慢病毒介导的稳定表达let-7a的睾丸卵黄囊瘤细胞株的建立

Construction of a stable human testicular yolk sac tumor cell line by over-expression of microRNAlet-7a

目的构建microRNAlet-7a重组慢病毒表达载体,建立稳定表达let-7a的睾丸卵黄囊瘤细胞株(TYST-1),并探索let-7a过表达后对睾丸卵黄囊瘤(TYST)细胞增殖的影响。方法根据miRBase数据库let-7a的序列,设计合适的引物序列,利用聚合酶链反应(PCR)钓取目的基因片段,经过扩增、酶切后,与慢病毒载体GV309连接。转化DH5α感受态细胞,阳性克隆进行PCR及测序鉴定。293T细胞中包装慢病毒,利用荧光法测定病毒滴度。将构建好的let-7a慢病毒载体感染睾丸卵黄囊瘤(TYST)细胞,经嘌呤霉素筛选并扩大培养后得到稳定克隆株,建立稳定表达let-7a的TYST-1。用Quantitative real-time PCR(qRT-PCR)检测let-7a表达水平,用Cell Counting Kit-8(CCK-8)检测let-7a过表达后对TYST细胞增殖的影响。结果构建的let-7a慢病毒载体经质粒酶切和测序鉴定正确;let-7a慢病毒感染组细胞内let-7a水平比阴性病毒对照组和空白对照组分别提高3.O倍和3.8倍(P<O.05)。CCK-8法细胞增殖试验结果显示,较空白组和阴性病毒组,let-7a慢病毒感染组细胞的增殖能力明显受到抑制(P<O.05)。结论成功构建let-7a慢病毒载体并感染TYST细胞,获得稳定过表达let-7a的TYST-1;并证实let-7a过表达后抑制了TYST细胞的增殖。

Objective To construct recombinant lentiviral vector of miRNA let-7a,establish a stable testicular tumor cell line (TYST-1)overexpressing let-7a and examine its effect on the cellular proliferation of testicular yolk sac tumor (TYST).Methods The primers of human miRNAlet-7a gene were designed for amplifying let-7agene fragments by polymerase chain reaction (PCR)according to miRBase database Let-7a sequence was amplified,purified and ligated with lentiviral vector plasmid (GV309).The DH5α competent cells were transformed and positive clones identified by PCR and sequencing.The recombinant lentivirus was packaged in 293T cells.Viral titer was detemined by fluorescence method.Then the vector was transfected into TYST.After puromycin selection and culture expansion,stable cell clones of overexpressing let-7a (TYST-I)were established.The expression of let-7a was detected by quantitative real- time polymerase chain reaction (qRT-PCR).And cell counting Kit-8(CCK-8)method was employed for examining the proliferation of TYST cell after transfection. Results The successful construction of let-7a lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing.The intracellular expressions of miRNA let-7a increased by 3.0and 3.8folds after transfecting with miR-let-7a than those in negative control and blank control groups respectively (P<0.05).CCK-8assay showed that cell proliferation of TYST in miR-let-7a group was significantly lower than those in negative control and blank control groups (P<0.05).Conclusions Let-7a recombinant lentiviral vector has beensuccessfully constructed.A stable testicular tumor cell line is established after vector transfection. And let-7a suppresses the TYST cell proliferation.