|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell-selective gene silencing in prostate cancer LNCap cells using prostate-specific membrane antigen promoter and enhancer in vitro and in vivo
Ranlu Liu, Jiantao Sun, Zhihong Zhang and Yong Xu1
Tianjin Institute of Urology and Department of Urology, Second Hospital, Tianjin Medical University, Tianjin, 300211, People's Republic of China
RNAi (RNA interference) has been widely used to silence specific genes. However, RNAi may also cause off-target silencing and elicit non-specific side effects. To achieve cell-specific gene silencing, a cell-selective promoter has to be used to drive RNAi expression. Furthermore, different terminators of cell-selective promoters may cause different silencing efficacies. In order to explore the best promoter and terminator combination and prove the cell-selective gene silencing effect of PSMAe/p (prostate-specific membrane antigen enhancer/promoter), we first constructed three plasmids by using PSMAe/p and three different terminators [poly(A), minipoly(A) and poly(U)] to explore the cell-selective driving ability of PSMAe/p by targeting EGFP (enhanced green fluorescent protein) in LNCaP, PC-3, EJ and HEK-293 (human embryonic kidney) cells. Then we chose NS (nucleostemin), an important endogenous gene of prostate cancer, and constructed the NS-targeting shRNA (small-hairpin RNA) expression plasmid by using PSMAe/p–poly(A) combination. Cell proliferation, cell cycle and early apoptosis in vitro and xenograft tumour growth in BALB/c nude mice in vivo were detected after NS knockdown. Results showed that PSMAe/p can drive EGFP silencing in LNCaP, not in PC-3, EJ and HEK-293 cells and PSMAe/p–poly(A) combination achieved the best silencing efficacy. Then PSMAe/p-shNS-poly(A) drives NS knockdown in LNCaP cells, not in PC-3, EJ and HEK-293 cells. Furthermore, RNAi-mediated NS knockdown not only reduces cell proliferation rate, reduces the percentage of S-stage cells and increases the percentage of G1-stage cells and increases the early apoptosis ratio in LNCaP cells in vitro, but also inhibited the LNCaP xenograft tumour growth in BALB/c nude mice in vivo by intratumoural injection. In conclusion, we have demonstrated that PSMAe/p–poly(A) combination is a promising delivery system for targeted RNAi gene therapy of prostate cancer. We showed one effective antitumour strategy by targeting NS protein, an important target in prostate cancer, with PSMAe/p-shNS-poly(A). These results serve as an important step for developing novel strategies to treat prostate cancer.
Key words: LNCap cell, prostate cancer, PSMA, RNA interference
Abbreviations: CMV, cytomegalomavirus, DAB, diaminobenzidine, EGFP, enhanced green fluorescent protein, FCM, flow cytometry, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, HEK-293 cells, human embryonic kidney cells, IHC, immunohistochemical, JNK, c-Jun N-terminal kinase, NS, nucleostemin, Pol II, polymerase II, Pol III, polymerase III, PSMA, prostate-specific membrane antigen, PSMAe/p PSMA enhancer/promoter,, RNAi, RNA interference, RT–PCR, reverse transcriptase–PCR, shRNA, small-hairpin RNA, siRNA, small interfering RNA, UPRT, uracil phosphoribosyl transferase
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Owing to its sequence specificity, RNAi (RNA interference) has been widely used to silence specific genes to investigate gene functions. In mammalian cells, this can be accomplished by introducing siRNAs (small interfering RNAs) (Elbashir et al., 2001) or shRNAs (small-hairpin RNAs) into cells (Shi et al., 2003). The shRNAs are usually delivered as gene constructs that are composed of a promoter, a short DNA sequence encoding shRNA and a transcription termination sequence. The structure of shRNAs mimics that of pre-micro RNAs, which are endogenously encoded natural shRNAs, and this structure renders shRNAs more stable silencing effects over longer periods of time (Paul et al., 2002; Sui et al., 2002). Most shRNA-delivery constructs use RNA Pol III (polymerase III) promoters, including U6, H1 and tRNA promoters. The main advantage of these promoters is that they direct high levels of shRNA expression, which in turn can achieve highly effective silencing. In addition, these gene constructs are small and simple and easily inserted into vectors. Unfortunately the application of these constructs is limited due to the fact that these promoters have no cell specificity and exceedingly high levels of shRNA expression increase the probability of off-target silencing and non-specific effects, including interferon response and cellular toxicity (Bridge et al., 2003; Jackson et al., 2003). CMV (cytomegalomavirus) promoters, a kind of RNA Pol II (polymerase II) promoters, are also used to synthesize shRNAs and different silencing efficacies have been reported (Xia et al., 2002; Zeng and Cullen, 2003; Ling and Li, 2004; Miyagishi et al., 2004). However, CMV promoters also lack cell specificity and thus produce undesirable effects in non-target cells.
