Multitarget therapy of malignant cancers by the head-to-tail tandem array multiple shRNAs expression system

Abstract

Coexpression of multiple shRNAs can simultaneously inhibit multiple genes or target multiple sites on a single gene. These approaches can be used for dissecting complex signaling pathways and even be applied to targeting multiple genes in cancer therapy. Here we established a simple and efficient multiple shRNAs expression system based on pSUPER, the most popular expression vector in mammalian cells. A series of head-to-tail tandem array multiple shRNAs expression vectors were constructed containing different combinations of six shRNA expression cassettes targeting genes involved in cell proliferation and survival pathways: Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB. In HeLa and HEK293 cells, the multiple shRNAs expression constructs could efficiently and simultaneously induce inhibition of all six genes. We further evaluated the inhibition effects of the multiple shRNAs expression vectors on the human prostate cancer cell line PC3, which contains different cell variants with distinct oncogenic signaling alterations. The results revealed that the multiple shRNAs expression system could inhibit all six genes and was much more efficient in inducing apoptosis in the PC3 cells. Our results suggest that the multitarget shRNAs expression system could be an effective strategy in cancer therapy and be applied to any other DNA vector-based shRNA expression system.

Introduction

RNAi-mediated gene silencing can be efficiently induced by siRNAs or shRNAs that are either chemically synthesized and exogenously introduced into cells or endogenously expressed from transfected vectors or infected viruses.1 Co-delivery or coexpression of multiple siRNAs or shRNAs can result in either inhibiting multiple genes simultaneously or targeting multiple sequences on a single gene at the same time. These approaches provide an extremely powerful tool for dissecting complex gene networks and treating complicated gene disorders.2 However, the efficacy of simultaneously inhibiting multiple genes and the capability of targeting multiple sequences on a single gene at the same time are mainly dependent on the co-delivery or co-transfection efficiency of various siRNA and shRNA molecules or expression vectors. Alternatively, using a delivery carrier that conjugates multiple siRNAs or shRNAs, or an expression vector that harbors multiple siRNA or shRNA expression cassettes can simultaneously introduce or transcribe multiple siRNAs or shRNAs in cells. In particular, construction of the multiple siRNAs or shRNAs expression vectors provides a simple and efficient strategy for inhibiting multiple genes or targeting multiple sequences simultaneously. Compared with siRNAs, shRNAs prove to be better effectors in the induction of RNAi-mediated gene silencing; in addition, shRNAs are easy to use and cost effective whether synthesized chemically or expressed endogenously.3, 4 Thus, a simple and efficient strategy for constructing multiple shRNAs expression systems derived from existing shRNA expression vectors would not only directly extend the utility of the vectors but also greatly increase the value of RNAi-based gene silencing techniques.

The concept of inhibiting cell proliferative and survival pathways as a means of RNAi-based tumor therapy has been well recognized and broadly investigated. Using the sequence-specific siRNA or shRNA molecule to inhibit a single therapeutic target may reduce tumor progression, but almost all cancer cells are likely not dependent on a single oncogenic signaling pathway.5 Moreover, many malignant tumors show striking genetic heterogeneities and phenotypic variations, suggesting that a single target therapy is not likely to eradicate cancer cells.6 To circumvent these serious problems in cancer therapy, an alternative strategy is to target multiple components simultaneously, which are essential for promoting and maintaining the malignant cells, by using multiple gene-specific siRNAs or shRNAs.7, 8, 9, 10, 11, 12, 13

To exploit the RNAi-mediated gene silencing technology, particularly in anticancer therapies, it is extremely useful to establish a cloning strategy not only for efficiently constructing the multiple shRNAs expression vectors but also for precisely mapping the cloned constructs. In this study, we have developed a novel and efficient cloning strategy for constructing a head-to-tail tandem array multiple shRNAs expression system, as well as designed a simple and reliable procedure for mapping the cloned constructs. To demonstrate the simplicity and efficiency of this system, we first constructed six single shRNA expression cassettes against the Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB genes,14, 15, 16, 17, 18, 19 and subsequently constructed a series of head-to-tail tandem array multiple shRNAs expression vectors that contained different combinations of the six single shRNA expression cassettes. To assess the inhibition effects of the multiple shRNAs expression constructs, we first examined the inhibition efficiencies induced by the six single shRNA expression cassettes and then the combined effects of the multiple shRNAs expression constructs in both HeLa and HEK293 cells. In addition, to evaluate the potential effects of multitarget inhibitions on cancer therapy, we further examined the inhibition effects induced by the multiple shRNAs expression constructs in human prostate cancer cell line PC3 that contains different cell variants with distinct oncogenic signaling alterations.

