本文選題：苦蕎凝集素 + miR-21��； 參考：《山西大學(xué)》2017年碩士論文

【摘要】：miRNA是一類由18-25個(gè)核苷酸組成的、可以對(duì)相應(yīng)靶基因進(jìn)行負(fù)調(diào)控的單鏈RNA分子。作為基因調(diào)控的方式,miRNA是在基因轉(zhuǎn)錄后階段進(jìn)行調(diào)控,人體內(nèi)大約有三分之一的基因都受到miRNA的調(diào)節(jié),因此miRNA對(duì)生命的調(diào)節(jié)是一個(gè)廣泛存在的過(guò)程。一些可以起到抑制癌癥的miRNA稱之為抑癌miRNA,與癌癥發(fā)生發(fā)展緊密相關(guān)的miRNA稱之為癌miRNA。越來(lái)越多的分子學(xué)研究證據(jù)表明,在許多腫瘤細(xì)胞中,miRNA的表達(dá)是失調(diào)的,而癌miRNA一般都過(guò)量表達(dá),因此癌miRNA可以作為癌癥治療的有效靶點(diǎn)。苦蕎凝集素(Tartary Buckwheat Lectin,TBL)是一類兼具核糖體失活蛋白N-糖苷酶活性和蘆丁水解酶活性的糖蛋白,具有顯著的靶向抑制結(jié)腸癌細(xì)胞增殖的功能。先前的研究表明,TBL可以通過(guò)下調(diào)miR-135ab的表達(dá),從而使miR-135ab失去對(duì)其靶基因APC的抑制,提高APC蛋白的表達(dá)量,對(duì)Wnt/b-catenin信號(hào)通路產(chǎn)生抑制,從而抑制結(jié)腸癌細(xì)胞的增殖。通過(guò)miRNA篩查,我們發(fā)現(xiàn)TBL可以下調(diào)或者上調(diào)諸多miRNA的表達(dá),其中對(duì)miR-21的下調(diào)最為顯著。miR-21是典型的癌miRNA,在目前發(fā)現(xiàn)的多種腫瘤細(xì)胞中均表現(xiàn)為上調(diào)。本研究以miR-21為線索,通過(guò)雙熒光素酶實(shí)驗(yàn)發(fā)現(xiàn)PTEN基因是miR-21的靶基因之一,使用TBL處理后,可以緩解miR-21對(duì)PTEN基因的抑制。在qRT-PCR實(shí)驗(yàn)中,我們發(fā)現(xiàn)TBL可以上調(diào)PTEN以及PTEN下游AKT m RNA的水平,下調(diào)PI3K p110亞基mRNA的水平。通過(guò)Western Blot實(shí)驗(yàn)表明,使用TBL處理后細(xì)胞中的PTEN蛋白顯著上調(diào),而AKT蛋白的表達(dá)量下調(diào)。細(xì)胞周期檢測(cè)發(fā)現(xiàn),TBL可以上調(diào)G1期細(xì)胞含量,下調(diào)S期和G2期的細(xì)胞含量。通過(guò)傷口愈合實(shí)驗(yàn),我們發(fā)現(xiàn)TBL可以顯著抑制人結(jié)直腸癌細(xì)胞HCT116的遷移。上述實(shí)驗(yàn)結(jié)果表明,來(lái)自苦蕎麥中的凝集素TBL抑制人結(jié)直腸癌細(xì)胞HCT116的一個(gè)可能機(jī)制是通過(guò)下調(diào)miR-21的表達(dá),上調(diào)其靶基因PTEN,進(jìn)而抑制PI3K/AKT信號(hào)通路,阻滯細(xì)胞周期的進(jìn)行以及抑制細(xì)胞的遷移能力。本實(shí)驗(yàn)內(nèi)容對(duì)深入開(kāi)展生物活性物質(zhì)在抗癌、抑癌中的作用方式,揭示相關(guān)的作用機(jī)理具有重要的價(jià)值。
[Abstract]:MiRNA is a single stranded RNA molecule composed of 18-25 nucleotides that can negatively regulate the target gene. As a way of gene regulation, miRNAs are regulated at the post-transcriptional stage of genes. About 1/3 genes in human body are regulated by miRNA. Therefore, the regulation of miRNA on life is a widespread process. Some miRNA, which can inhibit cancer, are called tumor suppressor miRNAs, and miRNA, which is closely related to the development of cancer, is called cancer miRNA. More and more molecular studies have shown that the expression of miRNAs is misaligned in many tumor cells, while miRNA is generally overexpressed in cancer cells. Therefore, cancer miRNA can be used as an effective target for cancer treatment. Tartary buckwheat lectin (Tartary buckwheat lectin) is a class of glycoproteins with both ribosomal inactivating protein N-glycosidase activity and rutin hydrolase activity. Previous studies have shown that TBL can inhibit the expression of miR-135ab, thus causing miR-135ab to lose the inhibition of its target gene APC, increase the expression of APC protein, inhibit the Wnt/b-catenin signaling pathway, and thus inhibit the proliferation of colon cancer cells. Through miRNA screening, we found that TBL can down-regulate or up-regulate the expression of many miRNA, among which the down-regulation of miR-21 is the most significant. MiR-21 is a typical cancer miRNAs. In this study, PTEN gene was found to be one of the target genes of miR-21 by double luciferase experiment based on miR-21. The inhibition of PTEN gene induced by miR-21 could be alleviated by TBL treatment. In the qRT-PCR experiment, we found that TBL could up-regulate the level of PTEN and PTEN downstream AKT m RNA, and down-regulate the mRNA level of PI3K p110 subunit. Western Blot assay showed that the expression of PTEN protein was up-regulated and the expression of AKT protein was down-regulated after TBL treatment. Cell cycle analysis showed that TBL could up-regulate cell content in G1 phase and down-regulate cell content in S phase and G2 phase. Through wound healing experiments, we found that TBL could significantly inhibit the migration of HCT116 in human colorectal cancer cells. These results suggest that one of the possible mechanisms by which lectin TBL from Tartary buckwheat inhibits HCT116 in human colorectal cancer cells is by down-regulating the expression of miR-21, upregulating its target gene, PTEN, and thus inhibiting the PI3K/AKT signaling pathway. Block cell cycle and inhibit cell migration. This experiment is of great value for the further development of biological active substances in anticancer and anti-cancer mode, and to reveal the related mechanism of action.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R73-36

