QKI-5调控WT1和Caspase8抑制猪ST细胞凋亡的研究

发布时间：2018-07-23 15:31

【摘要】：睾丸支持细胞位于睾丸曲细精管基底部,与各种不同发育状态的生精细胞共同构成曲细精管壁。支持细胞具有为生精细胞提供营养,启动精原干细胞分化,维持精子发生,吞噬精子发生过程中的残余胞质等重要功能。睾丸支持细胞的增殖决定了动物个体睾丸大小和成熟后精子质量和数量。并且成熟的支持细胞数量又取决于未成熟的支持细胞数量。因此,未成熟的支持细胞对动物睾丸发育和生精能力也有着至关重要的作用。QKI是一种RNA结合蛋白,属于高度保守的STAR家族成员,QKI主要表达QKI-5,QKI-6和QKI-7三种异构体。QKI能通过结合靶m RNA的QRE序列,影响RNA的代谢,从而参与细胞增殖、凋亡、分化等过程。本研究的目的是探讨QKI-5对ST细胞凋亡的影响以及QKI-5对WT1和Caspase8的转录后调控作用。首先,确认猪的QKI基因具有QKI-5、QKI-6和QKI-7这三种剪接模式。根据人的QKI-5,QKI-6和QKI-7序列分别设计三对特异性引物,以猪的c DNA为模版,成功克隆出三条目的片段。它们分别具有QKI-5、QKI-6和QKI-7特异的C末端,并且,通过分析发现QKI-5具有典型的核定位信号(NLS)。随后,研究了QKI-5对ST细胞凋亡的影响。首先成功构建QKI-5的过表达载体并合成了4条干扰载体(sh RNAs)。在ST细胞中进行过表达和沉默QKI-5,MTS结果表明,过表达QKI-5后,细胞数显著高于对照组,而干扰QKI-5后,细胞数出现显著降低。流式细胞术结果表明,过表达QKI-5时,细胞凋亡率为2.32±0.11%,而沉默QKI-5时,细胞凋亡率为25.64±0.68%,差异显著(p0.01),表明QKI-5具有抑制ST细胞凋亡的功能。然后,验证了QKI-5抑制细胞凋亡的分子机制。生物信息学分析发现Caspase8基因的3’UTR上有1个可被QKI结合的QRE元件(1009bp-1014bp,ACTAAC),而WT1有2个,分别是QRE-1(2046bp-2052bp,ACTAAC)和QRE-2(2211bp-2217bp,ACTAAC)。为了验证QKI-5是否与Caspase8和WT1的3’UTR结合,成功构建了Caspase8的野生型双荧光素酶报告基因载体Caspase8-3’UTR-WT和突变型载体Caspase8-3’UTR-MUT;以及WT1的野生型载体WT1-3’UTR-WT和WT1-3’UTR-MUT1,WT1-3’UTR-MUT2和WT1-3’UTR-MUT3三种突变型载体。双荧光素活性检测发现,QKI-5能与Caspase8的3’UTR结合,但只与WT1基因的QRE-1结合。最后,验证了QKI-5对Caspase8和WT1的转录后调控作用。实验结果表明,在加入放线菌素D 0-2h到6-8h期间,过表达QKI-5有助于WT1 m RNA的稳定,过表达QKI-5还能显著促进WT1及其下游基因Bcl-2的表达量;而且,当sh QKI3与pBI-WT1共转染ST细胞时,WT1基因可以弥补因沉默QKI-5引起的Bcl-2基因表达水平降低的现象。沉默QKI-5时,结果相反。在过表达QKI-5细胞中,加入放线菌素D 0-2h到6-8h期间,QKI-5能加快Caspase8的m RNA降解。能显著抑制Caspase8和Caspase3的表达水平,沉默QKI-5时,结果相反。结论:QKI-5具有抑制ST细胞的凋亡的功能。其分子机制是通过转录后调控维持WT1 m RNA稳定,促进WT1及其下游基因Bcl-2的表达量;QKI-5可加快Caspase8 m RNA的降解,并显著抑制Caspase8和Caspase3的表达水平,从而起到抑制ST细胞的凋亡功能。
[Abstract]:Testis supporting cells are located in the basement of the seminiferous tubule of the testis, and together with a variety of spermatogenic cells with different developmental states together to form the wall of the fine seminiferous tubule. Support cells have the important function of providing nutrition for spermatogenic cells, starting spermatogonial stem cell differentiation, maintaining spermatogenesis, phagocytosis and the important function of residual cytoplasm in the process of spermatogenesis. Colonization determines the size of the testis and the quantity and quantity of sperm after maturity. And the number of mature supporting cells depends on the number of immature support cells. Therefore, immature support cells also play a vital role in the development and spermatogenesis of animal testis..QKI is a kind of RNA binding protein, which is highly conserved STAR. Family members, QKI, mainly express QKI-5, QKI-6 and QKI-7 three isomers.QKI can affect the metabolism of RNA by combining the QRE sequence of the target m RNA, thus participating in the process of cell proliferation, apoptosis and differentiation. The purpose of this study is to explore the effect of QKI-5 on the apoptosis of ST cells and the post transcriptional regulation of QKI-5. The QKI gene has three splicing patterns of QKI-5, QKI-6 and QKI-7. According to human QKI-5, QKI-6 and QKI-7 sequences, three pairs of specific primers were designed respectively. The three target fragments were cloned successfully with C DNA as a template. They had QKI-5, QKI-6, and QKI-7 specific ends respectively. (NLS). Then, the effect of QKI-5 on the apoptosis of ST cells was studied. First, the overexpression vector of QKI-5 was constructed and 4 interference carriers (SH RNAs) were synthesized. The expression and silence of QKI-5 in ST cells showed that the number of cells was significantly higher than that of the control group after the overexpression of QKI-5, and the number of cells decreased significantly after the interference QKI-5. The cell apoptosis rate was 2.32 + 0.11%, and the cell apoptosis rate was 2.32 + 0.11%, while the cell apoptosis rate was 25.64 + 0.68%, the difference was significant (P0.01), indicating that QKI-5 had the function of inhibiting the apoptosis of ST cells. Then, the molecular mechanism of QKI-5 inhibition of apoptosis was verified. Bioinformatics analysis found that there were 1 on the 3 'UTR of the Caspase8 gene. QRE components (1009bp-1014bp, ACTAAC) that can be combined by QKI, and WT1 have 2, QRE-1 (2046bp-2052bp, ACTAAC) and QRE-2 (2211bp-2217bp, ACTAAC). E8-3 'UTR-MUT; and the three mutant vectors of WT1-3' UTR-WT and WT1-3 'UTR-MUT1, WT1-3' UTR-MUT2 and WT1-3 'UTR-MUT3. Double fluorescein activity detection found that QKI-5 can be combined with 3' but only combined with the transcriptional regulation of the genes. The results showed that overexpression of QKI-5 could contribute to the stability of WT1 m RNA during the addition of actinomycin D 0-2h to 6-8h, and the overexpression of QKI-5 also significantly promoted the expression of WT1 and its downstream gene Bcl-2. When silent QKI-5, the result is opposite. In over expression QKI-5 cells, QKI-5 can accelerate m RNA degradation of Caspase8 by adding actinomycin D 0-2h to 6-8h. It can significantly inhibit the expression level of Caspase8 and Caspase3, when silent QKI-5, the result is opposite. Controlling the stability of WT1 m RNA and promoting the expression of WT1 and its downstream gene Bcl-2; QKI-5 can accelerate the degradation of Caspase8 m RNA, and significantly inhibit the expression level of Caspase8 and Caspase3, thus inhibiting the apoptosis function of ST cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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