Materials and methods

Cell culture

The human cervical-carcinoma-derived cell line HeLa, the embryonic kidney epithelial cell line HEK293 and the prostatic-carcinoma-derived cell line PC3 were cultured and maintained in Dulbecco's modified Eagle's medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% antibiotic/antimycotic solution (Gibco BRL) at 37 °C in a humidified incubator with 5% CO2. These three cell lines were subcultured according to their growth rates generally 1–2 times a week after treatment with 0.1% trypsin (Biowhittaker Acambrex, Walkersville, MD).

Transfection

At 24 h before gene transfection, cells were seeded in six-well culture plates at 1 × 105 cells per well. The cells were transfected with 2 μg of the single shRNA or the multiple shRNAs expression construct by Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. After incubation for 48 h, the transfected cells were directly solubilized on six-well culture plates with radioimmunoprecipitation assay lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride) supplemented with protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany), and aliquots of the cell lysates containing equal amounts of protein were examined by western blot analyses with protein-specific antibodies. Total protein in the cell lysates was quantified by the BCA assay (Pierce, Rockford, IL) as recommended by the manufacturer.

Construction of the pSUPER/EGFP expression vector

Plasmid DNA was constructed by using standard molecular cloning techniques. The shRNA expression vector pSUPER/EGFP (Figure 1a) was generated and selected by inserting the enhanced green fluorescent protein (EGFP) expression cassette from blunt-ended (HindIII+EcoRI)-pCMV-EGFP into the blunt-ended (BamHI+NotI)-pSUPER vector (kindly provided by Dr R Agami, The Netherlands Cancer Institute, Amsterdam, the Netherlands). The pCMV-EGFP was constructed by inserting the EGFP DNA fragment from NotI+blunt-ended BamHI-pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA) into the NotI+blunt-ended XhoI-pCMVβ vector (BD Biosciences Clontech). The pSUPER/EGFP vector particularly contained three multiple cloning sites (MCSs), MCS1, MCS2 and MCS3, as indicated in Figure 1a. The restriction enzymes BglII and HindIII were used for cloning the shRNA coding sequences and HindIII and KpnI sites in the MCS3 were used for constructing another shRNA expression cassette in a head-to-tail tandem array manner as described in Figure 1c.

Figure 1
figure1

Design and strategy for constructing a head-to-tail tandem array multiple shRNAs expression system. (a, b) Constructs of the shRNA expression vectors. Both the pSUPER/EGFP (a) and pshXX (b) vectors contained the human H1 promoter (HsH1)-driven with or without shRNA (shXX) coding sequence (red bar) as well as the CMV promoter (PCMV)-driven and SV40 polyA signal (pA)-terminated enhanced green fluorescent protein (EGFP) expression cassette that serves as a reference protein expression system for the vectors. (c) Procedure for constructing the multiple shRNAs expression vectors. For preparation of vector or insert, the vector pshXX(v) or insert pshXX(i) plasmid is first digested with HindIII or EcoRI, then is blunt-ended by end-filling and finally digested with KpnI to produce a vector or insert DNA fragment with one blunt-ended and one sticky-ended termini. The KpnI+blunt-ended EcoRI-shXX(i) DNA fragment is cloned into the KpnI+blunt-ended HindIII-pshXX(v) vector. The resulting construct, designated as pshXX(v)/shXX(i), can serve as a new cloning vector for another construction.

Construction of the shRNA and head-to-tail tandem array multiple shRNAs expression vectors

The experimental procedure for cloning a single shRNA expression cassette involved ligating an annealed oligonucleotide duplex into the BglII and HindIII restriction sites of the pSUPER/EGFP vector. Oligonucleotides formed a short synthetic DNA fragments targeting the Bcl-2, Survivin, Akt1, Erk2, CyclinE, NFκB, Drosha, DGCR8 or Exportin-5 gene were purchased from commercial suppliers. Sequences of the synthetic DNA oligonucleotides used in this study are listed in Table 1. The simple and efficient strategy for constructing the head-to-tail tandem array multiple shRNAs expression vectors is shown in Figure 1c. The detailed procedure is described in the Results section.

Table 1 Sequences of the shRNAs for cell survival and miRNA processing genes

Mapping of the shRNA expression cassettes in constructed multiple shRNAs expression vectors

The number of shRNA expression cassettes cloned in the multiple shRNAs expression vectors could be precisely analyzed by simply using restriction enzyme mapping. The cloned constructs were first digested with the unique restriction enzymes, EcoRI and HindIII, and then analyzed by electrophoresis in a 0.8% agarose gel with an appropriate molecular weight marker. The digested products should contain two DNA fragments, the pBluescript II KS/EGFP vector and the shRNA expression cassette(s) insert. Because the length of a single shRNA expression cassette including human the H1 promoter and the shRNA coding sequence is about 291 bp, the size of the inserted shRNA expression cassette(s) is a multiple of 291 bp.

