Construction Of CD59 SiRNA Expression Vector And Effect Of Peptide Seals To CD59 On The Expression Of Apoptosis-related Genes In HeLa Cells

Objectives: To construct a recombinant retroviral vector pSUPER retro RNAi that target CD59 gene and which are essential to study the mechanism of CD59 in tumor cell apoptosis by RNA interference. We study the effect of peptide seals to CD59 which designed and synthesized by us in HeLa cells and investigate the mechanism of peptide seals to CD59 in inducing apoptosis of HeLa cells. Our study and research will give a new way in tumor therapy.Methods: The siRNA targeting CD59 gene was designed according to the quence of CD59 mRNA available in GenBank and designed rules of RNA interfere sequence. A pair of 60-nt oligonucleotides,each containing the sites of restriction endonuclease at both ends, were designed and synthesized which contains 19bp-target sequece(from 220 to 238). Oligonucleotides were annealed and ligated with linearized pSUPER retro neo+GFP by T₄ ligase. The recombinants were transferred into E coli JM109 and identified by enzyme digestion, PCR and sequencing. After ineraction with 50mg/L peptide seals to CD59 for 24 hours, The apoptosis of HeLa was detected by transmission electron microscope, The inhibitory ratio of cell proliferation was measured by MTT and the expression of Survivin, caspase-3 and bax was detected by immunohistochemistry.Results: Recombinant retroviral vector pSUPER retro RNAi that target CD59 gene were successfully constructed which were identificated by enzyme digestion, PCR and sequencing. The fragmented caryons of HeLa cells were detected by transmission electron microscope, The inhibitory ratio of HeLa cells with CD59-transfected gene+peptide seal group was higher than that of HeLa cells+peptide seal group by MTT, There was a significant difference in depressing the proliferation between two groups (P<0.01). Compared with HeLa cells+peptide seal group, the Survivin expression of HeLa cells with CD59-transfected gene+peptide seal group was decreased (P<0.05) while caspase-3 expression was increased(P<0.05). There was no significant difference in the expression of bax in four groups(P>0.05) by immunohistochemistry.Conclusions: Recombinant retroviral vector pSUPER retro RNAi that target CD59 gene were successfully constructed which were identificated by enzyme digestion, PCR and sequencing. The peptide seal to CD59 can down-regulate the expression of Survivin, activate caspase-3 and enhance the apoptosis of HeLa cells. Peptide seals to CD59 can induce apoptosis of HeLa cells and find a new way in tumor therapy.