Silencer® Pre-designed siRNAs (Ambion)
TaqMan® Gene Expression Assays (Applied Biosystems)
Delineating the Role of Survivin in Oncogenesis: An siRNA Study

In this study, targeted siRNAs were used to alter survivin expression, and a series of biochemical and cell-based assays were performed on the transfected cells to assess known indicators of apoptosis to better understand the role of survivin in cancer. Silencer® Pre-designed siRNAs targeting survivin (Ambion) were first evaluated for silencing using TaqMan® Gene Expression Assays (Applied Biosystems). The validated siRNAs were then used to study the functional effects of survivin down regulation in HeLa cells. Apoptosis was assessed by monitoring: a) cell survival/proliferation, b) nuclear condensation and chromosomal fragmentation, c) phosphatidyl serine externalization, and d) caspase activation. The magnitude of the induced biological effects were correlated with that of siRNA induced silencing at the mRNA and protein levels using a time course study. Together, the data indicates that reduction of survivin protein levels induced apoptosis-related events, although it failed to activate pro-caspase-3.

Introduction
Survivin (BIRC5) is a 17 kDa bifunctional protein that plays critical roles in the regulation of both cell division and survival [1]. Survivin expression is linked to a broad range of cancers [2]; all major cancer types express survivin while survivin is not expressed in noncancerous, differentiated cells [3].

In many cell types, survivin blocks apoptosis by inhibiting members of the caspase cysteine protease family, such as caspase-9 [4], caspase-3 and caspase-7 [5]. The phosphorylation of survivin at Thr 34 by the cyclin-dependent kinase cdc2 is believed to promote physical interaction between survivin and caspase-9, resulting in caspase-9 inhibition [6]. Interestingly, survivin and cdc2 proteins comigrate at the mitotic spindles and both appear to be critical for proper progression through the cell cycle [7]. Survivin, like other “inhibitors of apoptosis” (IAP) proteins, contains a single BIR (baculovirus IAP repeat) domain that facilitates binding to caspases as well as to HBXIP (hepatitis B X-interacting protein)--a proposed cofactor of survivin [1].

Based on their expression patterns and observed roles in blocking apoptosis, the survivin complex and its constituents appear to be very important for cancer cell survival and for apoptotic control. In spite of these observations, the exact mechanism by which overexpression of survivin promotes oncogenesis is unclear.

Testing of siRNAs

Experimental Design. The efficacy and potency of three distinct Silencer® siRNAs targeting survivin were tested by monitoring survivin mRNA levels in cells that had received a range of siRNA concentrations. Each of the siRNAs was reverse transfected [8] into HeLa cells in triplicate at multiple concentrations (range 0.05−100 nM) using siPORT™ NeoFX™ Transfection Agent (Ambion). Optimal transfection conditions had been previously established using the siPORT NeoFX Transfection Agent for reverse transfection of HeLa cells [9].

48 hours after transfection, TaqMan® Gene Expression Assays (Applied Biosystems) were used to monitor down regulation of survivin mRNA caused by the siRNAs as compared to that in cells treated with a nontargeting negative control siRNA (Silencer Negative Control #1 siRNA; Ambion).

Results. All three of the siRNAs tested gave potent silencing. For example, at just 1 nM siRNA concentration, all three siRNAs showed >85% survivin mRNA silencing (Figure 1). However, at very low siRNA concentrations, the IC50 values (concentration where the targeted mRNA is reduced by 50%, or inhibitory concentration) highlighted differences among these three siRNA molecules. Two of the siRNAs, #2646 and #121294, were selected for this study. The use of at least two siRNAs rather than a single, validated siRNA is necessary to confirm that the phenotypic results observed are due to silencing of the intended gene and not due to an siRNA sequence-dependent off-target effect. In our case, we purposely chose two siRNAs that provided different silencing efficiencies: the IC50 was at least 10-fold lower for #2646 than for #121294. The less efficacious siRNA, #121294, was included in this study to compare phenotypic results from siRNAs that provided different levels of survivin silencing.

