miR-34b在成肌分化过程中的功能及作用机制研究

发布时间：2018-01-04 18:31

本文关键词：miR-34b在成肌分化过程中的功能及作用机制研究　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:成肌分化过程包括成肌细胞的增殖,然后分化为成肌细胞,最后融合形成肌管。小鼠C2C12细胞是常用的研究成肌分化的细胞模型。microRNA(miRNA)是一类通过靶向靶基因mRNA的3′UTR,抑制其翻译或诱导其降解,在转录后水平调控基因表达的微小非编码RNA。已有研究表明,miRNA在成肌分化中起重要调控作用。最近,本研究鉴定了39个新的与成肌分化相关的miRNA,其中包括miR-34b。miR-34b是miR-34家族成员(包括miR-34a、miR-34b和miR-34c)之一,已有文献表明miR-34参与和促进多种细胞的分化,但其在成肌分化中的功能尚不清楚。目的:探究miR-34b在成肌分化过程中的功能及其调控机制。方法与结果:利用本实验室建立的S-Poly(T)Plus miRNA检测技术,发现miR-34b在成肌分化和肌肉损伤后再生过程中表达上调。在C2C12细胞中转染化学合成的miRNA类似物或抑制剂,发现miR-34b具有抑制成肌细胞增殖并促进成肌分化的功能。为探讨miR-34b调控成肌细胞增殖分化的分子机制,本研究利用isobaric tags for relative and absolute quantitation(iTRAQ)技术,在蛋白质水平定量分析了受miR-34b调控的基因,并构建了成肌细胞受miR-34b调控的网络图。在过表达miR-34b后下调的97个蛋白中,Ywhag、Ahcy和Ncl为miR-34b潜在的靶基因(生物信息学预测),并通过了3′UTR荧光素酶报告载体的验证。其中,NUCL蛋白的表达趋势与细胞分化状态密切相关。通过免疫染色和Western Blot检测,发现NUCL的表达和细胞内定位在成肌分化过程中呈现动态变化。利用慢病毒介导的shRNA技术感染C2C12细胞,发现干扰Ncl表达后能明显抑制成肌细胞增殖和成肌分化。为进一步验证Ncl在成肌分化中的作用,在C2C12细胞转染了多种靶向Ncl的siRNA,分析了nucleolin表达水平与myogenin之间的关系;结果表明,Ncl既可能促进肌肉分化,也可能抑制肌肉分化,具体功能依赖于Ncl的蛋白表达水平。另外,本研究还发现miR-34b在成肌分化过程中的显著上调受甲基化调控。结论:miR-34b在成肌过程中上调,抑制细胞增殖并促进成肌分化,该调控作用通过靶向一系列基因(包括Ywhag、Ahcy和Ncl)而实现。miR-34b-Ncl轴在成肌分化调控过程中扮演重要功能。Ncl以剂量依赖性的方式调节成肌分化,而miR-34b正是以微调的方式调控Ncl在成肌分化过程中的表达水平。
[Abstract]:Background: myogenic differentiation involves the proliferation of myoblasts and then differentiation into myoblasts. Mouse C2C12 cells are commonly used to study myogenic differentiation. MicroRNAs miRNAs are a class of 3UTRs that target the target gene mRNA. Inhibition of its translation or induction of its degradation, regulation of gene expression at the post-transcriptional level of the tiny non-coding RNA. recent studies have shown that miRNA plays an important role in the regulation of myogenic differentiation. This study identified 39 new miRNAs associated with myogenic differentiation, including that miR-34b.miR-34b is a member of the miR-34 family (including miR-34a). MiR-34b and miR-34c), it has been reported that miR-34 participates in and promotes the differentiation of many kinds of cells. But its function in myogenic differentiation is not clear. Objective: to explore the function and regulatory mechanism of miR-34b in myogenic differentiation. Plus miRNA detection technology. It was found that the expression of miR-34b was up-regulated during myogenic differentiation and regeneration after muscle injury. The chemically synthesized miRNA analogues or inhibitors were transfected into C2C12 cells. It was found that miR-34b could inhibit the proliferation of myoblasts and promote the differentiation of myoblasts. In order to investigate the molecular mechanism of miR-34b regulating the proliferation and differentiation of myoblasts. In this study, isobaric tags for relative and absolute quantitative analysis (iTRAQ) technique was used. The genes regulated by miR-34b were quantitatively analyzed at the protein level, and a network map of myoblasts regulated by miR-34b was constructed. Among 97 proteins down-regulated after overexpression of miR-34b. Ywhagar Ahcy and Ncl are potential target genes for miR-34b (bioinformatics predictor) and have been verified by 3TUTR luciferase report vector. The expression trend of NUCL protein was closely related to the differentiation of cells. The expression of NUCL protein was detected by immunostaining and Western Blot. It was found that the expression and intracellular localization of NUCL showed dynamic changes during myogenic differentiation. C2C12 cells were infected with shRNA mediated by lentivirus. It was found that interfering with the expression of Ncl could significantly inhibit the proliferation and myogenic differentiation of myoblasts. In order to further verify the role of Ncl in myogenic differentiation. Several siRNAs targeting Ncl were transfected into C2C12 cells, and the relationship between nucleolin expression and myogenin was analyzed. The results showed that NCL could promote or inhibit muscle differentiation, and its specific function depended on the protein expression level of Ncl. It was also found that the upregulation of miR-34b during myogenic differentiation was regulated by methylation. Conclusion: miR-34b is up-regulated during myogenesis, which inhibits cell proliferation and promotes myogenic differentiation. This regulation is mediated by targeting a series of genes (including Ywhag). Ahcy and NCL). The .miR-34b-NCL axis plays an important role in the regulation of myogenic differentiation. NCL regulates myogenic differentiation in a dose-dependent manner. MiR-34b regulates the expression of Ncl during myogenic differentiation in a fine-tuned manner.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3416

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