光素酶報告基因載體(-5229/-16)-AFP,(-3375/-16)-AFP,(-1865/-16)-AFP和(-225/-16)-AFP,酶切以及測序鑒定分子量序列正確,檢測分別加入P15和PDTC對不同長度的AFP啟動子轉錄活性的影響,結果顯示(-5184/+29)-AFP和(-3330/+29)-AFP變化最為顯著,即NF-κB的調控位點主要在(-5184,-1820)片段上;對報告基因活性變化最大的片段(-5184,-1820)進行預測,位點(-4717/-4726),(-4372/4381),(-3171/3180)可能是NF-κB與AFP啟動子結合并調控該片段的轉錄活性的位點;以全長AFP基因啟動子報告基因載體(-5229/-16)-AFP為模板,對上述位點進行點突變,并比較添加P15和PDTC前后報告基因載體突變對AFP基因轉錄活性的影響程度,結果顯示(-4717/-4726),(-4372/4381)突變后P15和PDTC不再能夠調節(jié)AFP的轉錄活性,即位點(-4717/-4726),(-4372/4381)是在p55PIK/NF-κB調控AFP轉錄活性的關鍵位點。結論:在肝癌細胞中,p55PIK介導的信號通路參與了肝癌生物標志蛋白AFP的表達;實驗結果還表明p55PIK對AFP表達的調控是通過NF-κB信號通路實現的;在AFP啟動子上游的NF-κB的作用位點(-4717/-4726),(-4372/4381)在p55PIK/NF-κB調節(jié)AFP轉錄活性中發(fā)揮重要作用。
[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.

【學位授予單位】：湖北工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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相關期刊論文 前10條

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