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目的建立稳定过表达miR-26a的肝癌细胞株。方法以CTE049质粒为模板,PCR扩增miR-26a序列418bp,片段两侧添加XbaⅠ和BamHⅠ酶切位点,In-Fusion克隆至pHAGE-fullEF1a-MCS-IZsGreen中,次日挑选单菌落,提取质粒并进行测序和酶切鉴定;所构建的载体命名为pLVT-miR26a。获得pLVT-miR26a后,按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带miR-26a和ZsGreen基因的慢病毒感染小鼠肝癌细胞(Hep1-6)、人肝癌细胞(BEL-7402、HepG2、Sk-Hep-1、HepG2-luc),以建立稳定过表达miR-26a的肝癌细胞系;qRT-PCR检测所建立肝癌细胞系中miR26a的表达水平。结果测序和酶切证实成功构建了pLVT-miR26a,按标准程序生产的携带miR-26a和ZsGreen基因的慢病毒上清成功感染小鼠及人肝癌细胞。结论成功建立稳定过表达miR-26a的肝癌细胞株,为相关后续研究打下了良好基础。
Objective To establish a hepatoma cell line stably overexpressing miR-26a. METHODS: The miR-26a sequence was 418bp amplified by PCR using the CTE049 plasmid as a template. XbaⅠ and BamHⅠ restriction sites were added to both sides of the fragment and in-Fusion was cloned into pHAGE-fullEF1a-MCS-IZsGreen. Single colonies were picked on the next day to extract the plasmid And sequencing and restriction enzyme digestion; the constructed vector was named as pLVT-miR26a. After pLVT-miR26a was obtained, the lentivirus was packaged according to the standard procedure recommended by Invitrogen and confirmed whether the lentivirus was successfully produced. The lentivirus carrying miR-26a and ZsGreen genes infect Hep1-6, BEL-7402, HepG2, Sk-Hep-1 and HepG2-luc) to establish miR-26a-stable hepatocellular carcinoma cell lines. QRT-PCR was used to detect the expression of miR26a in hepatoma cell lines. Results Sequencing and restriction analysis confirmed that pLVT-miR26a was successfully constructed and successfully transfected into mouse and human hepatocellular carcinoma cells by lentiviral supernatants carrying miR-26a and ZsGreen genes. Conclusion The successful establishment of stable hepatoma cell lines overexpressing miR-26a has laid a good foundation for subsequent studies.