[Inhibition of CD40 expression on CA46 cell lines by hairpin siRNA eukaryotic expression vectors]

To construct human CD40 hairpin siRNA eukaryotic expression vectors and investigate their effects on CD40 expression, proliferation and apoptosis of CA46 cells.

Two DNA oligonucleotides encoding CD40 hairpin siRNAs were synthesized, and cloned into pSilenCircle to construct recombinant plasmid siCD40/pSilenCircle that expressed hairpin siRNAs under the control of pol III U6 promoter. At the same time, antiCD40/pSilenCircle encoding the antisense CD40 RNA and siFly/pSilenCircle encoding siRNA against luciferase were constructed as control vectors. The recombinants were identified by restriction enzyme digestion analysis and sequencing. The recombinant plasmids were transfected into CA46 cells. CD40 expression on CA46 cells and apoptosis were detected by flow cytometry. The proliferation of transfected CA46 cells was detected by MTS colorimetry.

(1)siCD40/pSilenCircle, antiCD40/pSilenCircle and siFly/pSilenCircle had been constructed successfully. (2) CD40 expressions on siCD40/pSilenCircle- and antiCD40/pSilenCircle-transfected CA46 cells were significantly reduced, as compared with that on siFly/pSilenCircle-transfected CA46 cells (P<0.01 and P<0.05, respectively). Both hairpin siRNAs and antisense RNA had no influence on the proliferation and apoptosis of CA46 cells.

Constructed siCD40/pSilenCircle and antiCD40/pSilenCircle can significantly inhibit CD40 expression, but have no significant influence on the proliferation and apoptosis of CA46 cells.