Cell-specific targeting of siRNA is an important issue to consider in RNAi therapeutics (Robinson, 2004). To achieve cell-selective silencing, a cell-specific promoter has to be used to drive RNAi expression. However, cell-specific promoters are not perfect promoters either. First, the specificity of transcription has been shown to be negatively related to the level of transcription activity (Lu et al., 1999), which means that increasing transcription specificity may reduce the expression level of shRNAs. Secondly, the terminator of cell-specific promoters is poly(A) (polyadenylation). Compared with poly(U), poly(A) is more complex and makes the construction of shRNA expression vectors more difficult. Despite these disadvantages, several studies have used cell-specific promoters to successfully silence specific genes in Type II alveolar epithelial cells and HEK-293 cells (human embryonic kidney cells; Gou et al., 2004; Zhou et al., 2005). Furthermore, poly(U) and minipoly(A) (AATAAA) terminators are interchangeable for terminating shRNA transcription from CMV promoters (Song et al., 2004). To our knowledge, there have been no published reports on comparing the termination efficacy of different terminators combined with a cell-specific promoter in promoter-shRNA-terminator constructs.
A variety of specific tumour markers have been identified in prostate cancer, including PSA (prostate-specific antigen), PSMA (prostate-specific membrane antigen) and human kinase 2. Using these tumour antigen regulatory elements is an important method to solve the targeting problem of prostate cancer gene therapy. Among these tumour antigens, PSMA shows great potential to be used in gene therapy because of its high levels of prostate tissue-specific expression in >98% of patients with prostate cancer and almost all patients with prostate-cancer metastasis (Israeli et al., 1994a, b). Furthermore, its expression is actually strengthened after the treatment of hormone deprivation (Israeli et al., 1994a, b), which is important considering that most patients with advanced prostate cancer have undergone hormone-deprivation therapy. Therefore PSMAe/p (PSMA enhancer/promoter) constitutes one of the most promising gene delivery systems to specifically express therapeutic genes in prostate cancer cells. PSMAe/p could successfully drive the expression of EGFP (enhanced green fluorescent protein) and UPRT (uracil phosphoribosyl transferase) in the suicide gene system in prostate cancer cells. The tandem structure of a single enhancer and a promoter exerts the strongest driving effect with a driving capability 53 times of that of the PSMA promoter alone. Importantly, EGFP and UPRT are only expressed in PSMA-expressing LNCaP cells (Zeng et al., 2005). These results not only proved the cell-specific driving capabilities of PSMAe/p but also laid a solid foundation for targeted therapy of prostate cancer. However, Zhao et al. (2009) showed that, driven by PSMAe/p, RNAi effects were observed in both PC-3 and LNCaP cells upon transient transfection, which proved the tissue specificity and challenged the cell specificity of this system. One caveat is that the authors did not show whether PSMA is expressed in PC-3 cells, and thus further studies are required to solve this discrepancy. Furthermore, it has not yet been reported, as a cell-specific promoter, whether PSMAe/p can drive transcription of shRNAs and which termination sequence is most effective.
We have explored the cell-selective driving ability of PSMAe/p combined with three different terminators [poly(A), minipoly(A) and poly(U)] by evaluating the knocking down efficiency of EGFP shRNAs and chose the best PSMAe/p-terminator combination accordingly. Then with the best PSMAe/p-terminator combination, we showed that PSMAe/p–poly(A) causes cell-selective gene silencing in prostate cancer LNCap cells in vitro and in vivo by targeting NS (nucleostemin), an important endogenous gene in prostate cancer cells (Liu et al., 2008, 2010).
2. Materials and methods
pPSMAe/p was a gift from Professor Li Hong (West China Hospital of Sichuan University). This plasmid included a PSMAe/p sequence inserted into the pEGFP-1 plasmid (Zeng et al., 2005). pEGFP-C1 (GenBank® Accession No. U55763) carrying reporter gene EGFP was kept in our lab.
