Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells
2009; Springer Nature; Volume: 28; Issue: 12 Linguagem: Inglês
10.1038/emboj.2009.139
ISSN1460-2075
AutoresSara Napoli, Chiara Pastori, Marco Magistri, Giuseppina M. Carbone, Carlo V. Catapano,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle21 May 2009free access Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells Sara Napoli Sara Napoli Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Chiara Pastori Chiara Pastori Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Marco Magistri Marco Magistri Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Giuseppina M Carbone Giuseppina M Carbone Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Carlo V Catapano Corresponding Author Carlo V Catapano Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Sara Napoli Sara Napoli Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Chiara Pastori Chiara Pastori Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Marco Magistri Marco Magistri Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Giuseppina M Carbone Giuseppina M Carbone Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Carlo V Catapano Corresponding Author Carlo V Catapano Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Search for more papers by this author Author Information Sara Napoli1, Chiara Pastori1, Marco Magistri1, Giuseppina M Carbone1 and Carlo V Catapano 1 1Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland *Corresponding author. Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Via Vela 6, Bellinzona 6500, Switzerland. Tel.: +41 091 820 0365; Fax: +41 091 820 0397; E-mail: [email protected] The EMBO Journal (2009)28:1708-1719https://doi.org/10.1038/emboj.2009.139 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Small interfering RNAs (siRNAs) directed to gene promoters can silence genes at the transcriptional level. siRNA-directed transcriptional silencing (RdTS) was first described in plants and yeasts and more recently in mammalian cells. RdTS has been associated with the induction of epigenetic changes and the formation of complexes containing RNA interference and chromatin-remodelling factors. Here, we show that a promoter-targeted siRNA inhibits transcription of the c-myc gene. Transcriptional silencing of c-myc did not involve changes of known epigenetic marks. Instead, the c-myc promoter-targeted siRNA interfered with transcription initiation blocking the assembly of the pre-initiation complex. Transcriptional interference depended on Argonaute 2 and a noncoding promoter-associated RNA initiated upstream and overlapping the transcription start site. Silencing of c-myc led to growth arrest, reduced clonogenic potential and senescence of c-myc over-expressing prostate cancer cells with minimal effect on normal cells. RNA-directed transcriptional interference may be a natural mechanism of transcriptional control and siRNAs targeting noncoding RNAs participating in this regulatory pathway could be valuable tools to control expression of deregulated genes in human diseases. Introduction Noncoding small RNAs, like small interfering RNAs (siRNAs) and microRNAs (miRNAs), are emerging as important regulators of cellular functions controlling gene expression at multiple levels (Kim and Rossi, 2007; Filipowicz et al, 2008). Recent studies have shown that small double-stranded RNAs—here called promoter-targeted siRNAs to distinguish them from siRNAs complementary to mRNA—can silence genes at the transcriptional level when directed to gene regulatory regions. The phenomenon of siRNA-directed transcriptional silencing (RdTS) was first described in yeasts and plants, where it was shown to involve DNA and chromatin modifications in the targeted genomic region (Matzke and Birchler, 2005; Buhler and Moazed, 2007; Grewal and Elgin, 2007). More recently, RdTS was shown to occur in mammalian cells and, like in yeasts and plants, was associated with the induction of epigenetic events (Morris et al, 2004; Ting et al, 2005; Weinberg et al, 2006; Pulukuri and Rao, 2007). However, the underlying mechanisms are still poorly understood. In