RNA去甲基化酶FTO抑制剂对小鼠精原细胞增殖的调控研究

发布时间：2018-06-27 02:14

  本文选题：精原细胞 + N6甲基腺嘌呤　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：精子发生包括精原细胞的增殖与分化,减数分裂和精子细胞的变形成熟等过程,受到转录因子、组蛋白修饰和非编码RNA等表观调控因子调控。近期研究表明m6A是真核生物mRNA上最丰富的一种化学修饰,参与调控mRNA剪接,降解以及翻译等代谢过程,从而调控基因的表达;另据报道,m6A修饰参与精子发生的调控。FTO作为第一个被发现的m6A去甲基化酶,通过调控m6A修饰水平参与调控脂肪细胞的分化,癌细胞的增殖。MA2作为甲氯芬那酸(meclofenamic acid,MA)的乙酯形式,可以有效的竞争性结合到FTO的活性中心,从而抑制FTO的m6A去甲基化酶活性。为探究m6A修饰对精原细胞增殖的调控作用,本研究选择小鼠B型精原细胞和初级精母细胞之间阶段来源的GC-1 spg细胞作为研究对象,采用western blot、qRT-PCR、细胞流式、EdU、m6A dot blot等方法研究m6A对GC-1 spg细胞增殖的调控作用;此外,构建双荧光报告系统并验证m6A报告质粒的功能。获得结果如下:1、MA2处理不影响FTO蛋白的表达,mRNA的m6A修饰水平在MA2处理后显著上调,并且m6A修饰水平的增加与MA2处理浓度呈正相关。2、MA2处理导致GC-1 spg细胞活性的降低,多核细胞数目的增加,但是TUNEL阳性细胞比例没有改变;同时,Cleaved PARP、Cleaved Caspase-3,Bax和Bcl2的蛋白表达无显著变化。3、MA2处理GC-1 spg细胞导致EdU阳性细胞比例的下降,和增殖标记基因PCNA和ki67表达的下调。另外,MA2处理诱导GC-1 spg细胞周期G1/S阻滞。4、查询在线数据库(http://mirlab.sysu.edu.cn/rmbase/)得到含有m6A修饰的细胞周期调控基因;进一步的检测表明MA2处理上调CDK8的表达,但是下调CDK1,CDK2,CDK7,CDK9和CDK12的mRNA表达。5、针对CREBBP基因3’UTR区的3个m6A修饰位点成功构建了CREBBP-m6A报告系统,结果表明细胞中mCherry的荧光强度与m6A修饰水平呈负相关。构建了CDK2基因3’UTR m6A修饰位点的荧光报告质粒,并验证了CDK2 3’UTR区的m6A修饰可以调控CDK2转录本的表达。综上,通过FTO抑制剂MA2处理GC-1 spg细胞,增加精原细胞中mRNA的m6A修饰,并调节细胞周期调控基因的表达,进而诱导细胞周期G1/S阻滞,抑制细胞增殖。此外,建立了以CREBBP-m6A报告质粒为基础的m6A修饰水平变化的检测系统。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S814

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