miRNA-20a作为CHO-K1细胞抗凋亡调控靶标的研究
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摘要
通过分析miRNA表达谱发现,miRNA-20a在凋亡CHO-K1细胞中的表达降低。通过构建稳定表达miRNA-20a的重组细胞株,分析该重组细胞的抗凋亡能力。分别构建携带miRNA-20a前体序列或反义序列的重组慢病毒,转染CHO-K1细胞,得到miRNA-20a功能获得或功能抑制的重组细胞。无血清过度消耗培养液应激环境下培养重组细胞,分析靶基因E2F1的表达水平,通过细胞活力和Caspase3/7活性分析评价细胞的抗凋亡能力。结果显示,miRNA-20a过表达的细胞株,E2F1的表达受到显著抑制,在无血清过度消耗的应激环境下,细胞活力显著提高,Caspase3/7活性降低。相反,miRNA-20a抑制表达的细胞株中,E2F1表达增加,而细胞活性显著降低,抗凋亡能力显著下降。上述结果提示,miRNA-20a有望作为哺乳动物宿主细胞的遗传重构靶标,构建耐受应激环境的抗凋亡细胞。
The miRNA microarray analysis showed that the miR-20 aexpression was significantly downregulated in apoptotic CHO-K1 cells,which suggested that miRNA-20 awere potential target for genetic engineering cells to enhance apoptosis resistance.The genetic recombinant CHO-K1 cells with miRNA-20 astable expression or inhibiting expression were constructed using the recombinant lentivirus vectors with pre-miR-20 asequences or anti-miR-20 asequences.To evaluate anti-apoptosis ability of recombinant cells,the cells were cultured in spent medium without serum,then the expression level of target gene E2F1,cell viability and Caspase 3/7activity were analysed.The results show that the expression of E2F1 and Caspase 3/7activity are significantly inhibited,consequently the cells viability is improved.In contrast,the anti-apoptosis anbility is weakened in recombinant CHO-K1 cells with inhibited miR-20 aexpression.These results suggest that stable miR-20 aexpression is beneficial to maintain viability of cells exposed to full-stress culture and miR-20 amay become a target for developing new technologies in enhancing the resistance of mammalian cells to shear stress.
The miRNA microarray analysis showed that the miR-20 aexpression was significantly downregulated in apoptotic CHO-K1 cells,which suggested that miRNA-20 awere potential target for genetic engineering cells to enhance apoptosis resistance.The genetic recombinant CHO-K1 cells with miRNA-20 astable expression or inhibiting expression were constructed using the recombinant lentivirus vectors with pre-miR-20 asequences or anti-miR-20 asequences.To evaluate anti-apoptosis ability of recombinant cells,the cells were cultured in spent medium without serum,then the expression level of target gene E2F1,cell viability and Caspase 3/7activity were analysed.The results show that the expression of E2F1 and Caspase 3/7activity are significantly inhibited,consequently the cells viability is improved.In contrast,the anti-apoptosis anbility is weakened in recombinant CHO-K1 cells with inhibited miR-20 aexpression.These results suggest that stable miR-20 aexpression is beneficial to maintain viability of cells exposed to full-stress culture and miR-20 amay become a target for developing new technologies in enhancing the resistance of mammalian cells to shear stress.
引文
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[3]Kramer O,Klausing S,Noll T.Methods in mammalian cell line engineering:from random mutagenesis to sequence-specific approaches[J].Applied Microbiology and Biotechnology,2010,88(2):425-436.
[4]Xu X,Nagarajan H,Lewis N E,et al.The genomic sequence of the Chinese hamster ovary(CHO)-K1cell line[J].Nature Biotechnology,2011,29(8):735-741.
[5]Hackl M,Jakobi T,Blom J,et al.Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome:identification,annotation and profiling of microRNAs as targets for cellular engineering[J].Journal of Biotechnology,2011,153(1/2):62-75.
[6]Muller D,Katinger H,Grillari J.MicroRNAs as targets for engineering of CHO cell factories[J].Trends in Biotechnology,2008,26(7):359-365.
[7]Barron N,Sanchez N,Kelly P,et al.MicroRNAs:tiny targets for engineering CHO cell phenotypes?[J].Biotechnology Letters,2011,33(1):11-21.
[8]Jadhav V,Hackl M,Druz A,et al.CHO microRNA engineering is growing up:recent successes and future challenges[J].Biotechnology Advances,2013,31(8):1501-1513.
[9]Leung A K,Sharp P A.MicroRNA functions in stress responses[J].Molecular cell,2010,40(2):205-215.
[10]O′Donnell K A,Wentzel E A,Zeller K I,et al.c-Mycregulated microRNAs modulate E2F1expression[J].Nature,2005,435(7043):839-843.
[11]Gammell P,Barron N,Kumar N,et al.Initial identification of low temperature and culture stage induction of miRNA expression in suspension CHO-K1cells[J].Journal of Biotechnology,2007,130(3):213-218.
[12]Barron N,Kumar N,Sanchez N,et al.Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7[J].Journal of Biotechnology,2011,151(2):204-211.
[13]Jadhav V,Hackl M,Klanert G,et al.Stable overexpression of miR-17enhances recombinant protein production of CHO cells[J].Journal of Biotechnology,2014,175:38-44.
[14]张兵兵,鲜成玉,侯汝涛,等.miRNA-20a作为哺乳动物宿主细胞抗凋亡调控靶标的应用:中国,201310354271.2[P].2013-11-20.Zhang Bingbing,Xian Chengyu,Hou Rutao,et al.The application of minRNA-20aas a mammalian host cell apoptosis tegulation target:China,2013103542712[P].2013-11-20.(in Chinese)
[15]Olive V,Jiang I,He L.mir-17-92,a cluster of miRNAs in the midst of the cancer network[J].The International Journal of Biochemistry&Cell Biology,2010,42(8):1348-1354.
[16]Kumari A,Iwasaki T,Pyndiah S,et al.Regulation of E2F1-induced apoptosis by poly(ADP-ribosyl)ation[J].Cell Death and Differentiation,2015,22:311-322.
[17]Furukawa Y,Nishimura N,Furukawa Y,et al.Apaf-1is a mediator of E2F-1-induced apoptosis[J].The Journal of Biological Chemistry,2002,277(42):39760-39768.
[18]Nahle Z,Polakoff J,Davuluri RV,et al.Direct coupling of the cell cycle and cell death machinery by E2F[J].Nature Cell Biology,2002,4(11):859-864.
[19]Polager S,Ginsberg D.p53and E2f:partners in life and death[J].Nature Reviews Cancer,2009,9(10):738-748.
[20]Johnson D G,Degregori J.Putting the oncogenic and tumor suppressive activities of E2Fintocontext[J].Current Molecular Medicine,2006,6(7):731-738.
[21]Stanelle J,Putzer B M.E2F1-induced apoptosis:turning killers into therapeutics[J].Trends in Molecular Medicine,2006,12(4):177-185.
[22]Yu Z,Wang C,Wang M,et al.A cyclin D1/microRNA 17/20regulatory feedback loop in control of breast cancer cell proliferation[J].The Journal of Cell Biology,2008,182(3):509-517.