human cells, RdTS requires the formation of multi-protein complexes containing elements of the RNA interference (RNAi) machinery, like Argonaute (Ago) proteins (Janowski et al, 2006; Kim et al, 2006). Ago proteins have been shown to localize to siRNA-targeted promoters and their knock-down abolishes RdTS (Janowski et al, 2006; Kim et al, 2006). DNA and chromatin modifying activities, like DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs), are also recruited to the targeted promoter and participate in RdTS (Kim et al, 2006; Weinberg et al, 2006). However, transcriptional silencing by siRNAs can occur without DNA or histone modifications, suggesting that the process can be mediated by multiple, partially distinct mechanisms (Ting et al, 2005; Janowski et al, 2005a, 2006; Schwartz et al, 2008). Unclear is also the target of the siRNAs. It was proposed that siRNAs might bind to single-stranded DNA regions, such as those present at TSSs, forming RNA:DNA hybrids and physically block transcription (Janowski et al, 2005a). Recently, siRNAs have been shown to bind to promoter-associated RNAs (pRNAs) forming RNA:RNA hybrids and create docking sites for the recruitment of gene silencing complexes (Han et al, 2007; Schwartz et al, 2008). Although still not extensively investigated, RdTS could be a very effective strategy to knock-down expression of genes involved in pathological processes. siRNAs could be designed to act as gene-specific transcriptional repressors and knock-down selectively expression of cancer promoting genes. This approach might be particularly useful for targets, like transcription factors, frequently over-active in cancer cells but difficult to address with conventional small-molecule drugs (Darnell, 2002). In this study, we explored the possibility to use siRNAs to repress transcription of the c-myc gene. c-Myc is a transcription factor and a key regulator of cell proliferation and death. It is one of the most frequently affected oncogenes in human cancers, contributing to deregulated cell proliferation, differentiation, survival and angiogenesis (Pelengaris et al, 2002; Adhikary and Eilers, 2005). c-Myc over-expression is due to gene amplification, chromosomal translocation or activation by other oncogenic pathways. In prostate cancer, c-myc is amplified or over-expressed in 30–80% of cases and is a major player in prostate cancer progression and acquisition of the androgen-independent phenotype (Pelengaris et al, 2002; Adhikary and Eilers, 2005). We designed siRNAs directed to sequences overlapping the major TSS in the c-myc gene following a strategy first described by Corey's group (Janowski et al, 2005a, 2005b). Our study had a twofold objective. We wanted to assess the ability of the siRNAs to block c-myc transcription and define the underlying mechanism, and second to determine whether silencing of c-myc by this approach resulted in the induction of relevant and stable changes in the phenotype of cancer cells over-expressing the gene. Our study showed that promoter-targeted siRNAs could effectively inhibit c-myc transcription. Analysis of the mechanism revealed that transcriptional silencing did not involve modifications of epigenetic marks earlier associated with RdTS. Instead, the siRNA acted by preventing transcription initiation by a process that could be defined as promoter-specific RNA-directed transcriptional interference (RdTI). RdTI depended on Ago2 and relied on the binding of the siRNA to a pRNA transcribed upstream of the TSS. Silencing of c-myc by the siRNA led to inhibition of proliferation and clonogenic potential of c-myc over-expressing prostate cancer cells with minimal effect on low c-myc expressing normal cells, underscoring the therapeutic potential of this strategy. RdTI may be part of natural RNA-based transcriptional control mechanisms and siRNAs targeting noncoding pRNAs participating in these regulatory pathways could be valuable tools to control the expression of deregulated genes involved in human diseases. Results Inhibition of c-myc expression by promoter-targeted siRNAs c-Myc is one of the most frequently