Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology · 2015
[Recombinant adenovirus of microRNA-193b inhibits proliferation of K562 cells].
Аннотация на английскомСтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Shu Yan, Qi Jieyu, Wang Canwei, Yu Shujing, Tao Kun
Аннотация
OBJECTIVE: To construct a recombinant adenovirus vector containing human pre-miR-193b and investigate its effect on the proliferation of chronic myelocytic leukemia K562 cells.
METHODS: The cDNA of pre-miR-193b was obtained by chemical synthesis and inserted into the adenoviral shuttle vector pAdTrack-CMV. The recombinant shuttle plasmid was linearized by Pme I and transformed into AdEasier cells for homologous recombination in E.coli BJ5183 with the adenoviral backbone plasmid pAdEasy-1. The recombinant adenoviral plasmid was linearized by Pac I and then used for transfecting HEK293 cells. After package and amplification in HEK293 cells, the virus titer was determined by serial dilution assay. The expression level of miR-193b in K562 cells was detected by real-time quantitative PCR. The cell proliferation was observed by MTT assay.
RESULTS: The recombinant plasmid named pAd-miR-193b was confirmed by restriction enzyme analysis and sequencing. The recombinant adenovirus could infect K562 cells efficiently. Compared with control group, real-time quantitative PCR revealed that the expression of miR-193b was significantly up-regulated in K562 cells infected with pAd-pre-miR-193b, and the cell proliferation was suppressed significantly in K562 cells with up-regulated miR-193b expression.
CONCLUSION: The over-expression of miR-193b may suppress the proliferation of K562 cells.
METHODS: The cDNA of pre-miR-193b was obtained by chemical synthesis and inserted into the adenoviral shuttle vector pAdTrack-CMV. The recombinant shuttle plasmid was linearized by Pme I and transformed into AdEasier cells for homologous recombination in E.coli BJ5183 with the adenoviral backbone plasmid pAdEasy-1. The recombinant adenoviral plasmid was linearized by Pac I and then used for transfecting HEK293 cells. After package and amplification in HEK293 cells, the virus titer was determined by serial dilution assay. The expression level of miR-193b in K562 cells was detected by real-time quantitative PCR. The cell proliferation was observed by MTT assay.
RESULTS: The recombinant plasmid named pAd-miR-193b was confirmed by restriction enzyme analysis and sequencing. The recombinant adenovirus could infect K562 cells efficiently. Compared with control group, real-time quantitative PCR revealed that the expression of miR-193b was significantly up-regulated in K562 cells infected with pAd-pre-miR-193b, and the cell proliferation was suppressed significantly in K562 cells with up-regulated miR-193b expression.
CONCLUSION: The over-expression of miR-193b may suppress the proliferation of K562 cells.