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 陳萬(wàn)青;鄭榮壽;張思維;曾紅梅;左婷婷;賈漫漫;夏昌發(fā);鄒小農(nóng);赫捷;;2012年中國(guó)惡性腫瘤發(fā)病和死亡分析[J];中國(guó)腫瘤;2016年01期

2 賈喬瑾;崔曉東;王轉(zhuǎn)花;;苦蕎凝集素對(duì)結(jié)腸癌細(xì)胞增殖的抑制作用[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2015年04期

3 包龍龍;嚴(yán)妍;季衛(wèi)丹;沈舒文;吳孟超;蘇長(zhǎng)青;;MiR-21通過(guò)AKT/ERK途徑對(duì)肝癌BEL-7402細(xì)胞增殖和遷移的影響[J];腫瘤;2013年11期

4 吳共發(fā);黃綺亭;曾宇婷;林俊汕;葉梓瑩;胡純婷;;miR-21通過(guò)PTEN/AKT通路抑制白血病細(xì)胞K562的遷移和增殖[J];醫(yī)學(xué)研究生學(xué)報(bào);2013年10期

5 王立峰;陳靜宜;謝慧慧;王玉梅;石嘉懌;梁正昌;劉瑞海;鞠興榮;袁建;;薏米多酚細(xì)胞抗氧化及HepG2細(xì)胞毒性和抗增殖作用[J];中國(guó)農(nóng)業(yè)科學(xué);2013年14期

6 熊欣;鄭長(zhǎng)黎;;miRNA-214對(duì)胃癌細(xì)胞BGC823,MKN45的細(xì)胞周期和凋亡的影響[J];中國(guó)普通外科雜志;2010年12期

7 張研;;我國(guó)小雜糧生產(chǎn)現(xiàn)狀與發(fā)展策略[J];河北農(nóng)業(yè)大學(xué)學(xué)報(bào)(農(nóng)林教育版);2010年03期

8 李引平;;山西省小雜糧生產(chǎn)優(yōu)勢(shì)及發(fā)展思路[J];山西農(nóng)業(yè)科學(xué);2010年02期

相關(guān)碩士學(xué)位論文 前1條

1 張繼曼;黑豆紅花色苷的優(yōu)化提取及其降血糖作用的研究[D];安徽農(nóng)業(yè)大學(xué);2010年

，

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