over-expressed genes in human cancers (Pelengaris et al, 2002; Adhikary and Eilers, 2005). Like other oncogenic transcription factors, c-Myc is an ideal but difficult target for conventional drugs. To assess the ability of siRNAs to block c-myc transcription, we designed siRNAs directed to sequences overlapping the c-myc TSS (Figure 1A). The sequences targeted by myc9 and myc13 started at nucleotide-9 and -13 relative to the TSS, respectively, and encompassed the 11-nucleotide region from −9 to +2 that is unwound in the initiation complex and found to be important for transcriptional silencing (Holstege et al, 1997; Janowski et al, 2005a, 2005b). PC3 prostate cancer cells were transfected with siRNAs and harvested 72 h later to measure c-myc mRNA and protein level. c-myc mRNA was reduced in a dose-dependent manner in cells transfected with myc9 and myc13 compared with control siRNA-treated cells, with myc13 been more effective than myc9 as shown by RT–PCR and quantitative RT–PCR (qRT–PCR) (Figure 1B). Mismatched and scrambled control siRNAs (i.e. M3, M4, M5 and GL3) as well as siRNAs directed to sequences near the TSS (−24 to −59 from the TSS) but not overlapping the critical 11-nucleotide sequence did not have any effect on c-myc expression as shown by RT–PCR and qRT–PCR (Figure 1C). Consistently, c-Myc protein level was reduced in cells treated with active siRNAs (myc9 and myc13) and not affected in cells treated with control siRNAs as shown by immunoblotting (Figure 1C). c-myc expression was also reduced by the siRNAs in DU145 and LNCaP prostate cancer cells expressing high and intermediate levels of the gene (Supplementary Figure S1). As further demonstration of sequence and target specificity, transfection of active and control siRNAs in PC3 cells did not induce detectable levels of INF-α (Supplementary Figure S2), which could be a potential source of off-target effects (Hornung et al, 2005; Judge et al, 2005). Nuclear run-on assays were performed to determine whether reduced c-myc expression by myc13 was due to transcriptional inhibition. A decrease of nascent c-myc transcripts was seen in cells treated with the siRNA compared with control-treated cells, confirming reduced transcription of the gene (Figure 1E). siRNA generally induce a transient reduction of transcript level, which is reversed within few days (Kim and Rossi, 2007). To determine whether the promoter-targeted siRNAs had a similar behaviour, c-myc mRNA was measured by qRT–PCR at 2, 5 and 7 days after transfection. The effect of myc 9 and myc13 on c-myc RNA was similar at days 2 and 5, whereas it reversed partially at day 7 (Figure 1F), consistent with earlier data obtained using a similar approach (Janowski et al, 2005a). Under similar conditions, an siRNA targeting the mRNA reduced c-myc RNA to the same extent as myc13 and exhibited a similar kinetics of recovery, as shown earlier with a reporter gene system (Bartlett and Davis, 2006). Figure 1.Inhibition of c-myc gene expression by promoter-targeted siRNAs. (A) Position of the siRNA target sequences relative to the major transcription start site (+1) in the c-myc promoter. Myc9 and myc13 siRNAs were directed to sequences starting 9 and 13 nucleotide upstream to the TSS, respectively. (B) PC3 prostate cancer cells were transfected with 50 or 100 nM of siRNAs. Total RNA was extracted after 3 days and analysed by RT–PCR (left panel) or qRT–PCR (right panel). M4 was used as mismatched control siRNA. P<0.05 for my9 and myc13 compared with control-treated cells. (C) PC3 cells were transfected with 100 nM of myc13 along mismatched and control siRNAs (M3, M5 and GL3) or with siRNAs (si24 and si59) directed to sequences in the c-myc promoter not overlapping the TSS. Cells were harvested after 3 days and mRNA analysed by RT–PCR (top panels) or qRT–PCR (bottom panels). (C) Mock-transfected control cells. P<0.05 for myc13 compared with control-treated cells. (D) PC3 cells were transfected with 100 nM of the indicated siRNAs and harvested after 3 days to assess c-Myc protein by western blotting. (E) PC3 cells were transfected with 100 nM of siRNAs and