Figure 1. Validation of siRNAs Targeting Survivin. Three Silencer® Pre-designed siRNAs targeting human survivin and nontargeting Silencer Negative Control #1 siRNA (Ambion, Cat #AM4624) were reverse transfected into HeLa cells in triplicate at 5 concentrations using 0.3 µL siPORT™ NeoFX™ Transfection Agent (Ambion, Cat #AM4510). 48 hr post transfection, RNA was isolated using the MagMAX™-96 Total RNA Isolation Kit (Ambion, Cat #AM1830). cDNA was synthesized using MMLV Reverse Transcriptase (Ambion, Cat #AM2043) and 2 µL cDNA was used to amplify survivin mRNA in real time RT-PCR reactions with Survivin Hs00977611_g1 TaqMan® Gene Expression Assay (Applied Biosystems). Percent gene expression remaining is expressed as the relative amount of survivin mRNA in cultures transfected with survivin siRNAs vs cells transfected with the nontargeting control siRNA. TaqMan Gene Expression Assays against 18S rRNA were used to normalize for differences in total RNA concentration.

Correlating Silencing with Biological Effect
The phenotypic changes that follow transfection of cells with targeting siRNAs are the result of multistep processes that occur over time. siRNA reduction of mRNA levels causes a subsequent decrease in targeted protein. Protein concentration eventually reaches a threshold amount sufficient to trigger a phenotype change. It is therefore important to establish the time course of target mRNA and subsequent protein reduction, and correlate this period with observed phenotypic effects.

Silencing Time Course Methods

Experimental Design. To tie biological effects to silencing, it is critical to perform these assays during the time frame in which silencing is most profound. We defined the period of survivin mRNA and protein silencing by delivering survivin and nontargeting siRNAs to cells and measuring mRNA and protein levels at 24, 48, 72, 96, and 120 hour post transfection time points. mRNA levels were monitored using qRT-PCR and the same Applied Biosystems TaqMan Gene Expression Assay as was used for verifying siRNA induced silencing in Figure 1 (Figure 2). For survivin protein analysis, cells were harvested and lysed, protein was separated using SDS-PAGE and then transferred to PVDF membrane, and Western blot analysis was performed using the Applied Biosystems Western-Light™ Immunodetection System (Figure 3). The Western-Light System provides a sensitive, chemiluminescent based protein detection method.

Results. Transfection of survivin siRNA into HeLa cells reduced survivin mRNA greater than 80% compared to nontargeting negative control transfected cells. However, the patterns of silencing were not identical. Survivin siRNA #2646 knocked down survivin mRNA over 80% consistently over the time course of this study, while survivin siRNA #121294 was efficient at knocking down survivin mRNA during the first 48 hours, with survivin mRNA levels rising to 50% expression by 96 hours post transfection (Figure 2). The survivin protein expression pattern paralleled that of survivin mRNA expression (Figure 3), suggesting that survivin mRNA and protein expression are characteristic of knockdown dictated by the siRNA.

Figure 2. Time Course of Survivin mRNA Reduction in HeLa cells Transfected with Survivin siRNAs. Two Silencer® siRNAs [siRNAs #2646 (A) and #121294 (B)] were transfected in triplicate (30 nM siRNA) into HeLa cells using siPORT™ NeoFX™ Transfection Agent (0.3 µL; Ambion) in 96 well plates (4000 cells/well). Cells were lysed at the denoted times (X axis) and total RNA was subsequently isolated from each sample using the MagMAX™-96 Total RNA Isolation Kit (Ambion, Cat #AM1830). Survivin mRNA levels were measured using Survivin Hs00977611_g1TaqMan® Gene Expression Assay. Percent remaining gene expression was expressed as the relative amount of survivin mRNA in cultures transfected with survivin siRNA versus cells transfected with Silencer Negative Control #1 siRNA (Ambion, Cat #AM4635). A TaqMan Gene Expression Assay against the 18S rRNA subunit was used as a control for total RNA loading.

