Construction Of Human CD40 Hairpin SiRNA Expression Vectors And Their Inhibition Of CD40 Expression On CA46 Cell Line

Aim: To construct human CD40 hairpin siRNA expression vectors and investigate their RNAi effects on CD40 expression on CA46 cell line.Methods: Two CD40 sequence-specific DNA oligonucleotides were synthesized. Then the inserts were cloned to form siCD40/pSilenCircle vectors that express hairpin siRNAs under the control of the pol III U6 gene promoter. AntiCD40/pSilenCircle plasmid, which encodes the antisense CD40 RNA, and siFly/pSilenCircle plasmid, which contains unrelated sequences, were constructed in the same way. The recombinants were identified by restriction enzyme digestion and sequencing. The plasmids were transfected into CA46 cell line using FuGene6. CD40 expression on membrane and cell apoptosis were detected with flowcytometry; cell proliferation was detected with MTS.Result: 1,Human CD40 hairpin siRNA expression vectors—siCD40/pSilenCircle, antisense CD40 RNA vectors—antiCD40/pSilenCircle and control vector—siFly/pSilenCircle were constructed successfully. 2,In the presence of siCD40/pSilenCircle and antiCD40/pSilenCircle, CD40 expression was significantly reduced, compared to that of siFly/pSilenCircle (P<0.01 and P<0.05 respectively). No significant effects had been found on cell proliferation and apoptosis in the presence of siCD40/pSilenCircle and antiCD40/pSilenCircle. Conclusion: Human CD40 hairpin siRNA expression vectors were constructed successfully and can significantly inhibit CD40 expression, but no significant effects had been found on cell proliferation and apoptosis in the presence of siCD40/pSilenCircle and antiCD40/pSilenCircle. RNA interfering technology may serve as an effective means of gene regulation.