harvested after 3 days. Nuclei were incubated in the presence of biotin-UTP. Nascent RNA was purified using streptavidin-agarose beads and analysed by RT–PCR with primers specific for c-myc and GAPDH. Right panel, gel densitometry data (mean±s.d.) from three independent experiments. P<0.05 compared with control-treated cells. Left panel, gel scan of a representative experiment. (F) PC3 cells were transfected with 100 nM of Gl3, myc9, myc13 and a mRNA targeting siRNA and harvested after 2, 5 and 7 days. mRNA level was measured by qRT–PCR. Values are normalized for the level in mock-transfected cells at day 2. P<0.05 for myc9, myc13 and siRNA compared with mock-and GL3-transfected cells at days 2 and 5. Download figure Download PowerPoint Promoter-targeted siRNAs block assembly of the transcription pre-initiation complex The myc13 siRNA induced an efficient and stable block of c-myc transcription. To investigate the mechanism by which the siRNA acted on the c-myc promoter, we examined the effects on epigenetic marks earlier reported to be involved in RdTS in mammalian cells. Using chromatin immunoprecipitation (ChIP), we assessed the level of histone H3K9 dimethylation (H3K9me2) and H3K27 trimethylation (H3K27me3), two repressive epigenetic marks (Li et al, 2007) shown to increase as a result of RdTS (Ting et al, 2005; Kim et al, 2006). The level of both H3K9me2 and H3K27me3 in the c-myc promoter did not change on transfection of myc13 (Figure 2A). H3K9me2 and H3K27me3 were enriched, however, in the promoter of the transcriptionally silenced genes p16 and RARB2 in PC3 cells (Kondo et al, 2008), confirming the adequacy of the assay conditions (Supplementary Figure S3). Adjacent regions of the c-myc promoter region (Figure 2B) were also examined with no evidence of increased H3K9me2 or H3K27me3 up to 7 days after transfection (data not shown). The level of histone H3 acetylation, a marker of transcriptionally active chromatin (Li et al, 2007), was also unaffected by treatment with the siRNA (Figure 2A). Furthermore, treatment of PC3 cells with the DNMT inhibitor 5-azadeoxycytidine (5-azadC) or the pan HDAC inhibitor trichostatin (TSA) did not affect c-myc silencing, indicating that DNMTs and HDACs were unlikely to be involved in the process (Figure 2C). These results argued against the involvement of these epigenetic events in silencing of c-myc by the siRNA targeting the TSS and were in agreement with earlier reports indicating that RdTS could be independent of histone and DNA modifications (Ting et al, 2005; Janowski et al, 2005a, 2006). Figure 2.siRNA-directed inhibition of pre-initiation complex formation on the c-myc promoter. (A) PC3 cells were transfected with 100 nM of myc13 and GL3 and harvested after 3 days. Chromatin immunoprecipitation (ChIP) was performed with antibodies against dimethylated histone H3K9 (H3K9me2), trimethylated histone H3K27 (H3K27me3) and acetylated histone H3 (AcH3). Input and immunoprecipitated DNA were measured by PCR with primers amplifying the −83/+124 region of the c-myc promoter (left panel) and SYBR Green qPCR (right panel). (B) Map of the c-myc promoter showing the regions probed in ChIP experiments. (C) PC3 cells untreated or treated with 5-azadC (left panel) or TSA (right panel) were transfected with siRNAs. Total RNA was extracted after 3 days and c-myc mRNA was measured by RT–PCR. (D) PC3 cells were transfected with siRNAs and ChIP was performed after 24 h with an anti-RNA polymerase II (Pol II) antibody. PCR was performed with primer sets amplifying the −84/+124 region. (E) PC3 cells were transfected with siRNAs and ChIP was performed after 3 (left panel) and 7 (right panel) days using an antibody for RNA polymerase II. PCR was performed with primer sets amplifying the indicated regions of the c-myc promoter. (F) ChIP assays were performed after 1, 3 and 7 days from transfection of siRNAs and RNA Pol II binding assessed by SYBR Green qPCR. P<0.01 compared with control-treated cells. (G) ChIP was performed with an antibody directed to TFIIB and the amount of input and immunoprecipitated DNA determined by PCR with primers amplifying