Figure 3. Time Course of Survivin Protein Reduction in HeLa Cells Transfected with Survivin siRNAs. Two unique Silencer® siRNAs [siRNAs #2646 (A) and #121294 (B)] were transfected in triplicate (30 nM siRNA) into HeLa cells using siPORT™ NeoFX™ Transfection Agent (5 µL; Ambion Cat #AM4510) in 6 well plates (2.5x105 cells/well). Cell were harvested and lysed at the denoted times. Protein lysates (40 µg protein) were separated on SDS-PAGE followed by transfer to membrane. Survivin protein was detected by immunoblot using a rabbit polyclonal antibody to survivin (1:2000 dilution; Abcam Cat #AB469) and the Western-Light™ Immunodetection System (Applied Biosystems Cat #T1047). GAPDH was detected for use as a loading control (Ambion Cat #AM4300). NT=Nontransfected; NC=Silencer Negative Control siRNA #1 (Ambion Cat #AM4611).

Methods for Measuring Apoptosis

Experimental Design. A series of biochemical and morphological assays were performed to measure the effect of siRNA-mediated silencing of survivin on several apoptotic indicators over the time course described above. Cells transfected with survivin siRNAs #2646 and #121294 were harvested at various time points after transfection, and assessed for the following:

• Cell Survival. Apoptosis ultimately leads to cell death. Relative cell survival was measured using fluorescein diacetate (FDA), a fluorogenic nonspecific esterase substrate.
  
• Nuclear Condensation. Chromatin condensation is a late apoptosis indicator and was monitored by fluorescence microscopy after DAPI staining of the cells.
  
• Increased Phosphatidyl Serine Externalization. Induction of membrane asymmetry, as evidenced by phosphatidyl serine externalization, is an early apoptosis indicator and was measured by labeling with fluorescent annexin V and then assaying with an 8200 Cellular Detection System (Applied Biosystems Cat #4342920).
  
• Pro-caspase-3 Activation. Caspase-3 activation is an early to mid-stage apoptosis indicator and was assayed using a fluorogenic caspase-3 substrate.

Cell Survival Results. Survivin supports cell proliferation and, thus, silencing of survivin might be expected to result in a decrease in cell number. As compared to non-transfected cells, the two survivin siRNAs did not lead to significantly decreased cell numbers at any of the time points monitored (Figure 4).

Figure 4. Effect of Survivin Silencing by siRNA on Cell Number.Silencer® siRNAs #2646 and #121294 were transfected in triplicate (30 nM siRNA) into HeLa cells (4000 cells/well in 96 well plates) using siPORT™ NeoFX™ Transfection Agent (0.3 µL; Ambion Cat #AM4510). At various time points post transfection, cells were harvested and lysed in 125 µL cold cell lysis buffer (50 mM Na-HEPES pH 7.4, 40 mM NaCl , 0.5% NP-40, 0.5 mM EDTA) for 20 min at 4°C. Cell extract (8 µL) was added to 384 well plate wells containing 32 µL substrate solution (0.01 mg/ml fluorescein diacetate in 40 mM TrisCl pH 7.5, 20 mM NaCl, 5% acetonitrile). Relative cell number/well was determined by measuring the increase in fluorescence (ex=488 nm, em=529 nm) over 4 min at room temperature.

Nuclear Morphology Results. Specific silencing of survivin has been shown to cause apoptotic events, one of which is characteristic changes in nuclear morphology due to nuclear condensation. Nuclear morphology was therefore observed 48−72 hours post transfection by staining cells with DAPI. Nuclear changes were observed in HeLa cells transfected with each survivin specific siRNA. At 48 hours, many survivin siRNA transfected cells exhibited nuclear condensation, while Silencer Negative Control #1 siRNA transfected cells did not (Figure 5).

Figure 5. Survivin Silencing Causes Changes in Nuclear Morphology. HeLa cells transfected with (A) the survivin targeting siRNAs (30 nM) and (B) Silencer® Negative Control #1 siRNA (Ambion, Cat #AM4635), were fixed 48 hr post transfection and stained with DAPI. Nuclear morphology was assessed using an Olympus BX60 fluorescent microscope.

