Inhibited Effect On Human Prostate Carcinoma PC-3 Cell Line By RNA Interference-based Knockdown Of PTI-1 (PC-3)

RNA interference (RNAi) represents a phenomenon of double-stranded RNA (dsRNA)-mediated post-transcriptional gene silencing (PTGS). During this process, exogenous cellular dsRNA is cleaved by an ribonuclease, Dicer, to generate 19-23 bp small RNA fragments, referred to as small interfering RNA (siRNA). By joining an effector complex termed RISC (RNA-induced silencing complex), siRNA binds to the cellular RNA with homologous sequences, and contributes to the degradation of the corresponding RNA. At present, RNAi has been widely applied in the research of gene fuction andgene therapy as an efficient tool for specific gene silencing.Prostate carcinoma tumor-inducing gene 1 (PTI-1) cloned from a humanprostatic carcinoma cell line, LNCaP, was indicated as a dominant-acting, tumor-inducing gene. By using a cotransfection / nude mouse tumor assay and differential RNA display (DD) technology, Fisher PB initially detected this putative oncogene in nude mouse tumor-derived CREF-Trans6: 4NMT cells (CREF-Trans 6 cells transfected with LNCaP DNA), whereas it was notexpressed in parental CREF-Trans 6 cells. The 2106 bp full- length PTI-1 cDNA consists of three regions: a 5' unique 620 bp region (1-620 bp), an open reading frame from bp 621 to bp 1814 encoding a protein of 398 amino acids and a 3 'UTR (1818—2106 bp). The predicted protein coding moiety represents an truncated and mutate EF-1 α molecule. Compared with EF-1 α , the PTI-1 protein only contains two GTP binding domains. On the basis of sequence analysis, PTI-1 should encode a protein of 43.8 kDa. A predominant protein present after in vitro translation of PTI-1 has an M_r of-46 kDa. This value is larger than predicted and may result from protein modification, e.g. phosphorylation, in the rabbit reticulocyte lysate system. Based on the changes of its sequence and structure, PTI-1 probably acts as an oncogene effecting on the "genome stability" by the effect of "translational infidelity" to promote tumor growth and its process. PTI-1 's transcripts were overexpressed in various human tumor cell lines, prostate carcinoma tissues, as well as prostate carcinoma patient-derived blood samples, while not in normal proste tissues and BPH tissues. We highly proposed that PTI-1 acts as a domain-acting oncongene in tumorigeness and tumor process. We have cloned a mutant PTI-1, named PTI-1 (PC-3), in androgen-independent prostate carcinoma PC-3 cell line. Compared with EF-1 α , PTI-1 (PC-3) has 100% identity except for 9 bases at initiational code. The mutant PTI-1 was registered in GenBank with accession number AF397403. The PTI-1 (PC-3) has even higher homology to EF-1 α and the research on its function is more important.In principle, RNAi can be achieved by chemically synthesized RNA duplexes, or hairpin RNAs of 19-23 base pair stem-loop structures, which are expressed from an HI or U6 promoter of an eukaryotic expression vector, e.g. a pEGFP/U6 vector harboring a U6 promoter. Here we constructed the expression vectors of 3 siRNA, i.e. mP1, mP2, and mp3, which targeted to the transcript of the mutant PTI-1, PTI-1 (PC-3), respectively.The constructs were...