Western blot analysis of EGFP, Bcl-2, Survivin, Akt1, Erk2, CyclinE, NFκB p65, PARP, caspase-3, Drosha, DGCR8, Exportin-5 and β-actin

Equal amounts of total protein extracts were analyzed by electrophoresis in a 12% SDS–polyacrylamide gel and transferred onto an Immobilon-P membrane (Millipore, Billerica, MA) in a Semi-phor TE-70 unit (Hoefer Scientific, San Francisco, CA) by semidry electrophoretic transfer, and then incubated with mouse anti-GFP (B-2), anti-Survivin (D-8) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-CyclinE (clone HE12; Lab Vision Corp., Fremont, CA), anti-Exportin-5 (ab57491; Abcam, Cambridge, UK), anti-β-actin (Sigma Chemical, St Louis, MO) monoclonal antibodies, or mouse anti-Bcl-2, anti-Erk1 (which cross-reacts with Erk2 protein) polyclonal antibodies (Santa Cruz Biotechnology), or rabbit anti-Akt1/2/3 (H-136), anti-PARP (H-250) (Santa Cruz Biotechnology), anti-NFκB p65, anti-caspase-3 (Cell Signaling Technology, Beverly, MA), anti-Drosha (ab12286; Abcam) polyclonal antibodies, or goat anti-DGCR8 (N-19) polyclonal antibody (Santa Cruz Biotechnology) followed by incubation with horseradish-peroxidase-conjugated goat anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA), anti-rabbit, or donkey anti-goat (Santa Cruz Biotechnology) immunoglobulin G secondary antibodies. Bands were detected by using the enhanced chemiluminescence system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instruction.

Methyl thiazolyl tetrazolium cell growth assay

At 24 h before gene transfection, cells were seeded onto 96-well plates at a density of 1 × 104 cells per well in 200 μl complete medium. The cells were transfected with 0.25 μg of the single shRNA or multiple shRNAs expression construct by Lipofectamine 2000 according to the manufacturer's instruction. After incubation for 48 h or various time periods, the transfected cells were analyzed by the methyl thiazolyl tetrazolium (MTT) assay according to standard procedures. At 6 h before each incubation time point except day 0, the cells were treated with 10 μl of MTT solution (5 mg ml−1; Sigma Chemical) for 6 h, then dissolved in 200 μl of dimethyl sulfoxide (Sigma Chemical) and measured at 590 nm using an ELISA reader (Molecular Dynamics, Sunnyvale, CA).

Fluorescence-activated cell sorting (FACS) analysis

For detection of apoptotic cells characterized by DNA fragmentation, the transfected cells were fixed with 70% ethanol in phosphate-buffered saline for propidium iodide (Sigma Chemical) staining. The number of apoptotic cells was measured as the cell population at the sub-G1 phase by flow cytometry. The flow cytometry analyses were carried out on a FACSCalibur flow cytometer (Beckton Dickinson Biosciences, San Jose, CA).

Clonogenic survival assay

At 24 h after transfection, the transfected cells were harvested and re-seeded onto six-well culture plates at a density of 2000 cells per well in 2 ml complete medium. The medium was changed after 4 days. After incubation for 12 days, the colonies were directly stained with 0.5% crystal violet and photographed by using Nikon D80 digital camera. The number of colonies that contained more than 200 cells was counted to determine the survival rate.

Results

Strategy and experimental design for constructing the head-to-tail tandem array multiple shRNAs expression system

Figure 1 shows the design and strategy for constructing the head-to-tail tandem array multiple shRNAs expression system. On the basis of the most frequently used shRNA expression vector, pSUPER,20 we designed and established a simple and efficient procedure for constructing the head-to-tail tandem array multiple shRNAs expression vectors. To expand the utility of the pSUPER vector and fulfill the requirement of additional cloning capacity, we first constructed an shRNA expression vector, pSUPER/EGFP, containing the EGFP expression cassette and possessing three MCSs, MCS1, MCS2 and MCS3 (Figure 1a). On the basis of the molecular structure and the unique cloning sites located in MCS3, we designed a simple and efficient strategy for constructing the head-to-tail tandem array multiple shRNAs expression vectors and in particular also designed an easy and reliable protocol for mapping the cloned vectors.

The strategy for constructing the head-to-tail tandem array multiple shRNAs expression vectors is shown in Figure 1c. First, to prepare the cloning vector, the shRNA expression plasmid pshXX(v) was digested with HindIII, then an end fill-in was performed to generate a blunt-ended DNA fragment, and finally the plasmid was further digested with KpnI. Second, to prepare the inserting cassette, the shRNA expression cassette was isolated from the inserting plasmid pshXX(i) by digestion with EcoRI, then end fill-in was performed to produce a blunt-ended DNA fragment, and finally the cassette was digested with KpnI. The isolated DNA fragment containing the shRNA expression cassette was ligated into the purified cloning vector. The resulting plasmid was simply designated as pshXX(v)/shXX(i). The resulting construct pshXX(v)/shXX(i) could serve as a new cloning vector for a second construction cycle of inserting another shRNA expression cassette.

