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

【摘要】：目的:p55PIK是PI3K的一個(gè)調(diào)節(jié)亞基,在調(diào)節(jié)細(xì)胞周期以及炎癥進(jìn)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(dá),前期實(shí)驗(yàn)發(fā)現(xiàn)向肝癌細(xì)胞中添加p55PIK特異性抑制劑P15多肽,可以起到抑制肝癌細(xì)胞生長,以及下調(diào)肝癌診斷指標(biāo)AFPmRNA表達(dá)水平的效應(yīng),所以本課題的研究目的是闡明p55PIK調(diào)控AFP表達(dá)的分子機(jī)制。方法:以肝癌細(xì)胞系HepG2為研究對(duì)象,超表達(dá)和沉默p55PIK,然后利用實(shí)時(shí)定量PCR以及westernblot的方法檢測(cè)細(xì)胞中AFP的mRNA含量以及蛋白含量,明確肝癌細(xì)胞中p55PIK對(duì)AFP的調(diào)節(jié)效果;用p55PIK特異性抑制劑P15處理肝癌細(xì)胞,檢測(cè)NF-κB的活性,證實(shí)在肝癌細(xì)胞中p55PIK可以調(diào)節(jié)NF-κB的活性;以肝癌細(xì)胞系HepG2為研究對(duì)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(shí)時(shí)定量PCR以及westernblot的方法檢測(cè)細(xì)胞中AFP的mRNA含量以及蛋白含量,證實(shí)肝癌細(xì)胞中NF-κB對(duì)AFP的調(diào)節(jié)效果;通過同時(shí)上調(diào)p55PIK以及抑制NF-κB的方法,觀察抑制NF-κB是否可以抵消上調(diào)p55PIK導(dǎo)致的AFP上調(diào),證實(shí)P55PIK通過NF-kB信號(hào)路徑解調(diào)節(jié)AFP表達(dá);利用熒光素酶報(bào)告系統(tǒng),確定AFP基因啟動(dòng)子上受NF-κB調(diào)控的片段;利用生物信息學(xué)的手段,分析并預(yù)測(cè)AFP基因啟動(dòng)子上受NF-κB調(diào)控的片段中可能與NF-κB結(jié)合的位點(diǎn);利用點(diǎn)突變技術(shù),觀察上述DNA位點(diǎn)對(duì)p55PIK/NF-κB對(duì)AFP轉(zhuǎn)錄活性的影響,明確哪些位點(diǎn)在p55PIK/NF-κB調(diào)控AFP轉(zhuǎn)錄活性中發(fā)揮重要作用。結(jié)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(dá)量逐漸減少,并且沉默p55PIK后AFP的蛋白表達(dá)量顯著降低,超表達(dá)p55PIK后AFP的表達(dá)量顯著增加;向HepG2細(xì)胞中加入NF-κB抑制劑(PDTC)后,會(huì)起到下調(diào)AFP的mRNA及蛋白表達(dá)水平的效應(yīng);分別向HepG2細(xì)胞中加入P15和PDTC后,檢測(cè)NF-κB通路關(guān)鍵蛋白的表達(dá)情況,加入P15后p65表達(dá)量沒有顯著變化,p65(ser536)磷酸化水平顯著降低,IKBα表達(dá)量顯著升高,IKBα(ser36)磷酸化水平顯著降低,加入PDTC后p65表達(dá)量顯著降低,p65(ser536)磷酸化水平顯著降低,IKBα表達(dá)量顯著升高,IKBα(ser36)磷酸化水平顯著降低,觀察(control,p55PIK,p55PIK+PDTC)實(shí)驗(yàn)組,PDTC可以抵消由于過表達(dá)p55PIK而引起的AFP表達(dá)量升高;構(gòu)建得到AFP基因上游調(diào)控區(qū)不同片段的熒光素酶報(bào)告基因載體(-5229/-16)-AFP,(-3375/-16)-AFP,(-1865/-16)-AFP和(-225/-16)-AFP,酶切以及測(cè)序鑒定分子量序列正確,檢測(cè)分別加入P15和PDTC對(duì)不同長度的AFP啟動(dòng)子轉(zhuǎn)錄活性的影響,結(jié)果顯示(-5184/+29)-AFP和(-3330/+29)-AFP變化最為顯著,即NF-κB的調(diào)控位點(diǎn)主要在(-5184,-1820)片段上;對(duì)報(bào)告基因活性變化最大的片段(-5184,-1820)進(jìn)行預(yù)測(cè),位點(diǎn)(-4717/-4726),(-4372/4381),(-3171/3180)可能是NF-κB與AFP啟動(dòng)子結(jié)合并調(diào)控該片段的轉(zhuǎn)錄活性的位點(diǎn);以全長AFP基因啟動(dòng)子報(bào)告基因載體(-5229/-16)-AFP為模板,對(duì)上述位點(diǎn)進(jìn)行點(diǎn)突變,并比較添加P15和PDTC前后報(bào)告基因載體突變對(duì)AFP基因轉(zhuǎn)錄活性的影響程度,結(jié)果顯示(-4717/-4726),(-4372/4381)突變后P15和PDTC不再能夠調(diào)節(jié)AFP的轉(zhuǎn)錄活性,即位點(diǎn)(-4717/-4726),(-4372/4381)是在p55PIK/NF-κB調(diào)控AFP轉(zhuǎn)錄活性的關(guān)鍵位點(diǎn)。結(jié)論:在肝癌細(xì)胞中,p55PIK介導(dǎo)的信號(hào)通路參與了肝癌生物標(biāo)志蛋白AFP的表達(dá);實(shí)驗(yàn)結(jié)果還表明p55PIK對(duì)AFP表達(dá)的調(diào)控是通過NF-κB信號(hào)通路實(shí)現(xiàn)的;在AFP啟動(dòng)子上游的NF-κB的作用位點(diǎn)(-4717/-4726),(-4372/4381)在p55PIK/NF-κB調(diào)節(jié)AFP轉(zhuǎn)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(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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本文編號(hào)：1839420