苦荞凝集素通过下调miR-21的表达抑制结肠癌细胞增殖的研究

发布时间：2018-05-26 01:46

本文选题：苦荞凝集素 + miR-21　；参考：《山西大学》2017年硕士论文

【摘要】：miRNA是一类由18-25个核苷酸组成的、可以对相应靶基因进行负调控的单链RNA分子。作为基因调控的方式,miRNA是在基因转录后阶段进行调控,人体内大约有三分之一的基因都受到miRNA的调节,因此miRNA对生命的调节是一个广泛存在的过程。一些可以起到抑制癌症的miRNA称之为抑癌miRNA,与癌症发生发展紧密相关的miRNA称之为癌miRNA。越来越多的分子学研究证据表明,在许多肿瘤细胞中,miRNA的表达是失调的,而癌miRNA一般都过量表达,因此癌miRNA可以作为癌症治疗的有效靶点。苦荞凝集素(Tartary Buckwheat Lectin,TBL)是一类兼具核糖体失活蛋白N-糖苷酶活性和芦丁水解酶活性的糖蛋白,具有显著的靶向抑制结肠癌细胞增殖的功能。先前的研究表明,TBL可以通过下调miR-135ab的表达,从而使miR-135ab失去对其靶基因APC的抑制,提高APC蛋白的表达量,对Wnt/b-catenin信号通路产生抑制,从而抑制结肠癌细胞的增殖。通过miRNA筛查,我们发现TBL可以下调或者上调诸多miRNA的表达,其中对miR-21的下调最为显著。miR-21是典型的癌miRNA,在目前发现的多种肿瘤细胞中均表现为上调。本研究以miR-21为线索,通过双荧光素酶实验发现PTEN基因是miR-21的靶基因之一,使用TBL处理后,可以缓解miR-21对PTEN基因的抑制。在qRT-PCR实验中,我们发现TBL可以上调PTEN以及PTEN下游AKT m RNA的水平,下调PI3K p110亚基mRNA的水平。通过Western Blot实验表明,使用TBL处理后细胞中的PTEN蛋白显著上调,而AKT蛋白的表达量下调。细胞周期检测发现,TBL可以上调G1期细胞含量,下调S期和G2期的细胞含量。通过伤口愈合实验,我们发现TBL可以显著抑制人结直肠癌细胞HCT116的迁移。上述实验结果表明,来自苦荞麦中的凝集素TBL抑制人结直肠癌细胞HCT116的一个可能机制是通过下调miR-21的表达,上调其靶基因PTEN,进而抑制PI3K/AKT信号通路,阻滞细胞周期的进行以及抑制细胞的迁移能力。本实验内容对深入开展生物活性物质在抗癌、抑癌中的作用方式,揭示相关的作用机理具有重要的价值。
[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.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-36

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