本文選題：精原細胞 + N6甲基腺嘌呤　； 參考：《西北農(nóng)林科技大學》2017年碩士論文

【摘要】：精子發(fā)生包括精原細胞的增殖與分化,減數(shù)分裂和精子細胞的變形成熟等過程,受到轉錄因子、組蛋白修飾和非編碼RNA等表觀調(diào)控因子調(diào)控。近期研究表明m6A是真核生物mRNA上最豐富的一種化學修飾,參與調(diào)控mRNA剪接,降解以及翻譯等代謝過程,從而調(diào)控基因的表達;另據(jù)報道,m6A修飾參與精子發(fā)生的調(diào)控。FTO作為第一個被發(fā)現(xiàn)的m6A去甲基化酶,通過調(diào)控m6A修飾水平參與調(diào)控脂肪細胞的分化,癌細胞的增殖。MA2作為甲氯芬那酸(meclofenamic acid,MA)的乙酯形式,可以有效的競爭性結合到FTO的活性中心,從而抑制FTO的m6A去甲基化酶活性。為探究m6A修飾對精原細胞增殖的調(diào)控作用,本研究選擇小鼠B型精原細胞和初級精母細胞之間階段來源的GC-1 spg細胞作為研究對象,采用western blot、qRT-PCR、細胞流式、EdU、m6A dot blot等方法研究m6A對GC-1 spg細胞增殖的調(diào)控作用;此外,構建雙熒光報告系統(tǒng)并驗證m6A報告質粒的功能。獲得結果如下:1、MA2處理不影響FTO蛋白的表達,mRNA的m6A修飾水平在MA2處理后顯著上調(diào),并且m6A修飾水平的增加與MA2處理濃度呈正相關。2、MA2處理導致GC-1 spg細胞活性的降低,多核細胞數(shù)目的增加,但是TUNEL陽性細胞比例沒有改變;同時,Cleaved PARP、Cleaved Caspase-3,Bax和Bcl2的蛋白表達無顯著變化。3、MA2處理GC-1 spg細胞導致EdU陽性細胞比例的下降,和增殖標記基因PCNA和ki67表達的下調(diào)。另外,MA2處理誘導GC-1 spg細胞周期G1/S阻滯。4、查詢在線數(shù)據(jù)庫(http://mirlab.sysu.edu.cn/rmbase/)得到含有m6A修飾的細胞周期調(diào)控基因;進一步的檢測表明MA2處理上調(diào)CDK8的表達,但是下調(diào)CDK1,CDK2,CDK7,CDK9和CDK12的mRNA表達。5、針對CREBBP基因3’UTR區(qū)的3個m6A修飾位點成功構建了CREBBP-m6A報告系統(tǒng),結果表明細胞中mCherry的熒光強度與m6A修飾水平呈負相關。構建了CDK2基因3’UTR m6A修飾位點的熒光報告質粒,并驗證了CDK2 3’UTR區(qū)的m6A修飾可以調(diào)控CDK2轉錄本的表達。綜上,通過FTO抑制劑MA2處理GC-1 spg細胞,增加精原細胞中mRNA的m6A修飾,并調(diào)節(jié)細胞周期調(diào)控基因的表達,進而誘導細胞周期G1/S阻滯,抑制細胞增殖。此外,建立了以CREBBP-m6A報告質粒為基礎的m6A修飾水平變化的檢測系統(tǒng)。
[Abstract]:Spermatogenesis involves the proliferation and differentiation of spermatogonia, meiosis and deformational maturation of spermatocytes, and is regulated by transcriptional factors, histone modification and non-coding RNA. Recent studies have shown that m6A is one of the most abundant chemical modifications in eukaryote mRNA, which is involved in the regulation of mRNA splicing, degradation, translation and other metabolic processes, thus regulating gene expression. It is also reported that m6A modifies the regulation of spermatogenesis. FTO, as the first m6A demethylase found, regulates the differentiation of adipocytes by regulating m6A modification level. The proliferation of cancer cells, MA2, acts as an ethyl ester form of meclofenamic acidinic acid (MA). The m6A demethylase activity of FTO can be inhibited by competitive binding to the active center of FTO. In order to investigate the effect of m6A modification on spermatogonia proliferation, GC-1 spg cells derived from mouse B spermatogonia and primary spermatocytes were selected as the research objects. The effects of m6A on the proliferation of GC-1 spg cells were studied by western blottqRT-PCRand flow cytometry, and the function of m6A reporter plasmid was verified. The results showed that the m6A modification level of m6A was not affected by the treatment of MA2, and the increase of m6A modification level was positively related to the concentration of MA2, and the activity of GC-1 spg cells was decreased after treatment with M6A. The number of multinucleated cells increased, but the proportion of Tunel positive cells did not change, while the protein expression of Caspase-3, Bax and Bcl2 in Caspase-3 and Bcl2 was not significantly changed. 3MMA _ 2 treated GC-1 spg cells decreased the proportion of Edu positive cells, and down-regulated the expression of proliferative marker genes, PCNA and ki67. In addition, the cell cycle of GC-1 spg cells induced by MA2 treatment was blocked by G1 / S block .4. the cell cycle regulation gene containing m6A modification was obtained by querying online database (http://mirlab.sysu.edu.cn/rmbase/), and further detection showed that MA2 treatment up-regulated the expression of CDK8. However, down-regulation of mRNA expression of CDK7, CDK7, CDK9 and CDK12 was down-regulated. A CREBBP-m6A report system was successfully constructed for three m6A modification sites in the 3UTR region of CREBBP gene. The results showed that the fluorescence intensity of mCherry was negatively correlated with the m6A modification level. The fluorescent reporter plasmid of CDK2 gene 3- UTR m6A modification site was constructed, and it was proved that m6A modification of CDK23 UTR region could regulate the expression of CDK2 transcripts. In conclusion, GC-1 spg cells were treated with MA2, an inhibitor of FTO, to increase m6A modification in spermatogonial cells and to regulate the expression of cell cycle regulatory genes, thus inducing G1 / S arrest of cell cycle and inhibiting cell proliferation. In addition, a system for detecting the change of m6A modification level based on CREBBP-m6A reporter plasmid was established.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S814

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本文編號：2072276