Construction and mapping of the single shRNA and head-to-tail tandem array multiple shRNAs expression vectors

Following the procedure described in Figure 1c, we constructed a series of single shRNA (Figure 1b) and head-to-tail tandem array multiple shRNAs expression vectors, each of which contained different combinations of the six shRNA expression cassettes against the Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB genes (Figures 2a and b). These six shRNA-targeting sequences were selected and chosen based on previous studies.14, 15, 16, 17, 18, 19

Figure 2
figure2

Construction and mapping of the head-to-tail tandem array multiple shRNAs expression vectors. (a, b) Serial constructs of the multiple shRNAs expression vectors containing different combinations of shBcl-2, shSurvivin, shAkt1, shErk2, shCyclinE and shNFκB expression cassettes. (c, d) Restriction enzyme mapping of the multiple shRNAs expression constructs. The multiple shRNAs expression vectors as indicated in (a) and (b) were digested with EcoRI and HindIII, analyzed on a 0.8% agarose gel by electrophoresis and then photographed using an agarose gel imaging and analysis system.

To map the cloned constructs and to verify that they contained the correct inserts, the purified plasmid DNAs were digested with EcoRI and HindIII at two unique restriction sites and then analyzed by agarose gel electrophoresis. The analyses indicated that all the multiple shRNAs expression vectors possessed vector sizes identical to the original vector pSUPER/EGFP and contained the correct inserts according to the expected fragment sizes (Figures 2c and d).

Evaluation of the single shRNA and multiple shRNAs expression vectors against Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB genes

To evaluate the simplicity and efficiency of this procedure, we first tested the knockdown efficiency of the six single shRNA expression vectors, pshBcl-2, pshSurvivin, pshAkt1, pshErk2, pshCyclinE and pshNFκB, against the Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB genes, respectively, in HeLa and HEK293 cells. The results revealed that all six single shRNA expression constructs could specifically induce strong inhibition of their corresponding target gene expressions as compared with the vector pSUPER/EGFP in both the HeLa and HEK293 cells (Figures 3a and b). All six single shRNA-transfected HeLa and HEK293 cells, which were marked by the EGFP, displayed normal cell morphology and growth as compared with the mock and vector pSUPER/EGFP-transfected cells (Figures 3c and d).

Figure 3
figure3

Effects of the single shRNA-induced inhibition of Bcl-2, Survivin, Akt1, Erk2, CyclinE or NFκB expression in HeLa and HEK293 cells. (a, b) Western blot analysis of the single shRNA-transfected cells. HeLa (a) and HEK293 (b) cells were transfected with the single shRNA expression vectors as indicated. At 48 h after transfection, the expression levels of each corresponding target gene in the total protein extracts were determined by western blot analysis. The levels of enhanced green fluorescent protein (EGFP) and β-actin serve as reference proteins for transfection efficiency and loading control, respectively. (c) Morphological analysis of the single shRNA-transfected cells. HeLa and HEK293 cells were transfected with the single shRNA expression vectors as indicated. At 48 h after transfection, the transfected cells were photographed using an inverted fluorescence microscope. The cells transfected with the single shRNA expression vectors were marked by the EGFP. (d) Methyl thiazolyl tetrazolium (MTT) cell growth assay of the single shRNA-transfected cells at different incubation times. HeLa and HEK293 cells were transfected with the shRNA expression vectors as indicated. At 6 h before each incubation time point except the day 0 as indicated, the cells were treated with MTT for 6 h and then MTT cell growth assay was performed. The plotted data were averaged from three independent experiments and the bars represent mean±s.d.

To evaluate the inhibition effects of the multiple shRNAs expression vectors, HeLa and HEK293 cells were transfected with these multiple shRNAs expression constructs containing different combinations of shRNA expression cassettes. The results showed that all the multiple shRNAs expression constructs could specifically induce strong inhibition of their corresponding target gene expressions as compared with the vector pSUPER/EGFP in both the HeLa and HEK293 cells (Figure 4). All the multiple shRNAs expression constructs with different combinations of cassettes transfected HeLa and HEK293 cells, which were labeled by the EGFP, displayed normal cell morphology and growth as compared with the mock and vector pSUPER/EGFP-transfected cells (Figure 5).

Figure 4
figure4

Western blot analyses of the multiple shRNAs-induced simultaneous inhibitions of Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB expressions in HeLa and HEK293 cells. HeLa (a, c) and HEK293 (b, d) cells were transfected with the multiple shRNAs expression vectors with different combinations as indicated. At 48 h after transfection, the expression levels of each corresponding target gene in the total protein extracts were determined by western blot analysis. The levels of enhanced green fluorescent protein (EGFP) and β-actin serve as reference proteins for transfection efficiency and loading control, respectively.

