F9细胞中CHIR99021下调Dnmt3l基因的机制研究

发布时间：2018-03-31 09:41

本文选题：CHIR99021　切入点：Wnt　出处：《西北农林科技大学》2017年硕士论文

【摘要】：表观遗传是在不改变DNA的序列的情况下对DNA或染色体的修饰。DNA甲基化是主要的表观遗传修饰,具有重要的调控功能。DNA甲基转移酶(DNA methyltransferases,DNMTs)能够催化甲基从S腺苷甲硫氨酸转移到DNA。哺乳动物中的DNMT家族有五个成员:DNMT1,DNMT2,DNMT3A,DNMT3B,DNMT3L,其中DNMT3L因缺少催化结构域而不具有任何催化活性,但会与DNMT3A和DNMT3B相互作用促进它们的活性。Wnt信号通路是胚胎发育过程中重要的组成,其中经典Wnt通路依赖β-catenin转运到细胞核中,通过与T细胞因子/淋巴增强结合因子(T-cell factor/lymphoid enhancer-binding factor,TCF/LEF)结合,激活或抑制下游基因的表达。CHIR99021与PD0325901能够起到抑制细胞分化的作用,其中CHIR99021是常用的GSK3β的抑制剂,通过抑制此酶活性,使β-catenin在核内积累,达到Wnt后续的转录信号传递。本文首先利用实时定量PCR实验,确定了不同浓度CHIR99021对甲基转移酶家族基因的表达影响,发现对Dnmt3l基因有明显下调作用,而对于Dnmt3a,Dnmt3b等其余家族基因没有明显作用。利用蛋白免疫印迹实验确定了针对小鼠F9细胞,最适的CHIR99021处理浓度为10μM,并以此为重点进行后续研究,确定CHIR99021下调基因的具体机制。通过构建pCMV-Myc-β-catenin、pCDNA3.1-β-catenin和pCDNA3.1-β-catenin S37A载体,利用核质蛋白分离提取实验以及蛋白免疫印迹实验发现,CHIR99021对F9细胞处理后,β-catenin蛋白的总表达量有明显提高,并且细胞质和细胞核内的β-catenin含量都比对照组高,说明,CHIR99021可以通过抑制GSK3β来增加细胞质中β-catenin的稳定性,并且入核量也随之增高。通过双荧光素酶报告实验证明CHIR99021会对Wnt/β-catenin经典通路造成影响,激活该通路。过表达β-catenin后,Dnmt3l基因的表达出现下调,因此可得,在小鼠F9细胞中CHIR99021能够通过抑制GSK3β,使β-catenin在细胞核中积累,激活Wnt/β-catenin经典通路,最终导致Dnmt3l基因的抑制。β-catenin进入细胞核后,通常会与TCF/LEF复合体共同作用,并激活基因的转录,而对于本文研究基因Dnmt3l的抑制效果很少见。此复合体中起到抑制效果的只有TCF3蛋白,其余成员起转录激活效果,而近期研究对TCF3的抑制机制并不明确,而且对其在经典Wnt通路的作用没有准确和唯一的答案,因此,我对TCF3在此机制中的作用进行了研究。本实验利用RNA干扰,免疫荧光染色,实时定量PCR和蛋白免疫印迹实验,确定下调Tcf3(Tcf7l1)基因的表达会对Dnmt3l基因有上调的影响,并且Tcf3和β-catenin基因在经过CHIR99021处理24h后,都具有上调效果。综上,本研究证明了CHIR99021能够通过抑制GSK3β,使β-catenin在细胞核中积累,激活Wnt/β-catenin经典通路,β-catenin与核内的TCF3结合,共同抑制Dnmt3l基因的表达,为CHIR99021促进胚胎干细胞的自我更新能力提供理论依据。
[Abstract]:Epigenetics is the modification of DNA or chromosomes without changing the sequence of DNA. DNA methylation is the main epigenetic modification. DNMTs have an important regulatory function. DNMTs can catalyze the transfer of methyl from S-adenosylmethionine to DNA.The DNMT family in mammals has five members: DNMT1, DNMT2DNMT3AnMT3An, DNMT3ANMT3DNMT3L, in which DNMT3L does not have any catalytic activity due to its lack of catalytic domain. However, the interaction with DNMT3A and DNMT3B promotes their activity. Wnt signaling pathway is an important component of embryonic development, in which the classical Wnt pathway depends on 尾 -catenin translocation into the nucleus. By binding to T-cell factor/lymphoid enhancer-binding factor-T cell factor/lymphoid enhancer-binding factor-T cell / lymphoid enhancement binding factor TCF / LEF, activation or inhibition of downstream gene expression. CHIR99021 and PD0325901 can inhibit cell differentiation. CHIR99021 is a commonly used inhibitor of GSK3 尾, by inhibiting the activity of this enzyme. 尾 -catenin was accumulated in the nucleus to achieve the subsequent transcriptional signal transduction of Wnt. Firstly, the effects of different concentrations of CHIR99021 on the expression of methyltransferase family genes were determined by real-time quantitative PCR assay, and it was found that the effect of 尾 -catenin on the expression of Dnmt3l gene was down-regulated. However, there was no obvious effect on other family genes, such as Dnmt3ah3b and Dnmt3b. The optimal concentration of CHIR99021 for mouse F9 cells was determined by Western blotting assay, and the following study was carried out with emphasis on the treatment of Dnmt3aH, Dnmt3b and Dnmt3b. By constructing pCMV-Myc- 尾 -catenin pCDNA3.1- 尾 -catenin and pCDNA3.1- 尾 -catenin S37A vectors, we found that the total expression of 尾 -catenin protein was significantly increased after treated with CHIR99021 on F9 cells. The content of 尾 -catenin in cytoplasm and nucleus was higher than that in control group, indicating that CHIR99021 could increase the stability of 尾 -catenin by inhibiting GSK3 尾. The results of double luciferase report showed that CHIR99021 could affect the classical Wnt/ 尾 -catenin pathway and activate the pathway. After overexpression of 尾 -catenin, the expression of Dnmt3l gene was down-regulated, so the expression of Dnmt3l gene was down-regulated, and the expression of Dnmt3l gene was down-regulated after overexpression of 尾 -catenin. In mouse F9 cells, CHIR99021 can inhibit GSK3 尾, make 尾 -catenin accumulate in the nucleus, activate the classical pathway of Wnt/ 尾 -catenin, and eventually lead to the inhibition of Dnmt3l gene. 尾 -catenin usually works with TCF/LEF complex and activates gene transcription. However, the inhibitory effect of the gene Dnmt3l is rare. Only the TCF3 protein plays an inhibitory role in this complex, while the other members have transcriptional activation effect, but the mechanism of inhibition of TCF3 in recent studies is not clear. There is no accurate and unique answer to the role of TCF3 in the classical Wnt pathway. Therefore, I have studied the role of TCF3 in this mechanism. In this study, RNA interference, immunofluorescence staining, real-time quantitative PCR and Western blotting were used. It was determined that down-regulating the expression of Tcf3, Tcf7l1) gene would affect the up-regulation of Dnmt3l gene, and that both Tcf3 and 尾 -catenin genes could be upregulated after 24 hours of CHIR99021 treatment. In conclusion, this study demonstrated that CHIR99021 can make 尾 -catenin accumulate in the nucleus by inhibiting GSK3 尾. Activation of the classical Wnt/ 尾 -catenin pathway, 尾 -catenin binding to TCF3 in the nucleus, inhibition of the expression of Dnmt3l gene, which provides a theoretical basis for CHIR99021 to promote the self-renewal ability of embryonic stem cells.
【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】：Q78

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