目的研究肝癌细胞miRNA-138(miR-138)通过调控细胞周期蛋白D3(CCND3)、波形蛋白对肝癌细胞的影响。方法利用RT-PCR对肝癌组织及癌旁组织(对照)、4种肝癌细胞系(HepG2、HHCC、HUH7、BEL-7402)及正常肝细胞系HL-7702(对照)的miR-138的mRN...目的研究肝癌细胞miRNA-138(miR-138)通过调控细胞周期蛋白D3(CCND3)、波形蛋白对肝癌细胞的影响。方法利用RT-PCR对肝癌组织及癌旁组织(对照)、4种肝癌细胞系(HepG2、HHCC、HUH7、BEL-7402)及正常肝细胞系HL-7702(对照)的miR-138的mRNA表达进行检测;设计合成miR-138的模拟物(mimics)及抑制剂(inhibitor)。分别将空载体(对照)、miR-138的模拟物及抑制剂转染至HepG2细胞,与相应对照相比,分析miR-138对CCND3、波形蛋白表达及肝癌细胞活力的影响。应用CCK-8法、细胞创伤愈合试验和Transwell细胞迁移试验分别对转染空载体、miR-138 mimics 及inhibitor后的HepG2肝癌细胞增殖及迁移能力进行检测。Western blot检测上调或下调miR-138后miR-138相关靶蛋白CCND3及波形蛋白的表达。结果与相应对照相比,miR-138的表达水平在肝癌组织及肝癌细胞系中明显降低( P <0.01)。当miR-138 mimics转染肝癌细胞后,HepG2细胞的活力降低,创伤愈合能力较弱,迁移能力明显降低( P <0.01),经Western blot检测,波形蛋白和CCND3的表达水平降低( P <0.01)。转染miR-138 inhibitor 后,HepG2细胞的增殖能力升高,肝癌细胞的创伤愈合能力和迁移能力提升( P <0.01),与此同时,波形蛋白和 CCND3的表达水平升高( P < 0.01 )。结论通过调节波形蛋白和 CCND3的表达,miR-138在肝癌细胞的增殖活力、迁移能力的变化机制中发挥重要的作用。展开更多
Germinal centers （GC） of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antib...Germinal centers （GC） of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an important component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. In contrast, GC development in Ccnd3^-/- mice is markedly impaired, as is the T cell-dependent antibody response. Within the GC, although both switched and unswitched B cells are affected by cyclin D3 inactivation, the IgM^- pool is more severely reduced. Interestingly, despite a compensatory increase in cyclln D2 expression, a significant number of Ccnd3^-/- GC B cells accumulate in quiescent GO state. Lastly, although cyclin D3 inactivation did not disrupt BCL6 expression in GC B cells, it completely blocked the GC promoting effect of BCL6 overexpression, suggesting that cyclin D3 acts downstream of BCL6 to regulate GC formation. This is the first demonstration that cyclin D3 plays an important and unique role at the GC stage of B cell development.展开更多
文摘目的研究肝癌细胞miRNA-138(miR-138)通过调控细胞周期蛋白D3(CCND3)、波形蛋白对肝癌细胞的影响。方法利用RT-PCR对肝癌组织及癌旁组织(对照)、4种肝癌细胞系(HepG2、HHCC、HUH7、BEL-7402)及正常肝细胞系HL-7702(对照)的miR-138的mRNA表达进行检测;设计合成miR-138的模拟物(mimics)及抑制剂(inhibitor)。分别将空载体(对照)、miR-138的模拟物及抑制剂转染至HepG2细胞,与相应对照相比,分析miR-138对CCND3、波形蛋白表达及肝癌细胞活力的影响。应用CCK-8法、细胞创伤愈合试验和Transwell细胞迁移试验分别对转染空载体、miR-138 mimics 及inhibitor后的HepG2肝癌细胞增殖及迁移能力进行检测。Western blot检测上调或下调miR-138后miR-138相关靶蛋白CCND3及波形蛋白的表达。结果与相应对照相比,miR-138的表达水平在肝癌组织及肝癌细胞系中明显降低( P <0.01)。当miR-138 mimics转染肝癌细胞后,HepG2细胞的活力降低,创伤愈合能力较弱,迁移能力明显降低( P <0.01),经Western blot检测,波形蛋白和CCND3的表达水平降低( P <0.01)。转染miR-138 inhibitor 后,HepG2细胞的增殖能力升高,肝癌细胞的创伤愈合能力和迁移能力提升( P <0.01),与此同时,波形蛋白和 CCND3的表达水平升高( P < 0.01 )。结论通过调节波形蛋白和 CCND3的表达,miR-138在肝癌细胞的增殖活力、迁移能力的变化机制中发挥重要的作用。
文摘Germinal centers （GC） of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an important component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. In contrast, GC development in Ccnd3^-/- mice is markedly impaired, as is the T cell-dependent antibody response. Within the GC, although both switched and unswitched B cells are affected by cyclin D3 inactivation, the IgM^- pool is more severely reduced. Interestingly, despite a compensatory increase in cyclln D2 expression, a significant number of Ccnd3^-/- GC B cells accumulate in quiescent GO state. Lastly, although cyclin D3 inactivation did not disrupt BCL6 expression in GC B cells, it completely blocked the GC promoting effect of BCL6 overexpression, suggesting that cyclin D3 acts downstream of BCL6 to regulate GC formation. This is the first demonstration that cyclin D3 plays an important and unique role at the GC stage of B cell development.