the indicated regions of the c-myc promoter (left panel) and SYBR Green qPCR (right panel). P<0.05 compared with control-treated cells. Representative data at day 3 are shown. (H) PC3 cells were transfected with 100 nM of an siRNA targeting c-myc mRNA and harvested after 3 days to assess RNA Pol II binding to c-myc promoter by ChIP. PCR was performed with primers amplifying the −84/+124 region. Download figure Download PowerPoint To address the mechanism of transcriptional repression by the siRNA, we considered the possibility that it could interfere directly with PIC assembly at the TSS. To test this hypothesis, PC3 cells were transfected with siRNAs, and ChIP was performed to assess the binding of components of the PIC to the c-myc promoter. Binding of RNA Pol II was reduced in myc13-transfected cells as early as 24 h after transfection, indicating that it was an early event induced by the siRNA (Figure 2D). Decreased RNA Pol II binding was seen reproducibly with distinct primer sets and up to 7 days after transfection (Figure 2E). ChIP data on RNA Pol II binding were confirmed by qPCR with primers spanning the TSS (Figure 2F). Assembly of the PIC requires the binding of general transcription factors (GTFs) that direct RNA Pol II to the core promoter, stabilize the complex at the TSS and catalyse the steps necessary to initiate transcription (Hahn, 2004). To further determine the effects of the siRNA on PIC formation, we assessed binding of TFIIB. This GTF is a central component of PIC (Hahn, 2004; Deng and Roberts, 2007). TFIIB makes sequence-specific contact with the core promoter DNA and is absolutely required for RNA Pol II recruitment to the TSS (Hahn, 2004; Deng and Roberts, 2007). Binding of TFIIB to the c-myc promoter was reduced in myc13-transfected cells parallel to the decrease of RNA Pol II (Figure 2G). Thus, promoter occupancy by essential PIC components was reduced by the siRNA targeting the TSS according to a mechanism closely resembling transcriptional interference (Goodrich and Kugel, 2006; Martianov et al, 2007; Mazo et al, 2007). Unlike the promoter-targeted siRNA, the siRNA targeting c-myc mRNA did not affect RNA Pol II binding to the promoter, indicating that the two siRNAs acted by distinct mechanisms and reduced promoter occupancy by myc13 was not a consequence of reduced c-myc level (Figure 2H). RdTI and promoter-associated noncoding RNAs Noncoding RNAs have a prominent role in RdTS in plants and yeasts (Buhler and Moazed, 2007; Grewal and Elgin, 2007). Noncoding pRNAs have been shown recently to be the target of RdTS in mammalian cells (Han et al, 2007; Schwartz et al, 2008). Thus, we examined whether transcription occurred upstream and overlapping the c-myc TSS. RT–PCR with different primer sets showed the presence of low copy transcripts in the region −400 to +120 relative to the TSS (Figure 3A). Promoter-associated transcripts were much less abundant than c-myc mRNA as determined by semiquantitative RT–PCR (Figure 3B) and qPCR [∼50-fold lower than mRNA] (Figure 3C). The level of pRNAs correlated positively with c-myc mRNA expression, with higher levels in c-myc over-expressing PC3 cells than immortalized prostate epithelial LH cells (Figure 3C). Strand-specific RT–PCR was used to determine strand orientation of the promoter-associated transcripts. Sense transcripts were predominant over antisense transcripts in PC3, DU145 and LNCaP prostate cancer cells (Figure 3D) and no products were seen in no-RT reactions excluding contamination with genomic DNA (Supplementary Figure S4). 