DNA损伤应答：杂种细胞染色体不稳定及miR-214调控RNF8的研究

发布时间：2019-01-17 20:36

【摘要】：在大量的种间杂种细胞中,来自一个物种的染色体易发生丢失,而另一个物种的染色体则通常保持完整；例如,在人和小鼠的杂种细胞中,大量的人源染色体会被丢失。关于单一物种染色体丢失的机制目前仍知之甚少。在人工诱导或者有性杂交形成的杂种细胞研究中,对于染色体的丢失,前人曾提出一些假设,如物种间染色体分离不同步、核蛋白复制不同步及染色体空间隔离等；然而这些假设均源于对少量的固定细胞观察所得。活细胞实时摄影技术可追踪观察染色体在细胞周期进程中的行为,可准确确定染色体丢失的途径。本研究利用活细胞实时摄影和荧光原位杂交等技术,发现人鼠杂种细胞中人源染色体的丢失是渐进性的,以染色体断片的形式丢失。这种丢失与人源染色体上存在未修复的DNA损伤而细胞持续分裂有关。人源染色体上未修复的DNA损伤,可能是由于细胞发生融合后DNA损伤修复机制发生改变所致。此外,通过辐照增加人源染色体上的DNA损伤会加速杂种细胞中人源染色体的丢失,进一步表明,在人鼠杂种细胞中,人源染色体的丢失是因为人源染色体存在未修复的DNA损伤所致。综上,通过研究人鼠杂种细胞中染色体不稳定性,发现了一条新的引起染色体不稳定性的机制；该研究可为了解肿瘤发生提供参考。另外,本研究也提示：杂种细胞可作为深入研究DNA损伤应答分子机制的细胞模型。在多类肿瘤中,肿瘤的发生与DNA损伤应答的异常密切相关。DNA损伤应答信号通路包括感应DNA损伤、信号传递和促进DNA损伤修复等,其缺陷将导致肿瘤中普遍的特征之一染色体不稳定的发生。然而,目前对于这条信号通路仍不甚了解。泛素化连接酶RNF8介导着H2AX的泛素化,从而促进53BP1和BRCA1被募集到DNA损伤部位,进而促进DNA损伤修复,防止染色体不稳定。虽然RNF8是DNA损伤应答通路中的关键蛋白,但是对其表达的调控目前尚不清楚。本研究发现,在人卵巢癌细胞株中,miR-214可以通过结合3’UTR来抑制RNF8的表达,从而对DNA损伤应答造成干扰。同时,抑制miR-214的功能则可增加RNF8的表达,增强细胞的DNA损伤修复能力。另外,高表达不带有3’UTR的RNF8可以拯救miR-214过表达的效应。综上,这些结果表明,miR-214介导的RNF8的下调将干扰对DNA损伤的应答,进而导致卵巢癌染色体不稳定；该研究将有助于认识染色体不稳定性发生机制。
[Abstract]:In a large number of interspecific hybrid cells, chromosomes from one species tend to be lost, while chromosomes from another species are usually intact; for example, in human and mouse hybrids, a large number of human chromosomes are lost. Little is known about the mechanism of chromosome loss in a single species. In the study of hybrid cells induced by artificial or sexual hybridization, some hypotheses have been put forward for chromosome loss, such as dissynchronous chromosome separation between species, non-synchronization of nucleoprotein replication and chromosome spatial isolation, etc. However, these hypotheses are derived from observations of a small number of fixed cells. Live cell real-time photography can track the behavior of chromosomes during cell cycle and determine the pathway of chromosome loss. In this study, real-time photography of living cells and fluorescence in situ hybridization techniques were used to find that the loss of human chromosomes in the hybrid cells of human and mouse was progressive and was lost in the form of chromosome fragments. This loss is associated with unrepaired DNA damage and cell division on human chromosomes. The unrepaired DNA damage on human chromosomes may be due to the alteration of the repair mechanism of DNA damage after cell fusion. In addition, the loss of human chromosomes in hybrid cells was accelerated by increasing DNA damage on human chromosomes by irradiation, which further indicated that in human and mouse hybrids, The loss of human chromosomes is due to unrepaired DNA damage on human chromosomes. In conclusion, a new mechanism of chromosomal instability was found by studying chromosome instability in human mouse hybrid cells, which may provide a reference for the understanding of tumorigenesis. In addition, this study also suggests that hybrid cells can be used as a cell model for further study of the molecular mechanism of DNA damage response. In many kinds of tumors, the occurrence of tumor is closely related to the abnormal DNA damage response. The signal pathway of DNA damage response includes induction of DNA damage, signal transduction and promotion of DNA damage repair, etc. Its defects will lead to the occurrence of chromosomal instability, one of the most common characteristics of the tumor. However, this signal pathway is still not well understood. Ubiquitin ligase RNF8 mediates the ubiquification of H2AX, which promotes the recruitment of 53BP1 and BRCA1 to DNA damage sites, and further promotes DNA damage repair and chromosome instability. Although RNF8 is a key protein in DNA damage response pathway, the regulation of its expression is unclear. In this study, we found that in human ovarian cancer cell line, miR-214 can inhibit the expression of RNF8 by binding to 3'UTR, thus interfering with the DNA damage response. At the same time, inhibiting the function of miR-214 can increase the expression of RNF8 and enhance the ability of DNA damage repair. In addition, high expression of RNF8 without 3'UTR can save the effect of miR-214 overexpression. In conclusion, these results suggest that the down-regulation of RNF8 mediated by miR-214 will interfere with the response to DNA damage and lead to chromosomal instability in ovarian cancer. This study will be helpful to understand the mechanism of chromosomal instability.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.31

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