The interference sequence targeting EGFP was designed as previously reported (Xia et al., 2002). The sequence, which targets 418–438 bp in EGFP cDNA, was as follows: sense: 5′-CACAAGCTGGAGTACAACTAC-3′, loop: 5′-TTCAAGAGA-3′ and antisense: 5′-GTAGTTGTACTCCAGCTTGTG-3′. The three terminators were as follows: poly(A): the effective 62 bp sequence used in pSilencer™ 4.1-CMV neo (Ambion) (5′-AATAAAGGATCTTTTATTTTCATTGGATCTGTGTGTTGGTTTTTTGTATGCGGCCGCTAGCT-3′); minipoly(A): the core sequence of poly(A) (5′-AATAAA-3′); and Pol III terminator poly(U): TTTTTT . The EGFP shRNA-terminator was synthesized by TaKaRa Biotechnology Co. Ltd as SalI-Sense-Loop-AntiSense-terminator-BamH I and inserted into the pPSMAe/p vector (Figure 1). The three resulting plasmids pPSMAe/p-shEGFP-poly(A), pPSMAe/p-shEGFP-minipoly(A) and pPSMAe/p-shEGFP-poly(U) were confirmed by sequencing.
NS-targeting shRNA expression plasmid was constructed by replacing the sequence targeting EGFP in the pPSMAe/p-shEGFP-poly(A) plasmid with the sequence targeting NS. NS-specific shRNAi sequence targeted NS cDNA 300–320 bp (Genebank accession no. NM_014366) and had been proved effective before (Sijin et al., 2004; Liu et al., 2008, 2010). The shNS-poly(A) was synthesized as SalI-Sense-Loop-AntiSense-poly(A)-BamH I by TaKaRa Biotechnology. The resulting plasmid, named as pPSMAe/p-shNS-poly(A), was confirmed by sequencing.
2.2. Cell lines
Human prostate cancer LNCaP and PC-3 cells, bladder cancer EJ cells and embryonic kidney HEK-293 cells were bought from Cell Resource Center of Shanghai Institutes of Biological Sciences. All cells were maintained at 37°C in a humidified air atmosphere with 5% CO2 in RPMI 1640 (Gibco) supplemented with penicillin/streptomycin [100 I.U. (international units)/ml and 100 μg/ml respectively] and 10% fetal bovine serum (United star biotech Co. Ltd).
2.3. Cell transfection
For EGFP knockdown, pEGFP-C1 and pPSMAe/p-shEGFP were co-transfected into LNCaP, PC-3, EJ, and HEK-293 cells using Lipofectamine™ 2000 (Invitrogen). Each cell line was divided into four groups for transfection: Group A: pEGFP-C1+pPSMAe/p-shEGFP-poly(A); Group B: pEGFP-C1+pPSMAe/p-shEGFP-minipoly(A); Group C: pEGFP-C1+pPSMAe/p-shEGFP-poly(U); and Group D: pEGFP-C1+pPSMAe/p (empty plasmid control). The molecular mass ratio of pEGFP-C1 and the interference plasmid was 1:2. The molecular mass ratio of plasmids and Lipofectamine™ 2000 was 1:2.5.
NS knockdown was performed similarly. Briefly, LNCaP and PC-3 cells were divided into three groups, the silencer group: transfected with pPSMAe/p-shNS-poly(A); the vector group: transfected with empty pPSMAe/p; and the control group: transfected with PBS. Every group was in triplicate.
2.4. FCM (flow cytometry)
LNCaP cells were analysed by FCM to determine the EGFP expression levels. One day after transfection, cells were digested and washed twice with PBS. The cells were resuspended at 106/ml and measured by FACSCalibur™ (BD Bioscience). The analysis was repeated thrice.
