Survivin-targeted Apoptosis And Gene Silencing In Prostate Cancer

Carcinoma of the prostate, one of the most prevalent malignant tumors in older males, accounts for approximately one third of all cancers diagnosed in men in the United States and remains the second most common cause of cancer death in this group. The incidence rate has been increasing year by year with the change of average life span and dietary structure in China. The survival and growth of prostate cancer cells is initially dependent on the presence of androgens, and virtually all prostate cancer patients respond when first treated with androgen ablation. However, resistance to hormone blockade ultimately results in the recurrence of highly aggressive and metastatic prostate cancer that is androgen-independent . Unfortunately, Except operation, there is no effective therapy is yet available for androgen-independent prostate cancer patients. and it's very necessary to explore some new strategies for this malignant disease.Gene therapy offers a new approach to treatment of prostate cancer. Despite limited clinical success, tumor-targeted gene therapy remains a promising approach to the treatment of malignant diseases. It is currently being actively evaluated throughout the world.Survivin gene encodes the survivin protein, a member of the IAP protein family, which plays an important role in the survival of cancer cells and the progression of malignancy. Survivin is essential for cell division, and as an inhibitor of apoptosis. With dual roles in promoting cell proliferation and preventing apoptosis. Survivin expression is undetectable in most normal adult tissues, but is overexpressed in virtually every human tumor that has been studied. The survivin promoter is a promising tumor-specific promoter for transcriptional targeting based on its activation.Caspases are a conserved family of cysteine proteases. They play diverse roles in inflammatory responses and apoptotic pathways. Among the critical cysteine protease family members, caspase-3 plays an essential role in the cleavage of diverse Cellular proteins. On the molecular level, increased expression of caspases or direct introduced activity caspases, could increase caspase activity.RNA interference (RNAi) is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of the cognate double stranded RNA. It has rapidly developed into one of the most widely applied technologies with therapeutic potential in molecular and cellular research. MicroRNAs (miRNAs), widely distributed, small regulatory RNA genes, target both mRNA degradation and suppression of protein translation based on sequence complementarity between the miRNA and its targeted mRNA.First , in order to study survivin promoter transcriptional activities in human tumor cell and normal cel1. The fragment of survivin promoter S26830 and S2681 were acquired by PCR amplification and inserted into pGL3-Basic and pPRIME vectors. Then the reconstructed plasmid was transiently transfected into human prostate cancer cell lines PC3,LNCap,uterine cervix cancer cell lines Hela and normal Chang liver cell. The transcriptional activities of survivin promoter in various cel1s was determined by measuring the expression of luciferase and Green fluorescent protein (GFP). Survivin promoter had transcriptional activities in all tumor cell lines and the transcriptional activity of the S26830 was much higher than the S2681 and reached a level of one third of the transcriptional activity of the CMV promoter. However, All survivin promoters expressed scarcely in normal Chang liver cell. Our results descript that survivin promoter is tumor-specific.Second ,to design and construct microRNA expression vectors and observe its inhibitive effects on survivin gene.Based on survivin gene sequence, Three targeted pre-microRNA oligonucleotides was designed and synthesized. The fragment amplified by PCR was cloned into established microRNA expression vector pPRIME, then PC-3 cell was transiently transfected with correct construct pPRIME-SV1,pPRIME-SV2 and pPRIME-SV3, and the inhibition effect was determined by using PT-PCR and Western blot. The survival curve of PC-3 cells were detected by MTT method. Double enzyme digestion analysis and DNA sequencing results confirmed that the sequence of survivin specific microRNA expression vector was correct. Survivin mRNA and protein expression level in PC-3 cells were specifically suppressed after transfection with pPRIME-SV1 and pPRIME-SV2 plasmids. The proliferation of PC-3 cells become slowly and the cells number decreased about 40% compared with control groups.In order to observe the effects of targeting of survivin promoter and increased apoptotic molecule in PC-3. We choose pPRIME-SV2 that identified have better inhibit effect, using S26830 replaced CMV promoter and activity caspases-3 replaced GFP fragment . A series of recombinant plasmids were constructed and transfected into PC-3 cells. The apoptosis index of PC-3 cells were detected by flow cytometry and electron microscope. The results of flow cytometry indicated that the apoptosis index increased obviously in recombinant plasmids with activity caspases-3 and enhance sensitivity of platinol. The results of cellular structure and cell organ in electron microscope displayed corresponding change.In summary, based on the tumor tissue specificity, survivin-targeted apoptosis and gene silencing system was constructed. Observed its lethal effect in prostate cancer cell lines PC3. The advantage of the system is enhance the apoptosis in prostate cancer cell specificity by removed the inhibitory action of survivin in apoptotic and increasing the expression of activity caspases-3 simultaneously. The study explore a novel strategy of target gene therapy on prostate cancer.