5′ rapid amplification of cDNA ends (5′RACE) confirmed the presence of multiple transcripts initiating upstream of the major TSS (Figure 3E). Cloning and sequencing of the 5′RACE products showed that the promoter-associated transcripts initiated within 300 and 800 bp from the major TSS (Supplementary Figure S5). Figure 3.Detection of promoter-associated transcripts in the c-myc gene. (A) Total RNA was isolated from PC3 cells and amplified using distinct primer sets shown in the left panel to identify promoter-associated transcripts in the region surrounding the major c-myc transcription start site (P2). (B) Total RNA and genomic DNA from PC3 cells were amplified with primers specific for c-myc mRNA and pRNA (F−83/R+124). Genomic DNA was amplified in parallel to control for amplification efficiency. (C) RNA isolated from PC3 and normal prostate epithelial (LH) cells was examined by real time RT–PCR to assess c-myc mRNA and pRNA levels. (D) Total RNA was extracted from PC3, DU145 and LNCaP cells and analysed by strand-specific RT–PCR with F−83/R+124 primers to identify sense (S) and antisense (A) promoter-associated transcripts. (E) 5′RACE products from PC3 cells were amplified with a nested gene-specific primer and the Abridged Universal Amplification Primer (Invitrogen). The marked PCR products were cloned and sequenced to confirm their identity. Download figure Download PowerPoint As proposed for other noncoding RNAs (Mattick and Makunin, 2006; Prasanth and Spector, 2007) pRNAs in the c-myc promoter could have a regulatory function, perhaps maintaining promoter accessibility to transcription factors and assisting in transcription initiation. The presence of pRNAs also argued in favour of a model in which siRNAs bound to the noncoding transcript and formed a complex that switched off transcription as recently proposed (Han et al, 2007; Schwartz et al, 2008). In favour of this hypothesis, the level of the c-myc pRNA was reduced, although only partially, in myc13-treated cells indicating an interaction between the two RNA species (Figure 4A). The physical interaction was confirmed by biotin-linked siRNA pull-down assays in which cells were transfected with an siRNA having either the sense or antisense strand labelled with biotin. The biotin-labelled antisense strand of myc13 was found to bind to the pRNA, whereas no signals were detected with the biotin-labelled sense strand or unlabelled siRNA (Figure 4B). Furthermore, no signal was detected in biotin pull-down samples by PCR after RNase treatment, indicating the absence of contaminating genomic DNA (Figure 4B). Figure 4.Interaction of promoter-directed siRNA with promoter-associated RNA. (A) PC3 cells were transfected with siRNAs and total RNA extracted after 3 days. RT–PCR was performed with primers specific for pRNA (F−217/R+124) and β-actin. (B) PC3 cells were transfected with the siRNA myc13 with biotin-labelled sense (S) or antisense (A) strand and control (C) siRNA. Cells were harvested after 24 h and RNA bound to the biotin-labelled siRNA was isolated with streptavidin-agarose beads and examined by RT–PCR with primers specific for the c-myc pRNA (top panel). Input and pull-down samples were subjected to PCR after RNAse treatment to exclude the presence of contaminating DNA in pull-down samples (bottom panel). (C) PC3 cells were transfected with myc13 along with GL3 and siRNAs targeting Ago1 or Ago2. Cells were harvested after 3 days to measure c-myc, Ago1, Ago2 and β-actin mRNA by RT–PCR. (D) PC3 cells were transfected with the fully complementary siRNA (myc13) or the siRNA with central mismatches relative to the target sequence (myc13cm). Cells were harvested after 3 days to measure c-myc and β-actin mRNA by RT–PCR. (E) PC3 cells were transfected with myc13, myc13 cm and the indicated positive and negative control siRNAs and harvested after 3 days to assess of c-Myc protein level by western blot. (F) PC3 cells were transfected with 100 nM of a scrambled (SCR) or antisense (ASO) oligonucleotide directed to the pRNA. c-myc mRNA and pRNA level was assessed by RT–PCR. Download figure Download PowerPoint Ago 2 mediates RdTI in the c-myc gene To establish whether the RNAi machinery was involved in RdTI, we knocked-down Ago1 and Ago2 and examined the effect on siRNA-directed c-myc silencing. Ago proteins are integral components of the RNAi pathway (Kim and Rossi, 2007; Tolia and Joshua-Tor, 2007) and are involved in RdTS in mammalian cells (Janowski et al, 2006; Kim et al, 2006). Efficient silencing of both Ago1 and Ago2 was achieved using specific siRNAs both in single- (Supplementary Figure S6) and double-transfection experiments (Figure 4C). In the latter, knock-down