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

Abstract

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 determined by fluorescence method. Then the vector was transfected into TYST. After puromycin selection and culture expansion, stable cell clones of overexpressing let-7a (TYST-1) 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 1et-7a lentiviral vector was confirmed by plasmid enzyme digestion and DNA sequencing. The intracellular expressions of miRNA let-7a increased by 3.0 and 3.8 folds after transfecting with miR-let-7a than those in negative control and blank control groups respectively (P<0.05). CCK-8 assay 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 been successfully constructed. A stable testicular tumor cell line is established after vector transfection. And let-7a suppresses the TYST cell proliferation. Key words: microRNAs; Endodermal sinus tumor; Lentiviralvector; Cell proliferation