| It is now recognized that the occurrence and development of prostate cancer was significantly associated with tumor cell dysfunction of proliferation and apoptosis regulated by a variety of related genes including Livin/ML-IAP/KIAP (hereinafter referred to Livin). Livin is recently described as a cancer-associated member of inhibitor of apoptosis proteins family, highly expressed in prostate cancer cell lines and tissues. Livin gene encodes two splicing variants termed as Livin-αand Livin-β, that could possibly play different roles in prostate cancer. In most cases, Livin expression only in tumor cells but not or low expressed in the corresponding tissues, while the two isoforms of Livin expressed differently in different tissues and even in different cell lines from the same tissue. Livin is highly present in prostate cancer. We founded that only Livin-αwas expressed in the androgen-dependent prostate cancer cell lines, LNCaP, while in androgen-independent prostate cancer cell lines, PC3, Livin-a and Livin-βwere highly present. Livin may play an important role in prostate cancer development and progression. Livin regulating apoptosis has been recognized by most researchers. Nowadays, it was generally known that anti-apoptosis of Livin was mainly mediated in indirect pathway of XIAP inhibiting activity of Caspases 3,7,9 leading to apoptosis blocked. In addition, it was also found that the high affinity of Livin with SMAC will strongly interact with the XIAP-SMAC competitedly, strongly combined with SMAC to suppress apoptosis and prevent SMAC on Caspases by the XIAP-mediated inhibition of apoptosis. Furthermore, Livin can also activate JNK1 in TAK1/TAB1 signaling cascade pathway, and JNK1 activation is essential to protect from apoptosis of TNF-αand inter-leukin-converting enzyme. Although Livin-a andβare very similar in structure but different in function. Although both Livin-αand P can protect cells from tumor necrosis factor a (TNF-α) and anti-CD95-induced apoptosis, blocking Livin-βnot Livin-αcan inhibit the formation of HeLa cells, and promote cells to sensitize different apoptotic by stimula such as UV radiation, TNF-αor etoposide. Closely like Survivin in structure, Livin can regulate cell cycle of melanoma cell lines, LiBr. In Hela cells, Livin locates mainly in nucleus, partly in cytoplasm in filamentous proteins form, as Survivin distributes in Hela cells. We founded in prostate cancer cell lines LNCaP and PC3 that Livin-a can promote G1-S cell cycle transition, while Livin-βsignificantly inhibited apoptosis. MicroRNA (miRNA) is an endogenous non-coding single-stranded RNA molecule, a non-protein coding RNA posttranscriptionally regulating gene expression. It was estimated that at least 30% of the human gene were regulated by miRNAs. In our study, miRNA was founded associated with the posttranscriptional inhibition of Livin. We believe that offset between tumor cell proliferation and apoptosis was partly caused by expression of Livin, and Livin expression was regulated by the miRNA. The studies of Livin isoforms in cell proliferation, apoptosis and miRNA regulating Livin in occurrence and progression mechanism of prostate cancer, provides new research directions and important basis.Therefore, to learn the influence of Livin on cell cycle progression, especially in early event of prostate cancer, we employed an specific RNAi and overexpression of Livin isoforms ato assess the individual roles of Livin for the regulation of the cell cycle in androgen-dependent prostate cancer cell lines LNCaP clone FGC (LNCaP).Objective:This study aims to explore correlation Livin with proliferation, apoptosis and mechanism of miRNA regulating Livin expression in prostate cancer, for the further study of related miRNA, providing experimental evidence for targeted therapy of prostate cancer.Materials and Methods:1. The expression of Livin isoforms was investigated by Western blot in human prostate cancer cell lines LNCaP and PC3. Mainly the role of Livin-a in vitro was further studied in LNCaP. Using Livin siRNA and overexpression models, cell cycle analysis, Ki-67 immunocytochemistry and MTT assay were porformed respectively. Cyclins was also analyzed. 2. The expression of Livin isoforms was investigated by Western blot in several human prostate cancer cell lines. PC3 cells in vitro as a model, using Livin isoforms-specific RNAi technology, Livin-αorβRNAi respectively was performed, followed by Annexin V apoptosis detection and MTT cell survival assay, then observe Livin effects on apoptosis and sensitivity to apoptosis stimulus.3. Livin expression were detected by RealTime RT-PCR, Western blot detection in three prostate cancer cell lines; dual luciferase reporter gene expression vector carrying 3’UTR of Livin gene mRNA was built, Livin-specific miRNA predicted by MiRNA software, filtered and identified in vitro by dual luciferase reporter gene assay system experiments.Results:1. Livin-αPromotes Cell Proliferation by Regulating G1-S Cell Cycle Transition in Prostate CancerLivin-αwas expressed in both LNCaP and PC3:meanwhile; Livin-βwas only detected in the PC3. Livin-αsiRNA not only resulted in G1-S cell cycle arrest, but also strongly correlated with the descended proliferation index and survival rate in LNCaP. In comparison, overexpression of Livin-αresulted in an accelerated S phase entry combined with elevated proliferation index and survival in LNCaP. In addition, the subsequent changes of Cyclins level implied that Livin-αmay regulate the cell cycle via cell cycle regulators in LNCaP. Meanwhile, Livin-βdidn’t affect the proliferation index and lacked any influence on Cyclins alterations although promoting the survival in LNCaP.2. The Effects on Apoptosis by Livin RNAi in PC3 Prostate Cancer Cell LinesWe found that Livin-αand Livin-βin PC3 cell lines were highly expressed. In PC3 cells, by following Annexin V-FITC detection after Livin-αorβknockdown, it found that although the reduction in Livin-αand Livin-βcould increase the survival rate of PC3 cells, campared with the negative control group, Livin-βknockdown clearly led to a significant increase in apoptosis and sensitized TNF-αand etoposide in PC3 cells. Livin-αknockdown led to PC3 cells sensitize TNF-αand staurosporine, although Livin-αalso increased apoptosis in PC3 cells, but no significant difference compared with the negative control.3. Livin Regulated by microRNA in Prostate CancerBy RealTimeRT-PCR, Western blot detected in prostate cancer cell lines, LNCaP and DU145 in the presence Livin gene transcriptional inhibition; by transfection of antisense-specific MiRNA found that the Livin gene transcriptional inhibition by specific inhibition of Livin mRNA caused by the miRNA; by cloning the gene was successfully constructed Livin mRNA in the 3’UTR region dual luciferase reporter gene expression vector; predicted by computer MiRNA software to screen several candidates for Livin mRNA MiRNA; detected by Western blot and in vitro dual luciferase reporter gene show miR-198 is one pair of Livin gene 3’UTR target sites produce specific inhibition of MiRNA.Conclusions:1. Livin-αmay promote cell proliferation by regulating G1-S cell cycle transition and possibly play an important part in initiation of prostate cancer.2. Proper combination of Livin RNAi and some apoptotic stimuli may entail potential benefits in the treatment of prostate cancer.3. Livin was derepressed upon inhibition of miR198 in cells. |
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