of Ago2 prevented c-myc silencing by myc13, whereas knock-down of Ago1 and a control siRNA did not have any effect (Figure 4C). This indicated that c-myc silencing relied mainly on Ago2, whereas previously both Ago1 and Ago2 had been shown to mediate RdTS (Janowski et al, 2006; Kim et al, 2006). As Ago2 has slicer activity (Kim and Rossi, 2007), we examined whether cleavage of the pRNA was required for RdTI. For this purpose, we used an siRNA (myc13cm) with three central mismatched bases relative to the target sequence, which would abrogate target RNA cleavage (Filipowicz et al, 2008). Silencing of c-myc was not significantly affected by the presence of central mismatches as myc13cm was as effective as myc13 and the mRNA targeting siRNA in decreasing c-myc mRNA and protein (Figure 4D and E), indicating that RdTI did not depend on Ago2 slicer activity. Thus, binding of the siRNA to the pRNA rather than its elimination was required for silencing of c-myc. Consistent with this hypothesis, an antisense oligonucleotide that almost completely knocked-down the pRNA did not affect c-myc transcription (Figure 4F). Taken together, these data indicated that formation of the siRNA:pRNA complex with the contribution of Ago2 was responsible for reduced assembly of the functional PIC and blockade of transcription initiation in the c-myc promoter. c-myc promoter-targeted siRNAs are effective inhibitors of cancer cell proliferation Myc is a key regulator of cell proliferation and death (Pelengaris et al, 2002; Adhikary and Eilers, 2005). Cells that over-express c-myc may depend on the continuous production of the protein and be highly sensitive to its down-regulation. To determine whether cell proliferation was affected as a consequence of c-myc knock-down induced by promoter-targeted siRNAs, we monitored growth of siRNA-transfected PC3 cells over a 12-day period. Untreated and control-treated cells behaved identically, whereas proliferation of cells treated with myc9 and myc13 was markedly reduced (Figure 5A). In fact, growth of myc13-treated cells was almost completely arrested. At day 8, growth was reduced by 40 and 80% and after 12 days by 60 and 90% for myc9- and myc13-treated cells, respectively, relative to control cells. Overall, the extent of cell growth inhibition by the two siRNAs correlated well with their relative potency as c-myc transcriptional repressors. Similar results were seen in clonogenic assays with myc13 (see below), consistent with persistent silencing of the c-myc gene. Figure 5.Reduced proliferation and clonogenic potential of siRNA-transfected prostate cancer cells. (A) PC3 cells were transfected with 100 nM of siRNAs in 12-well plates and counted with an automated cell counter over a 12-day period. P<0.005 compared with control cells at days 5, 8 and 12 with myc13, and days 8 and 12 with myc9. (B) DU145 (top panel) and LNCaP (middle panel) prostate cancer cells and normal human fibroblasts (bottom panel) were transfected with 100 nM of siRNAs and counted over a 7-day period as indicated above. P<0.005 compared with control cells at days 5 and 7 for DU145 and LNCaP cells treated with myc13. (C) PC3, DU145 and LNCaP cells were transfected with 100 nM of siRNAs and plated at clonal density in six-well plates. Colonies were stained with crystal violet (left panels) and counted after 12 days using an automated colony counter (right panels). P<0.005 for PC3 and DU145, and P<0.05 for LNCaP cells. Download figure Download PowerPoint Variations in the transcriptional activity of a gene may affect the ability of siRNAs to bind to the promoter and inhibit its transcription. The combination of high targeting efficiency and cell addiction to the target may make this approach highly selective towards cancer cells with deregulated expression of the target, while sparing normal cells with a physiological expression level. We explored this concept by examining the effects of siRNAs in two other prostate cancer cell lines (DU145 and LNCaP) and normal human fibroblasts (NHFs). Prostate cancer cells with high and interm
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