Objective： To research the silence of NBS1 after transfection microRNA expressing eukaryotic recombinants and the changes of telomerase activation in telomerase-positive cell line Hela. Methods： According to the seq...Objective： To research the silence of NBS1 after transfection microRNA expressing eukaryotic recombinants and the changes of telomerase activation in telomerase-positive cell line Hela. Methods： According to the sequence of NBS1 mRNA, the NBS1 pre-microRNA was designed and synthesized, then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cells. The integrity of the insert fragment was verified through colony PCR and sequencing analysis. The NBS1 gene expression of NBS1 microRNA recombinants was detected by Real-Time PCR and western blot. Telomerase activity in Hela cells was assayed by TRAP-PCR-EB. Results： Sequences of insert fragment in microRNA expressing recombinants were correct. The NBS1 gene expression was decreased, and the telomerase activation of Hela cell reduced. Conclusion： NBS1 microRNA inhibits NBS1 gene expression, and depresses telomerase activation of Hela cells. This confirms that there is relevance between NBS 1 gene and telomerase activity.展开更多
基金supported by grants from National Basic Research Program of China(2005CB522404,2006CB910506)The Program for Changjiang Scholars and Innovative Research Team(PCSIRT)in Universities(IRT0519)The National Natural Science Foundation of China(30771232,30671184)~~
文摘Objective： To research the silence of NBS1 after transfection microRNA expressing eukaryotic recombinants and the changes of telomerase activation in telomerase-positive cell line Hela. Methods： According to the sequence of NBS1 mRNA, the NBS1 pre-microRNA was designed and synthesized, then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cells. The integrity of the insert fragment was verified through colony PCR and sequencing analysis. The NBS1 gene expression of NBS1 microRNA recombinants was detected by Real-Time PCR and western blot. Telomerase activity in Hela cells was assayed by TRAP-PCR-EB. Results： Sequences of insert fragment in microRNA expressing recombinants were correct. The NBS1 gene expression was decreased, and the telomerase activation of Hela cell reduced. Conclusion： NBS1 microRNA inhibits NBS1 gene expression, and depresses telomerase activation of Hela cells. This confirms that there is relevance between NBS 1 gene and telomerase activity.