Figure 5
figure5

Effects of the multiple shRNAs-induced simultaneous inhibitions of Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB expressions in HeLa and HEK293 cells. (a, b) Morphological analysis of the multiple shRNAs-transfected cells. HeLa and HEK293 cells were transfected with the multiple shRNAs expression vectors with different combinations as indicated. At 48 h after transfection, the transfected cells were photographed using an inverted fluorescence microscope. The cells transfected with the multiple shRNAs expression vectors were labeled by enhanced green fluorescent protein (EGFP). (c) Methyl thiazolyl tetrazolium (MTT) cell growth assay of the multiple shRNAs-transfected cells at different incubation times. HeLa and HEK293 cells were transfected with the multiple shRNAs expression vectors as indicated. At 6 h before each incubation time point except the day 0 as indicated, the cells were treated with MTT for 6 h and then MTT cell growth assay was carried out. The plotted data were averaged from three independent experiments and the bars represent mean±s.d.

Assessment of the inhibition effects of the single shRNA and multiple shRNAs expression vectors on human prostatic carcinoma cell line PC3

Because prostate cancer is an important cancer that exhibits biological heterogeneity, we used the PC3 prostate cancer cell line, which contains multiple sublines that have various signaling alterations,21 as a demonstration of the effectiveness of our technique. To assess the inhibition effect of single shRNA, PC3 cells were transfected with the six single shRNA expression constructs. The results indicated that all six single shRNA expression constructs could specifically induce strong inhibition of their corresponding target gene expressions as compared with the vector pSUPER/EGFP (Figure 6a). In contrast to the six single shRNA-transfected HeLa and HEK293 cells, a large number of the six single shRNA-transfected PC3 cells, which were marked by EGFP, displayed small round morphologies as compared with the mock and vector pSUPER/EGFP-transfected cells (Figure 7a). To evaluate the effect of these single shRNA-induced morphological changes, the transfected cells were further analyzed by MTT cell growth assay. The results revealed that all of the six single shRNA-transfected cells displayed a small or no inhibition effect on cell growth as compared with the mock and vector pSUPER/EGFP-transfected cells (Figure 7b).

Figure 6
figure6

Western blot analyses of the single shRNA- and multiple shRNAs-induced inhibitions of Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB expressions in PC3 cells. PC3 cells were transfected with the single shRNA (a) or multiple shRNAs (b) expression vectors as indicated. At 48 h after transfection, the expression levels of each corresponding target gene in the total protein extracts were determined by western blot analysis. The levels of enhanced green fluorescent protein (EGFP) and β-actin serve as reference proteins for transfection efficiency and loading control, respectively.

Figure 7
figure7

Effects of the single shRNA-induced inhibition of Bcl-2, Survivin, Akt1, Erk2, CyclinE or NFκB expression in PC3 cells. (a) Morphological analysis of the single shRNA-transfected cells. PC3 cells were transfected with the single shRNA expression vectors as indicated. At 48 h after transfection, the transfected cells were photographed using an inverted fluorescence microscope. The cells transfected with the single shRNA expression vectors were marked by enhanced green fluorescent protein (EGFP). (b) Methyl thiazolyl tetrazolium (MTT) cell growth assay of the single shRNA-transfected cells. At 42 h after transfection, the transfected cells were treated with MTT for 6 h and then MTT cell growth assay was performed. The plotted data were averaged from three independent experiments and the bars represent mean±s.d. (c) Western blot analysis of the poly(ADP-ribose) polymerase (PARP) and caspase-3 expressions. At 48 h after transfection, the expression patterns of both the PARP and caspase-3 in the total protein extracts were determined by western blot analysis. The levels of β-actin serve as a loading control.

To assess the inhibition effect of the multiple shRNAs on malignant prostate cancer, PC3 cells were transfected with the multiple shRNAs expression constructs. The results revealed that all the multiple shRNAs expression constructs could specifically trigger strong inhibition of their corresponding target gene expressions as compared with the vector pSUPER/EGFP (Figure 6b). In particular, a large number of the multiple shRNAs-transfected PC3 cells, which were labeled by the EGFP, displayed small round morphologies as compared with the mock and vector pSUPER/EGFP-transfected cells (Figure 8a). In addition, almost all of the multiple shRNAs-transfected cells, even those containing only two shRNAs, shBcl-2 and shSurvivin, exhibited significant reduction of cell numbers as compared with the mock, vector pSUPER/EGFP- or pshBcl-2-transfected cells. To further examine whether the reduction of cell numbers in multiple shRNAs-transfected cells caused by apoptosis, the transfected cells were fixed and stained with propidium iodide, as well as analyzed by flow cytometry. The results revealed that the number of cells at the sub-G1 phase was increased dramatically in the multiple shRNAs-transfected cells as compared with the mock, vector pSUPER/EGFP- or pshBcl2-transfected cells (Figure 8b). To evaluate directly the effect of the multiple shRNAs-induced morphological changes, the transfected cells were analyzed by MTT cell growth assay. The results showed that all the multiple shRNAs-transfected cells exhibited a very high inhibition effect on cell growth as compared with the mock, vector pSUPER/EGFP- and pshBcl-2-transfected cells (Figure 8c). Notably, the level of cell growth inhibition in transfected cells significantly increased as the number of shRNA expression cassettes increased in the multiple shRNAs expression constructs.

