Abstract:

Objective To construct the sphingosine kinase 1 （SPHK1） overexpression lentiviral vector， and to establish the SKOV3 lentiviral stable transfection cell line. Methods According to the SPHK1 data information provided by the National Center for Biotechnology Information （NCBI） database， the primers were designed and synthesized， the target gene was amplified， and connected to the GV492 plasmid treated with BamHⅠ and AgeⅠ restriction enzymes to construct the SPHK1 overexpression lentiviral vector； the positive clones were selected for PCR and sequencing identification； the lentiviral plasmid and the lentiviral packaging auxiliary plasmid were co-transfected into the HEK-293T cells for packaging and titer determination； according to the measured optimal multiplicity of infection （MOI） of 10， the corresponding lentiviral amounts in various groups were transfected into the SKOV3 cells， and the SKOV3 cells were divided into blank group （without treatment）， GV492 control group （GV492 control lentivirus infected SKOV3 cells）， and GV492-SPHK1 overexpression group （GV492-SPHK1 overexpression lentivirus infected SKOV3 cells， ov-SPHK1 group）； the optimal concentration of 2 mg·L^-1 puromycin was used to screen the stably transfected SKOV3 cell line； after 48 h， the medium was changed and replaced with 1 mg·L^-1 puromycin for screening for 14 d； the morphology and fluorescence expression of the cells were observed under fluorescence microscope； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of SPHK1 mRNA in the SKOV3 cells in various groups； Western blotting method was used to detect the expression level of SPHK1 protein in the SKOV3 cells in various groups. Results The PCR sequencing results showed that the gene sequence of the SPHK1 overexpression lentiviral vector was completely consistent with the target sequence， and the SPHK1 overexpression lentiviral vector was successfully constructed； the titer determination results showed that the lentiviral titers in GV492 control group and ov-SPHK1 group were 5×1011 and 8×1011 TU·L?1， respectively； the SKOV3 cells in GV492 control group and ov-SPHK1 group were in good state and showed strong fluorescence expression， suggesting that the SKOV3 stable transfection cell line overexpressing SPHK1 was successfully established； the RT-qPCR results showed that compared with blank group and GV492 control group， the expression level of SPHK1 mRNA in the SKOV3 cells in ov-SPHK1 group was significantly increased （P<0.01）； the Western blotting results showed that compared with blank group and GV492 control group， the expression level of SPHK1 protein in the SKOV3 cells in ov-SPHK1 group was significantly increased （P<0.01）. Conclusion The SPHK1 overexpression lentiviral vector is successfully constructed， and the SKOV3 stable transfection cell line is established.

Key words: Sphingosine kinase 1, SKOV3 cells, Lentivirus, Stable transfection cell line, Ovarian neoplasms