探究内质网应激在异倍体胚胎干细胞分化障碍中的作用机制

Aneuploidy, a karyotype of unbalanced chromosome gains or losses that is not a multiply of the haploid component, is the leading cause of severe developmental defects and is also a hallmark of cancer. However, whether aneuploidy is a driving cause or a consequence of tumor formation remains controversial. In our previous study, we established several aneuploid mouse embryonic stem (ES) cell lines, including trisomy 6, 8, 11, 12 or 15, and found that aneuploidy impaired differentiation of ES cells and increased their neoplastic potential. Co-culture of wild-type and aneuploid ES cells or supplementation with extracellular Bmp4 rescued the differentiation defects of aneuploid ES cells. It indicates that aneuploidy might dysregulate the secretion of extracellular factors and the dysregulation of secretion impedes the proper differentiation of ES cells. In-depth understanding of the mechanisms underlying differentiation defects of aneuploid ES cells is necessary. Studies on aneuploid yeasts, MEFs and human cell lines showed that aneuploidy caused protein stoichiometry imbalances and increased the load on the protein quality control system, which also lead to increasing sensitivity to proteotoxic compounds including Cycloheximide，MG132，Geldanamycin and 17-AAG. In this study, we will first analyze the protein quality control pathway in the four trisomic ES cell lines (Ts6, Ts8, Ts11, Ts15). Protein levels of several genes mapped on trisomic chromosomes will be detected by Western blot. The abilities of protein folding and degradation will be tested according to the expression of Hsp70, Hsp90 and Hsf1 and the accumulation of protein aggregates. Undifferentiated wild-type ES cells contain high levels of carbonylated proteins and advanced glycation end products (AGEs). Upon cell fate specification, removal of oxidatively damaged proteins is triggered and the proteasome activity is elevated. The levels of carbonylated proteins and AGEs during differentiation of aneuploid ES cells will be tested. To investigate whether proteasome in aneuploid cells can further be stimulated to adapt the cell-fate commitment, we will detect the proteasome subunits expression and proteasome activity. The deficiency of proteasome activity will result in protein overload and subsequently continuous endoplasmic reticulum (ER) stress, which activates unfolded protein response (UPR) and leads to diminished protein secretion. The expression of genes in UPR pathway such as XBP1, ATF4, ATF6, CHOP, GRP78, GRP94 will be tested by RT-qPCR and Western blot. Electronic microscopy will be used to observe the morphlogy of ER and vesicle transportation. By mass spectrometry-based profiling, the secretomes during aneuploid ES cell differentiation will be analyzed to discover the abnormal secreted factors. The final point is to determine the causality among the damaged proteasome, ER stress and differentiation defects of aneuploid ES cells. Proteasome activator Oleuropein, ER stress inhibitor 4-PBA and some extracellular factors will be tested to rescue the differentiation defects of aneuploid ES cells. Overall, our study will focus on the protein quality control system and ER-associated abnormal secretome during differentiation of aneuploid ES cells, which might underlie the cell differentiation defect. The candidate genes, factors, and pathways discovered in this study will possibly benefit cancer therapy in the future.

异倍体，即细胞内染色体数目的非整倍性变异。它是自发性流产和精神发育迟滞的主要原因，也是肿瘤细胞的重要特征。然而异倍体在肿瘤发生发展中的作用还存有争议。前期工作中，我们利用精心设计的分子遗传学策略成功获得了6、8、11、12、15号染色体分别三体的小鼠胚胎干（ES）细胞株系。发现这些三体细胞株生长增殖较快，克隆形成率提高，而细胞分化能力显著下降，移植后形成畸胎瘤的能力增强且分化程度较低。研究提示这可能与异倍体细胞不能正常分泌细胞外因子有关，然而异倍体削弱ES细胞分化的分子机制尚待阐明。本课题中，我们将研究异倍体ES细胞分化过程中细胞内蛋白降解系统的活性及内质网应激水平，并利用定量蛋白质谱分析技术确定异倍体细胞分化时外泌蛋白组分的改变。建立“异倍体-蛋白稳态失衡-内质网应激-外泌蛋白组分变化-ES细胞分化障碍”的因果链，找出影响异倍体ES细胞分化的关键因子，从而为肿瘤分化治疗策略提供新的线索。

异倍体是肿瘤细胞的重要特征，然而它在肿瘤发生发展中的作用还存有争议。前期工作中，我们成功获得了6、8、11、12、15号染色体分别三体的小鼠胚胎干细胞（ESC）株系。发现这些三体细胞株生长增殖较快，细胞分化能力显著下降，移植后形成畸胎瘤的能力增强且分化程度较低。然而异倍体削弱ESC分化的分子机制尚待阐明。本课题中，我们检测了异倍体细胞内的蛋白表达水平，发现三体染色体上的蛋白表达水平呈上升趋势。在ESC分化早期，异倍体细胞内羰基化蛋白水平较野生型细胞增加，而蛋白酶体活性不能被有效调动，异倍体细胞内蛋白酶体亚基Pa28α、β5i以及pomp 的表达水平下降，出现冗余蛋白堆积。随后，我们使用RT-qPCR和Western blot检测了非折叠蛋白反应（UPR）相关基因Bip, Perk, eIF2a, Atf4, Chop, Atf6, Xbp1在mRNA和蛋白水平的表达，发现异倍体细胞UPR相关基因的表达水平较野生型细胞更高，存在更为严重的UPR。为明确异倍体ESC分化过程中异倍体细胞外泌蛋白组分的变化，我们收集了EB分化早期的条件培养基，进行了定量蛋白质谱分析，发现与mRNA加工、RNA 剪切、蛋白折叠、细胞间粘附、抗原呈递、蛋白水解以及蛋白翻译等相关的生物学过程被富集。细胞外泌体、细胞间粘附、蛋白酶体复合物被下调。提示细胞外泌组分的变化是异倍体细胞分化缺陷的重要原因。为研究异倍体引起的蛋白酶体活性下降以及内质网过度应激与ESC分化缺陷之间是否存在因果关系，我们在异倍体细胞分化的过程中分别添加了蛋白酶体激活剂oleuropein和内质网应激的抑制剂4-PBA，发现添加oleuropein可以通过上调异倍体细胞内蛋白酶体活性部分回复异倍体EB的分化缺陷，添加4-PBA可以通过抑制异倍体细胞内的内质网应激压力部分回复异倍体EB的分化障碍。体内实验表明oleuropein和4-PBA可以降低异倍体ESC的畸胎瘤形成能力，促进三胚层分化。总之，该课题明确了异倍体细胞的蛋白稳态失衡/内质网过度应激与ESC分化障碍之间存在因果关系，为肿瘤的分化治疗策略提供了依据。