Figure 8
figure8

Effects of the multiple shRNAs-induced simultaneous inhibitions of Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB expressions in PC3 cells. (a) Morphological analysis of the multiple shRNAs-transfected cells. PC3 cells were transfected with the multiple shRNAs expression vectors as indicated. At 48 h after transfection, the transfected cells were photographed using an inverted fluorescence microscope. The cells transfected with the multiple shRNAs expression vectors were labeled by enhanced green fluorescent protein (EGFP). (b) Fluorescence-activated cell sorting (FACS) analysis of the multiple shRNAs-transfected cells. At 48 h after transfection, the transfected cells were harvested and fixed with 70% ethanol, as well as stained with propidium iodide and analyzed by flow cytometry. The number of apoptotic cells was measured as the cell population at the sub-G1 phase. (c) Methyl thiazolyl tetrazolium (MTT) cell growth assay of the multiple shRNAs-transfected cells. At 42 h after transfection, the transfected cells were treated with MTT for 6 h and then MTT cell growth assay was carried out. The plotted data were averaged from three independent experiments and the bars represent mean±s.d. (d) Western blot analysis of the poly(ADP-ribose) polymerase (PARP) and caspase-3 expressions. At 48 h after transfection, the expression patterns of both the PARP and caspase-3 in the total protein extracts were determined by western blot analysis. The levels of β-actin serve as a loading control.

To correlate directly the changes of cell morphology and number in the single shRNA- and multiple shRNAs-transfected cells with cell death by apoptosis, the cleavage patterns of death substrate poly(ADP-ribose) polymerase (PARP), which is cleaved into a specific 85 kDa form during apoptosis by caspase-3, as well as the activation levels of apoptosis effector caspase-3, which is activated proteolytically from a 32 kDa precursor into a 20 and a 10 kDa heterodimer, in the transfected cells were examined by western blot analysis.22, 23 The results revealed that the 85 kDa PARP cleavage form and the 20 kDa caspase-3 proteolysis product were clearly seen in the multiple shRNAs-transfected PC3 cells but absent or faintly observed in the single shRNA-transfected PC3 cells as well as the mock and vector pSUPER/EGFP-transfected cells (Figures 7c and 8c). In addition, the levels of both the 85 kDa PARP and 20 kDa caspase-3 products were significantly increased as the number of shRNA expression cassettes increased in multiple shRNAs expression constructs.

To assess the kinetic effects of the multiple shRNAs-induced cell growth inhibition in PC3 cells, the single shRNA- and multiple shRNAs-transfected cells were analyzed by MTT cell growth assay at different incubation times. The results showed that all the single shRNA-transfected cells displayed similar cell growth curves compared with the mock and vector pSUPER/EGFP-transfected cells (Figure 9a). In contrast, all the multiple shRNAs expression constructs indeed induced high levels of cell growth inhibition in transfected cells compared to the mock, vector pSUPER/EGFP- and pshBcl-2-transfected cells (Figure 9b). In addition, to further evaluate the long-term effects of inhibiting genes involved in cell proliferation and survival pathways in PC3 cells, the single shRNA- and multiple shRNAs-transfected cells were analyzed by clonogenic survival assay. The results revealed that all of the transfected cells including the single shRNA- and multiple shRNAs-transfected cells displayed a large inhibition on colony formation as compared with the mock and vector pSUPER/EGFP-transfected cells (Figures 9c and d).

Figure 9
figure9

Time-dependent growth effects of the single shRNA- and multiple shRNAs-induced inhibitions of Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB expressions in PC3 cells. (a, b) Methyl thiazolyl tetrazolium (MTT) cell growth assay of the single shRNA- and multiple shRNAs-transfected cells at different incubation times. PC3 cells were transfected with the single shRNA (a) or multiple shRNAs (b) expression vectors as indicated. At 6 h before each incubation time point except the day 0 as indicated, the cells were treated with MTT for 6 h and then MTT cell growth assay was performed. The plotted data were averaged from three independent experiments and the bars represent mean±s.d. (c, d) Clonogenic survival assay of the single shRNA- and multiple shRNAs-transfected cells. At 24 h after transfection, the transfected cells were re-plated in six-well culture plates at 2000 cells per well. After incubation for 12 days, the colonies were stained and photographed, as well as counted to determine the survival rate. The plotted data were averaged from three independent experiments and the bars represent mean±s.d.