Annexin V Assay Results. One of the earliest hallmarks of apoptosis is phosphatidyl serine externalization. To see whether survivin silencing would cause an increase in phosphatidyl serine externalization, cell labeling with fluorescent Annexin V was measured. Annexin V is a phospholipid binding protein with very high affinity to phosphatidyl serine, labeled Annexin V is routinely used in apoptosis detection assays. HeLa cells were transfected with either the survivin targeting siRNAs or a nontargeting negative control siRNA. 72 hours after transfection, fluorescent Annexin V labeling was assayed in homogeneous format using the 8200 Cellular Detection System (Applied Biosystems, Cat #4342920). The 8200 instrument enables mix-and-read assays with live cells and beads. As with cell number, the observed magnitude of the effect varied somewhat between the two siRNAs (Figure 6).

Figure 6. Phosphatidyl Serine Externalization Caused by Silencing of Survivin. (A) HeLa cells were transfected at 4000 cells/well with 30 nM each of Silencer® Survivin-targeting siRNAs #2646, #121294, and a nontargeting negative control siRNA (Negative Control #1 siRNA; Ambion, Cat #AM4635) in 6 replicates using siPORT™ NeoFX™ Transfection Agent (0.3 µL; Ambion Cat #AM4510). Nontransfected cells (NT) were also used as a negative control. Cells were harvested at five post transfection time points (24, 48, 72, 96, and 120 hr; 72 hr time point shown) and assayed for phosphatidyl serine externalization using fluorescent labeled Annexin V (blue) and CentriRed® DNA binding dye (pink) to enumerate cells. Cells were scanned on the Applied Biosystems 8200 Cellular Detection System and percent Annexin V positive cells was reported for each well using the 8200 System software. (B) Images of cells transfected with nontargeting negative control siRNA and Silencer siRNA #2646.

Caspase-3 Activation Results. A series of caspases are typically activated in the early stages of apoptosis. These proteases cleave key structural and nuclear proteins, which leads to chromosomal cleavage and nuclear condensation. Caspase-3 is generally the last caspase activated in the caspase cascade. We monitored the effect of survivin knockdown on activated caspase-3 levels. siRNA-mediated survivin silencing caused little or no effect in the pro-caspase-3 activation assay (Figure 7). Our observation agrees with published reports stating that either inhibition or silencing of survivin expression results in the activation of apoptosis events but does not induce caspase activation [5, 6].

Figure 7. Caspase Activation as a Measure of Apoptosis Induction. Cells were transfected with siRNAs as in (A). At various time points after transfection, cells were harvested and lysed in 125 µL cold cell lysis buffer (50 mM HEPES pH 7.4, 40 mM NaCl , 0.5% NP-40, 0.5 mM EDTA) for 20 min at 4°C. After mixing, 40 µL of cell extract was added to 96 well plate wells containing 120 µL substrate solution [10 µM acDEVDafc substrate in caspase buffer (20 mM Na-HEPES, pH 7.5, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 5 mM DTT)]. Caspase-3 activity was determined by measuring the increase in fluorescence (ex=400 nm, em=505 nm) over 2 hr at 30°C, and is normalized to cell number; y axis=acDEVDafc hydrolysis/cell number.

Discussion
In this study, survivin siRNAs were used to address whether survivin plays a role in apoptosis by assessing several biological processes previously associated with the apoptotic pathway. The time course of siRNA silencing of survivin mRNA and protein correlated well with decreased cell survival/proliferation; changes in nuclear membrane shape and chromosomal condensation and fragmentation; and phosphatidyl serine externalization; which are all indicative of apoptosis. This correlation suggests that specific silencing of survivin induces these apoptotic events. The lack of pro-caspase-3 activation by siRNA-mediated survivin silencing (Figure 7) agrees with published reports stating that either inhibition or silencing of survivin results in the activation of apoptosis events but does not cause caspase activation [10, 11]. The lack of this activation suggests that caspase-3 activity is not essential for apoptosis in HeLa cells. This conclusion is in accord with an emerging body of evidence suggesting that mammalian cells contain both caspase-dependent and caspase-independent apoptotic pathways [12].

For more information about the products used to study gene function through RNAi, see the companion article, Ensure RNAi Success. This article details the family of tools Applied Biosystems and Ambion supply for gene silencing experiments.

Scientific Contributors:
Lesslie Beauchamp, Diana M. Batten, Susan Magdaleno, Joe Krebs, Angie Cheng, Lance Ford • Ambion
Melissa Gee, Carol Khodier • Applied Biosystems, Bedford, MA