Construction and identification of ST8Sial gene over-expression vector and its effect on proliferation of human melanoma cells / 中国药理学通报

ABSTRACT

Aim To construct different over-expression vectors of ST8Sial gene and establish a cell line with stable ST8Sial over-expression , and to check the proliferation of cells with ST8Sial over-expression. Methods Polymerase chain reaction ( PCR) was used to amplify the code region of ST8Sial. The amplified ST8Sial fragment and pEGFP-Cl vector, pHBLV-CMV-MCS-3flag-EFl-ZsGreen-T2A-Puro vector were digested with restriction enzymes. The target gene fragment and the different vectors were ligated to obtain the ST8Sial-pEGFP-Cl recombinant vector and the recombinant lentiviral vector, respectively. PCR and DNA sequencing were used to identify recombinant vectors. Melanoma cells WM451 were transfected with different vectors. Cell line with stable ST8Sial over-expressing was established. ST8Sial mRNA level was checked by real-time PCR. STSSial protein expression level was checked by Western blot. Proliferation of the stable cells was assayed by CCK-8 method. The clony formation of stable cells was also performed. Results Both ST8Sial-pEGFP-Cl recombinant plasmid and STSSial over-expression lentiviral vectors were successfully constructed. The transfection efficiency of ST8Sial over-expression lentiviral vector was much higher than that of ST8Sial-pEGFP-Cl recombinant plasmid. A WM451 cell line with stable ST8Sial over-expression lentiviral vectors was established. Results showed that the over-expression of ST8Sial promoted the proliferation and colony formation of cells. Conclusions ST8Sial over-expression vectors are successfully constructed. The over-expression of ST8Sial promotes the proliferation and colony formation of WM451 cells.