2.5. RT–PCR (reverse transcriptase–PCR)
RNA was extracted using TRIzol® Reagent (Dingguo Biotech Co. Ltd). The synthesis of cDNA and PCR were performed using one-step mRNA Selective PCR Kit Ver.1.1 (TaKaRa Biotechnology). The PCR primer sequences and product lengths were as follows. EGFP (278 bp product): forward: 5′-AGTGCTTCAGCCGCTACCCC-3′ and reverse: 5′-GATGCCGTTCTTCTGCTTGTC-3′. PSMA (490 bp product): forward: 5′-ACCACATTTAGCAGGAAC-3′ and reverse: 5′-TGGATAGGACTTCACCC-3′. NS (565 bp product): forward: 5′-TAGTCCGAGCTTCATCGTATC-3′ and reverse: 5′-TTCCTGGTCACTGTCTTTGTC-3′. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (205 bp product): forward: 5′-GGATTTGGTCGTATTGGG-3′ and reverse: 5′-GGAAGATGGTGATGGGATT-3′. PCRs for EGFP and PSMA were 30 cycles of 85°C for 1 min, 50°C for 45 s and 72°C for 1 min. GAPDH was used as an internal control and its PCR was performed as 30 cycles of 85°C for 45 s, 50°C for 30 s and 72°C for 30 s.
2.6. Western blot
Total proteins from tissues and cells were extracted with SDS lysis buffer, separated by SDS/10% PAGE, and transferred to a nitrocellulose membrane. The membranes were probed with the specific primary antibody against NS (Cat# AF1638, R&D Systems) and followed by secondary peroxidase-conjugated rabbit anti-goat IgG (Boster Biological Engineering Co. Ltd). Western blots were developed with DAB (diaminobenzidine). GAPDH was used as an internal control.
2.7. Determination of cell proliferation in vitro
LNCaP and PC-3 cells were plated at 1.0×104/well in 24-well plates. Cells were transfected 24 h (for PC-3 cells) or 48 h (for LNCaP cells) after seeding, and divided into the silencer group, the vector group and the control group for transfection as described above. Every group was in triplicate. PC-3 cells were counted at 24, 36, 48, 60 and 72 h after transfection with a haemacytometer. LNCaP cells were counted at 48, 72, 96, 120 and 144 h after transfection to follow growth.
2.8. Detection of cell cycle
LNCaP and PC-3 cells were transfected as described above, collected 48 h later, fixed with 70% ethanol for >18 h, and dyed with propidium iodide. The stage percentages of G0/G1and S were determined by FCM. The experiment was repeated thrice.
2.9. Detection of early apoptosis
LNCaP and PC-3 cells were transfected as described above, collected 48 h later, and fixed with 70% ethanol for the detection of early apoptosis. Cells were dyed according to the instructions of Annexin V-EGFP cell apoptosis detection kit (Keygentec Co. Ltd). The percentage of early apoptosis was determined by FCM. The experiment was repeated thrice.
2.10. Assessment of LNCaP xenograft tumour growth in vivo
All experimental procedures conducted on animals were approved by the ethics committee of Second Hospital of Tianjin Medical University. Tumour growth assessment was performed as described before (Hattori and Maitani, 2006). To generate LNCaP tumour xenografts, 2×107 cells in 100 μl PBS were inoculated subcutaneously into the right flank of male BALB/c nude mice at 6 weeks of age [Beijing Experimental Animal Center of Chinese Academy of Medical Sciences, Beijing, China, permit number: SCXK (Jing) 2009-0007]. The tumour volume was calculated as: V = 1/2×LW2, where L (long axis) and W (short axis) are the larger and smaller diameters respectively. When the average tumour volume reached 150 mm3 (day 0), the mice were divided into three groups as follows and each group consisted of four tumours. The silencer group: intratumoural injection with 100 μl of the mixture containing 10 μg of pPSMAe/p-shNS-poly(A) and 25 μl of liposome; the vector group: intratumoural injection with 100 μl of mixture containing 10 μg of pPSMAe/p and 25 μl of liposome; and the control group: intratumoural injection with 100 μl of mixture containing 25 μl of liposome and PBS.
Intratumoural injection was given every 3 days for five times. Tumour growth was observed regularly every 3 days and tumour diameters were measured by a vernier caliper for calculating the tumour volume. Four days after the final injection, the nude mice were killed by cervical dislocation. Xenograft tumours were removed, and their size and weight measured. Tumours were kept in 10% neutral formaldehyde, embedded in paraffin and prepared for H/E (haematoxylin/eosin) staining and subsequent IHC (immunohistochemical) staining.