Evaluation of the shRNA expression vectors against Drosha, DGCR8 and Exportin-5 genes on PC3 cells

To investigate whether the induction of apoptosis in multiple shRNAs-transfected cells is caused by impairment of miRNA processing in PC3 cells, we directly examined the effects of silencing Drosha, DGCR8 and Exportin-5 expressions. To knockdown effectively the expressions of Drosha, DGCR8 and Exportin-5, we designed and constructed three shRNA expression vectors, pshDrosha, pshDGCR8 and pshExportin-5, using the siRNA validation system as described previously.24 To test their inhibition effects, PC3 cells were transfected with the three shRNA expression constructs. The results revealed that all three shRNA expression constructs could specifically induce strong inhibition of their corresponding target gene expressions as compared with the vector pSUPER/EGFP (Figure 10b). In contrast to the previous multiple shRNAs-transfected cells, the three shRNA-transfected cells, which were marked by the EGFP, exhibited normal cell morphology and growth as compared with the mock and vector pSUPER/EGFP-transfected cells (Figures 10a and d). In addition, western blot analysis of the PARP and caspase-3 expressions showed that only the intact forms of both the PARP and caspase-3 expressions were observed in the mock, vector pSUPER/EGFP- and three shRNA-transfected cells (Figure 10c).

Figure 10
figure10

Effects of the shRNA-induced inhibition of Drosha, DGCR8 or Exportin-5 expression in PC3 cells. (a) Morphological analysis of the shRNA-transfected cells. PC3 cells were transfected with the shRNA expression vectors as indicated. At 48 h after transfection, the transfected cells were photographed using an inverted fluorescence microscope. The cells transfected with the shRNA expression vectors were marked by the enhanced green fluorescent protein (EGFP). (b) Western blot analysis of the Drosha, DGCR8 and Exportin-5 expressions. At 48 h after transfection, the expression levels of the Drosha, DGCR8 and Exportin-5 in the total protein extracts were determined by western blot analysis. The levels of β-actin serve as a loading control. (c) Western blot analysis of the poly(ADP-ribose) polymerase (PARP) and caspase-3 expressions. At 48 h after transfection, the expression patterns of both the PARP and caspase-3 in the total protein extracts were determined by western blot analysis. (d) Time-dependent growth effect of the shRNA-induced inhibition of Drosha, DGCR8 or Exportin-5 expression. PC3 cells were transfected with the shRNA expression vectors as indicated. At 6 h before each incubation time point except the day 0 as indicated, the cells were treated with methyl thiazolyl tetrazolium (MTT) for 6 h and then MTT cell growth assay was carried out. The plotted data were averaged from three independent experiments and the bars represent mean±s.d.

Discussion

In this study, we demonstrated that a head-to-tail tandem array multiple shRNAs expression system could be easily constructed to target several genes at the same time and demonstrated a much better induction of apoptosis in the human prostate cancer cell line PC3. To the best of our knowledge, this is the first report of a multitarget shRNA expression system applicable to prostate cancer therapy.

To evaluate the efficiency of this system, we first constructed six single shRNA expression cassettes against the Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB genes. These six genes were chosen because they are not only involved in either cell proliferative or survival signaling pathways but also have decisive functions in both mechanisms.14, 15, 16, 17, 18, 19 By using these six single shRNA expression cassettes, we subsequently constructed a series of head-to-tail tandem array multiple shRNAs expression vectors that contained different combinations from a single shRNA expression cassette to as many as all six shRNA expression cassettes (Figure 2). Using both the HeLa and HEK293 cells, we demonstrated that not only the six single shRNA expression constructs but also the multiple shRNAs expression constructs exhibited high inhibition activities on each of target gene expressions (Figures 3a, b and 4). We further showed that the multiple shRNAs expression constructs could efficiently induce simultaneous inhibition of their corresponding target genes in the malignant prostate cancer cell line PC3, resulting in different levels of cell apoptosis (Figure 8). In particular, the levels of apoptosis induced by the multiple shRNAs expression vectors were increased as the combined number of shRNA expression cassettes increased in multiple shRNAs expression constructs. These results clearly indicated that this head-to-tail tandem array multiple shRNAs expression system provides not only an efficient and simple procedure for constructing multiple shRNAs expression vectors but also an extremely powerful tool for establishing combinational therapies.

