宫颈癌细胞中miR-101对let-7a-1成熟的调控作用与机制研究

发布时间：2018-06-05 00:27

本文选题：miR-101 + let-7a-1　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的微小RNA(miRNA)参与不同的生物过程,包括胚胎发育,代谢,分化等生理和病理过程。它们主要是通过与靶mRNA的3'UTR中的互补序列结合在转录和转录后水平调控基因表达发挥生物学功能。Let-7靶定特异性的癌基因,通过多种信号通路参与肿瘤的发生发展等过程,功能上起到抑癌基因的作用。有报道表明,在乳腺癌细胞中,组蛋白去甲基化酶直接抑制let-7a的转录,多能性因子Lin28也可以结合其初级和前体转录本中的茎环结构影响Dicer对let-7前体(pre-let-7a)的剪切,从而抑制let-7成熟体的生成。本研究的目的是探寻在宫颈癌细胞内,是否存在能够靶定let-7a-1前体的茎环loop结构的miRNAs及其对let-7a-1的成熟体、前体(pre-)和原始转录本(pri-)生成的影响,并确定参与这个过程的调控蛋白。本研究我们试图阐明miRNAs间的调节,以及miRNA成熟过程中前体loop结构起到的重要作用。方法1.首先利用生物信息学预测能够与pre-let-7a-1的loop结构中27个碱基可能(不完全)互补匹配的miRNAs。构建过表达和封闭上述miRNAs的质粒或合成抑制miRNAs表达的寡核苷酸,RT-qPCR实验检测宫颈癌细胞中改变上述miRNAs表达水平对let-7a-1成熟体、前体及原始转录本的影响。2.选择上述miRNAs中调控let-7a-1成熟体形成的miRNAs,通过EGFP荧光载体报告系统检测该miRNAs与let-7a-1的loop结构的靶定关系。3.Western blot检测miR-101对let-7a-1下游直接靶基因Aurora蛋白表达水平的影响。4.RIP实验检测miR-101通过结合pre-let-7a-1的loop结构对其成熟体形成调节依赖于Ago蛋白。结果1.生物信息学预测到miR-148a、miR-181b和miR-101是与pre-let-7a-1 loop在不同位点形成不完全互补配对,过表达和封闭上述miRNAs实验表明仅miR-101能够抑制let-7a-1成熟体表达,对其前体及原始转录本的表达水平无明显影响。2.通过EGFP荧光报告载体系统发现miR-101可以抑制具有let-7a-1前体loop结构的EGFP荧光水平和蛋白水平的表达。3.Western blot验证了miR-101对let-7a-1下游直接靶基因Aurora的上调作用是通过let-7a-1发挥作用的。4.RIP实验验证miR-101结合pre-let-7a-1调节let-7a-1成熟体形成依赖于Ago蛋白结论本研究发现miR-101通过结合pre-let-7a-1的loop结构抑制let-7a-1的成熟,该作用的发挥依赖Ago2蛋白。这对于miRNA形成的调控提供新的理论解释及实验依据。
[Abstract]:Objective small RNAs miRNAs are involved in various biological processes, including embryonic development, metabolism, differentiation and other physiological and pathological processes. They play a biological role in regulating gene expression at the transcriptional and post-transcriptional level by combining with complementary sequences in the 3'UTR of target mRNA, and by targeting specific oncogenes in Let-7, and participate in tumorigenesis and development through a variety of signal pathways. Function plays the role of tumor suppressor gene. It has been reported that histone demethylase directly inhibits the transcription of let-7a in breast cancer cells, and that the pluripotent factor Lin28 can also affect the splicing of let-7 precursor pre-let-7a by combining the stem ring structure in its primary and precursor transcripts. Thus, the formation of mature bodies of let-7 was inhibited. The aim of this study was to investigate the presence of miRNAs targeting the loop structure of the stem ring of let-7a-1 precursors and its effects on let-7a-1 maturation, precursor pre-formation and pripri-production in cervical cancer cells, and to identify the regulatory proteins involved in this process. In this study, we try to elucidate the regulation of miRNAs and the important role of precursor loop structure in the process of miRNA maturation. Method 1. First, bioinformatics was used to predict the possible (incomplete) matching miRNAs with 27 bases in the loop structure of pre-let-7a-1. Construction of plasmids that overexpressed and blocked the miRNAs or synthesis of oligonucleotide reverse transcriptase (RT-qPCR) to inhibit the expression of miRNAs were used to detect the effect of changes in the expression of miRNAs on let-7a-1 maturation, precursor and original transcripts in cervical cancer cells. Select miRNAss, which regulate the formation of let-7a-1 maturation in miRNAs, and detect the relationship between the loop structure of miRNAs and let-7a-1 by EGFP fluorescence vector reporting system. 3. Western blot detection of miR-101 on the expression level of Aurora protein downstream direct target gene of let-7a-1 .4.RIP experiment MiR-101 is dependent on Ago protein to regulate its maturation by binding to loop structure of pre-let-7a-1. Result 1. Bioinformatics predicted that miR-148a miR-181b and miR-101 formed incomplete complementary pairing with pre-let-7a-1 loop at different sites. Overexpression and blocking of the above miRNAs experiments showed that only miR-101 could inhibit the expression of let-7a-1 mature body, but had no significant effect on the expression level of its precursors and original transcripts. EGFP fluorescence report vector system showed that miR-101 could inhibit the expression of EGFP fluorescence level and protein level with let-7a-1 precursor loop structure. 3. Western blot confirmed that miR-101 upregulated let-7a-1 downstream direct target gene Aurora through let-7a-1. 4. Rip experiments demonstrated that miR-101 combined with pre-let-7a-1 regulates the formation of let-7a-1 maturation dependent on Ago protein. Conclusion in this study, miR-101 inhibits let-7a-1 maturation through loop structure binding to pre-let-7a-1. This role is dependent on Ago2 protein. This provides a new theoretical explanation and experimental basis for the regulation of the formation of miRNA.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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