XIAP Gene Small Interfering RNA Reverses The Multidrug Resistance Of Ovarian Carcinoma

IntroductionOvarian carcinoma is among the most lethal of all malignancies in women.Cisplatin-centered chemotherapy is the currently preferred treatment modality in human ovarian carcinoma,yet chemoresistance severely limits treatment success.Recent evidence suggests that deregulation of key pro- and anti-apoptotic pathways is a key factor in the onset and maintenance of chemoresistance.Furthermore,the discovery of novel interactions between these pathways suggests that chemoresistance may be multi-factorial.Technologies for gene knockout,including antisense oligonucleotides and ribozyme,have been frequently used to explore new functions of genes, but their low frequencies have limited their applications.Fortunately,the emergence of gene ablation technologies,based upon the RNA interference (RNAi) phenomenon,has provided new opportunities for experimental biology.Its blocking action on gene expression has been successfully observed in invertebrate,plant,and mammalian cells,and the knockdown of genes in cells has been achieved.Effective and highly specific,RNAi has become a new technique in knocking down genes,and it plays an important role in the study of gene function and gene therapy of diseases.To date,X-linked inhibitor of apoptosis protein(XIAP),a novel member of the inhibitors(IAP family),has been identified as the most potent one of caspases' inhibitors.Several recent reports have demonstrated that XIAP is an important regulator in apoptosis,triggered by various apoptotic stimulations, and that it is also widely recognized to play an important role in tumor formation and invasion/metastasis in both animal models and cancer patients. In a present study,XIAP has been shown to be one of the important regulators in cisplatin-induced apoptosis in ovarian cancer cells and that downregulation of XIAP sensitizes cells to cisplatin.The mechanisms of cisplatin resistance in ovarian carcinoma,however,remain poorly understood,and all the previous studies,including our work,are only in vitro.We hypothesized that knockdown of XIAP would inhibit ovarian tumor information and invasion/metastasis and that XIAP would be a good molecular target for cancer therapy.In this study,we hypothesized that manipulation of short hairpin RNA(shRNA)-induced gene silencing of XIAP would induce therapeutic apoptosis and sensitize drug-resistant human ovarian cancer cells to chemotherapy.To examine our hypothesis,we silenced XIAP expression in human ovarian cancer cells,A2780/DDP,by stable expression of an XIAP-specific siRNA.The knockdown of XIAP expression by siRNA in A2780/DDP cells showed a marked increase in chemosensitivity,besides an obvious inhibition of cell proliferation,tumorigenicity and a notable induction of cell apoptosis.Objective:To design small interfering RNA(siRNA) targeting to human XIAP gene,construct recombinant adenovirus(pAd-siRNA-XIAP) and transfer it to the human ovarian carcinoma cells A2780/DDP,and observe the effects of siRNA on the gene expression,proliferation,apoptosis, tumorigenicity and chemosensitivity.Study design:1.The psiRNA-XIAP expression plasmids targeting to human XIAP gene were designed,synthesized and identified: mRNA sequences of XIAP gene were got from Genbank.According to the design principles of siRNA,three specific siRNA sequences were selected by BLAST.The template DNA was constructed with these sequences.The recombinant plasmids psiRNA-XIAP1,2,3 were obtained by annealing and cloning the template DNA into a blank vector pSilencer.Then they were transferred into the strain DH5α,and identified by bacterial colonies PCR,restriction enzyme and sequencing of nucleic acid.The recombinant plasmids psiRNA-XIAP1,2,3 were transferred into the A2780/DDP cells.Twenty-four hours later,the interference effects of them were identified by RT-PCR and Western blot.2.The recombinant adenovirus pAd-siRNA-XIAP was constructed,identified and modified:By digestion of XbaⅠand XholⅠ,the siRNA-XIAP fragment was derived from the best effective psiRNA-XIAP,and cloned into the shuttle plasmid pAdTrack.After the digestion and linearization of PmeⅠ, pAdTrack-siRNA-XIAP was transfected into the E.coli strain BJ5183,which