Although previous studies have established several cloning strategies for constructing multiple shRNAs expression vectors,7, 8, 9, 10, 11, 12, 13 the system described in this study is built on the most popular shRNA expression vector, pSUPER,20 and in particular also provides a simple and easy method for validating the number of shRNA expression cassettes in isolated clones. Thus, currently available validated or published pSUPER-based shRNA expression vectors can be directly used for constructing the multiple shRNAs expression constructs according to the protocol described in this report. In addition, to simply identify or precisely estimate the efficiencies of the multiple shRNAs expression constructs, the pSUPER vector was particularly reconstructed to contain the EGFP expression cassette that provides an easily detectable and sensitive reporter system. Because the number of the multiple shRNA expression cassettes can be correctly mapped by simply digesting with the unique restriction enzymes, EcoRI and HindIII, the multiple shRNAs expression system can be easily transferred from the pSUPER/EGFP plasmid to any other gene delivery vehicles, including viral systems. Although this system can be theoretically used to construct a vector containing an unlimited number of shRNA expression cassettes, it is important to note that when the RNA-induced silencing complex is overexhausted by a large number of siRNA molecules, severe side effects can result from impaired miRNA functions.25

Prostate cancer is the most common and the second leading cause cancer-related death among men in Western countries. It is characterized by clinical and biological heterogeneities that display complex karyotypic abnormalities and harbor many specific genetic alterations.6 Current antitumor modalities such as surgery, chemotherapy and radiotherapy have only limited success in treatment of this cancer and many new therapeutic strategies are under development, in particular, target-directed therapies for prostate cancer that focus molecules and pathways that contribute to the cell proliferation and survival have great potential. However, the therapeutic application of target molecules or pathway inhibitors to prostate cancer has been limited because no common dominant oncogenic mutation affecting a signal kinase activation has yet been identified. Thus, development of combinational treatments including chemo-, radiation or novel RNAi therapeutics is important for complete eradication of prostate cancer cells. PC3, a malignant androgen-independent prostate cancer cell line, contains different cell variants with distinct oncogenic signaling alterations that contribute to cell proliferation and survival.21 In our study, using a single shRNA expression cassette to inhibit genes essential for either cell proliferation or survival, such as Bcl-2, Survivin, Akt1, Erk2, CyclinE or NFκB, in PC3 cells resulted in visible morphological changes but no significant cell death (Figures 7 and 9a). However, transfection of the PC3 cells with the multiple shRNAs expression constructs harboring different combinations of the six important genes caused not only visible morphological changes but also severe cell death by apoptosis (Figures 8 and 9b). These results clearly indicated that the multiple shRNAs expression constructs could effectively induce apoptosis in the highly malignant prostate cancer cells. Thus, development of the RNAi-based gene silencing therapies simultaneously inhibiting multiple genes in the cell proliferative and survival signaling pathways provides an extremely powerful therapeutic strategy for prostate cancer.26

The multiple shRNAs expression system may also provide a good strategy for antiviral therapy. The siRNA-induced inhibition of viral infection or replication by either directly targeting viral genes essential for replication and virion structure or specifically inhibiting host genes required for virus infection and propagation has already proved to be an extremely effective strategy for antiviral therapy.27, 28, 29, 30, 31 However, the existence of viral sequence diversities or emergence of resistant virus variants poses an important obstacle for using siRNA as a therapeutic agent.28, 32, 33, 34 One particular strategy to counteract the viral escape or resistance is to apply multiple siRNAs or shRNAs simultaneously targeting several different conserved regions of viral genomes.2, 35, 36, 37, 38

In summary, the results presented in this study clearly demonstrate that the head-to-tail tandem array multiple shRNAs expression system not only provides a simple and efficient procedure for constructing and mapping the multiple shRNAs expression constructs but also advances a great step in using combinational therapies for complicated malignancy such as prostate cancer. Although previous studies have described the usefulness of the RNAi-based combinational therapies by co-delivery or coexpression of multiple siRNAs or shRNAs,7, 8, 9, 10, 11, 12, 13 this study reported an easy-to-use and cost-effective shRNA expression vector, pSUPER/EGFP, which can be used for constructing the multiple shRNAs expression vectors. In addition, the multiple shRNAs expression vector containing six shRNA expression cassettes exhibited strong inhibition activities of all their corresponding target genes including Bcl-2, Survivin, Akt1, Erk2, CyclinE and NFκB simultaneously, indicating that functional RNAi activities are still supported for coexpression of as many as six shRNAs under the control of the human H1 promoter in HeLa, HEK293 and PC3 cells.

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Acknowledgements

This work was supported by grant NSC-95-2752-B-006-004-PAE from the National Science Council, Taiwan, ROC (to WT Chang) and grant DOH-TD-B-111-004 from the Department of Health, Taiwan, ROC (to CY Chou).

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Correspondence to W T Chang.

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Cheng, T., Teng, C., Tsai, W. et al. Multitarget therapy of malignant cancers by the head-to-tail tandem array multiple shRNAs expression system. Cancer Gene Ther 16, 516–531 (2009). https://doi.org/10.1038/cgt.2008.102

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Keywords

  • RNAi
  • multiple shRNAs expression system
  • human H1 promoter
  • pSUPER
  • cancer therapy
  • biological signaling pathways

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