摘要
目的:构建表达miR-192的重组腺病毒,感染HepG2细胞建立miR-192高表达的细胞模型,以研究miR-192在肝细胞中的功能。方法:将miR-192前体基因插入腺病毒穿梭载体pAdTrack,构建miR-192穿梭载体pAdTrack-miR-192,经线性化和同源重组,构建重组腺病毒载体pAd-miR-192;线性化后,在QBI-293A细胞中进行病毒包装;用包装成功的重组腺病毒感染HepG2细胞,72 h后收集细胞,提取总RNA,逆转录后进行定量PCR,检测miR-192和潜在靶分子视网膜母细胞瘤基因1(RB1)mRNA的变化。结果:获得670 bp的miR-192前体基因,经过穿梭载体后重组构建表达miR-192的腺病毒载体pAd-miR-192;将pAd-miR-192转染QBI-293A细胞,第8 d细胞病变及荧光的表达结果表明重组腺病毒成功包装;感染HepG2细胞后,定量PCR检测表明miR-192的表达较对照增加了835.87倍,同时检测到细胞中RB1 mRNA的表达显著降低。结论:构建了高效表达miR-192的腺病毒,建立了miR-192高表达的肝细胞模型,证明在肝细胞中抑制的RB1是miR-192靶分子,为后续miR-192在肝细胞中功能的研究奠定了基础。
Objective： To construct recombinant miR-192-expressing adenovirus and establish a high-level miR- 192 expression cell model by infecting HepG2 for miR-192 function study. Methods： miR-192 gene was ampli- fied from the HepG2 cell genomic DNA, and it was inserted into the shuttle vector pAdTrack. The constructed shuttle vector was linearized and transformed into E.coli BJ5183 to construct the recombinant miR-192-expressing adenovirus by homologous recombination. The recombinant adenovirus vector was linearized and packaged by trans- fected into QBT-293A cells, and then the virus particles were used to infect HepG2 cells. The RNA was extract- ed from the 72 h cultured cells, and the relative expression of miR-192 and retinoblastoma I（RB1） mRNA were detected by subsequent reverse transcription and q-PCR. Results： Gene of pre-miR-192 with 760 bp was ac- quired, and the shuttle vector pAdTrack-miR-192 and adenovirus vector pAd-miR-192 was constructed subsequent- ly. Cytopathic effect and fluorescence detection indicated that the adenovirus was successfully packaged 8 d follow- ing pAd-miR-192 infected QBI-293A ceils. The qPCR results indicated that the expression of miR-192 increased 835.87 times, and the expression of RB1 mRNA was markedly decreased. Conclusion： We successfully construct- ed the recombinant miR-192-expressing adenovirus and the miR-192 high level expressed cell model. Our results also suggested RB1 is one target of miR-192 in hepatoma carcinoma cell. The work lay the platform for further studying the function of miR-192 in ceils.
出处
《生物技术通讯》
CAS
2013年第1期8-12,共5页
Letters in Biotechnology
基金
国家自然科学基金青年基金(30900753)
