[The influence on expression of cyclin D1 by anti-sense oligonucleotide expression vector]

To construct eurokaratic expression vectors of pcDNA3.1-cyclin D1 and pcDNA 3.1-Anti-cyclin D1, and evaluate their influence on the expression of cyclin D1 in stable transfected Tca8113/CDDP cells.

The full length of cyclin D1 and its antisense fragments were acquired from Tca8113/CDDP cells by RT-PCR. These cDNA fragments were inserted into pUCm-T vector and the full length sequenced. Induced by IPTG, the vector could express cyclin D1 protein in vitro. After double digested by Hind III and EcoRI at the two ends of these cDNA fragments, these cDNA fragments were inserted into pcDNA3.1 vector and transfected into Tca8113/CDDP cells with the help of Lipfectamin 2000. Stable cell lines were acquired after continually selected with G418 for 4 weeks. The expression of cyclin D1 was detected with RT-PCR and immunohistochemical staining.

pcDNA3.1-cyclin D1, pcDNA3.1-Anti-5' and pcDNA3.1-Anti-3' vectors were constructed and named as pcDNA3.1(CO), pcDNA3.1-Anti-cyclin D1 5'(C5'), pcDNA3.1-Anti-cyclin D1 3'(C3') and pcDNA3.1-cyclin D1(CD1), respectively. The expression of cyclin D1 in Tca8113/CDDP cells transfected with C3' was significantly inhibited and the inhibition rate was 58.8%. While C5' could not inhibit the expression of cyclin D1. The protein level of cyclin D1 was strongly positive in Tca8113/CDDP-C0, Tca8113/CDDP-CD1 and Tca8113/CDDP-C5' cells. It was obviously repressed in Tca8113/CDDP-3' cells.

We successfully constructed pDNA3.1-cyclin D1 vector and pcDNA3.1-Anti-cyclin D1 vectors. Detected with RT-PCR and immunohistochemical staining, the mRNA level and protein expression of cyclin D1 were significantly inhibited in C3' transfected Tca8113/CDDP cells, which may provide a good experimental tool for further studying in reversing the multidrug resistance of Tca8113/CDDP cells.