has a adenovirus plasmid pAdEasy-1.Then the homologous recombinant plasmid pAd-siRNA-XIAP was screened by kanamycin resistance test,cut by enzyme BamHⅠ,and identified by gel electrophoresis.After an amplification of electroporation transformation and a digestion and linearization of PmeⅠ, pAd-siRNA-XIAP was transferred into the 293 ceils with liposome Lipofectamine 2000,to modify and construct the recombinant adenovirus Ad-siRNA-XIAP.After ultracentrifugation and purification,the titer of Ad-siRNA-XIAP was identified by dilution assay.3.The Ad-siRNA-XIAP was transfected into A2780/DDP cells.Then the cell proliferation,cell cycle,ultrastructure,apoptotic rate and multidrug resistance were measured or observed by MTT assay,FCM,DNA Gel assay and AO stain assay.4.The total RNA of ovarian carcinoma and normal tissue were extracted. Then the mRNA were isolated and purified,and were reverse-transcribed to construct a cDNA probe,which was marked with fluorescent molecule Cy3/Cy5.This probe was hybridized with a gene chip which contains 8464 cDNA genes.The genes,differently expressed after the transfection of Ad-siRNA-XINP to A2780/DDP cell,was identified and analyzed.Results:1.The recombinant plasmids psiRNA-XIAP1,2,3 were successfully constructed,which were proved by PCR,restriction enzyme and sequencing of nucleic acid.24h after the three siRNA expression plasmids were transfected into A2780/DDP cells;it was shown that the expression level of XIAP mRNA declined obviously.And results from Western blot assay indicated that the psiRNA-XIAP1 has the most remarkable effect,so it was used to construct the recombinant adenovirus.2.The recombinant adenovirus Ad-siRNA-XIAP was identified to be successfully constructed.There was bright green fluorescence in the transfected 293 cells.And a high virus titer was got.3.Transfected by the recombinant plasmid pAdEasy-siRNA-XIAP,the strain A2780/DDP could show some morph changes;its clonic grow could remarkably decrease;its cell cycle could be changed;its apoptosis rate could significantly increase;and the results of DNA Gel assay appear the typical "DNA ladder".4.Transfected by the recombinant plasmid pAdEasy-siRNA-XIAP,the A2780/DDP cells were sensitized to cisplatin remarkably.Associated with the manipulation of pAdEasy-siRNA-XIAP,Cisplatin can obviously promote the effect of chemotherapy and induce therapeutic apoptosis of tumor cells. According to the research results,this potentiation of pAdEasy-siRNA-XIAP would be relevant to the status and function of XIAP gene in ovarian cancer cells.5.Some genes were differently expressed after the transfection of Ad-siRNA-XIAP to A2780/DDP cell.After they were identified and analyzed, the research results showed that these genes belonged to different functional groups,including both signal and protein transmission,both oncogene and proto-oncogene,apoptosis genes,and so on.Conclusion:1.The recombinant plasmids pSilencer-XIAP1,pSilencer-XIAP2 and pSilencer-XIAP3 were successfully constructed.They could remarkably decrease the mRNA expression level of XIAP gene when transfected to A2780/DDP cells.Of all the three plasmids,pSilencer-XIAP1 has the most significant effect.2.The recombinant adenovirus Ad-siRNA-XIAP was successfully constructed by pSilencer-XIAP1 and adenovirus pAdEasy.A high titer of virus was got by this concise method,in which merits of RNA polymeraseⅢand promoterH1 were combined.3.The recombinant adenovirus Ad-siRNA-XIAP obviously inhibited the expression of XIAP gene in A2780/DDp cells.And it has more remarkable effect than the recombinant plasmid psiRNA-XIAP1.4.Induced by the recombinant adenovirus,RNAi technology can remarkably increase the chemosensitivity of human ovarian cancer cells, A2780/DDP,to cisplatin.XIAP would be a good molecular target for reversing the multidrug resistance of ovarian carcinoma and inducing ovarian cancer cells to apoptosis.5.Gene chip and RNAi technology are powerful tools to analyze functions of genes.Through them,we have successfully gotten the gene map of genes inhabited by XIAP gene's expression,and gotten the possible positions of XIAP gene's functional accesses.This will be very useful for further researches to clear the mechanisms of multidrug resistance in ovarian carcinoma.