2.11. IHC staining
The paraffin-embedded tissues were sliced into 4 μm sections. IHC staining was performed using the labelled streptavidin–biotin method. Briefly, following deparaffinization, antigen unmasking and inactivation of endogenous peroxidase, slides were first incubated with goat anti-human NS antibody (R&D Systems) at 4°C overnight, and with secondary biotin-conjugated rabbit anti-goat antibody (Boster Biological Engineering Co. Ltd) and HRP (horseradish peroxidase)-labelled streptavidin at 37°C for 20 min. The slides were finally developed with DAB, counterstained with haematoxylin/eosin, observed under a light microscope and analysed with the NIS-Elements freeware (Nikon).
To determine the NS-staining positive rate, at least four high-power microscope fields were randomly selected. Cells with pale-yellow to yellow-brown staining in cytoplasm or nucleus were considered positive. The positive rate was calculated by dividing the number of positive cells by the total cell number. Staining was determined as negative (−): no or weak staining with a positive rate of ≤5%; weak positive (+): pale-yellow staining with a positive rate of 5–25%; positive (++): staining intensity between weak positive and strong positive with a positive rate of 25–50%; strong positive (+++): yellow to yellow-brown staining with a positive rate of ≥50%.
2.12. Statistical analysis
SPSS 11.5 statistical software was used for statistical analysis. All results were presented as means±S.D. and the differences between means were analysed by one-way ANOVA. P<0.05 was considered statistically significant.
3.1. PSMA is specifically expressed in LNCaP cells
We confirmed by PCR that PSMA was specifically expressed in LNCaP cells, but not in PC-3, EJ and HEK-293 cells.
3.2. PSMAe/p-shEGFP-poly(A) drives greatest silencing of EGFP in LNCaP cells
To compare the silencing efficacies of PSMAe/p combined with three terminators [poly(A), minipoly(A) and poly(U)] in different cells, we transfected LNCaP, PC-3, EJ and HEK-293 cells with pEGFP-C1+pPSMAe/p-shEGFP-poly(A) (Group A), pEGFP-C1+pPSMAe/p-shEGFP-minipoly(A) (Group B), pEGFP-C1+pPSMAe/p-shEGFP-poly(U) (Group C), or pEGFP-C1+pPSMAe/p (empty plasmid control, Group D). One day after transfection, EGFP expression was observed by fluorescence microscopy (Figure 2a). In LNCaP cells, compared with the fluorescence intensity of Group D, the intensity of Groups A, B and C were reduced. No obvious change in EGFP fluorescence intensity was seen in PC-3, EJ or HEK-293 cells.
This EGFP silencing effect was confirmed by FCM analysis (Figure 2b). In LNCaP cells, the EGFP expression rates of Groups A, B, C and D were 5.7, 9.6, 20.4 and 45.5%, respectively. Compared with Groups B (P<0.05), C (P<0.01) and D (P<0.01), Group A has the lowest EGFP expression rates. In PC-3, EJ and HEK-293 cells, no significant change was observed (P>0.05).
We further compared the EGFP mRNA and protein levels between the four groups in these four cell lines. EGFP mRNA levels were measured by RT–PCR and quantified by the Image ProPlus analysis software (Figures 3a and 3b). EGFP protein levels were compared by Western blotting (Figures 3c and 3d). GAPDH was used as an internal control in both analyses. Results from both mRNA and protein expression analyses are consistent with these from fluorescence microscopy and FCM analyses. In PC-3, EJ, and HEK-293 cells, both EGFP mRNA and protein levels were not significantly different between the four groups (P>0.05). In contrast, in LNCaP cells, compared with the EGFP mRNA and protein levels of Group D, the levels of Groups A, B and C were significantly reduced (P<0.01). Group A displayed a greater reduction in mRNA and protein levels than Groups B and C (P<0.01 for mRNA levels and P<0.05 for protein levels). The residual EGFP mRNA and protein levels were significantly lower in Group B than in Group C (P<0.05).
In conclusion, PSMAe/p in combination with the terminator poly(A) induces greatest silencing of EGFP in LNCaP cells.
3.3. PSMAe/p-shNS-poly(A) drives NS knockdown in LNCaP cells
Since PSMAe/p in combination with the terminator poly(A) shows the best silencing effects on EGFP in LNCaP cells, we constructed the vector pPSMAe/p-shNS-poly(A) for NS knockdown. LNCaP and PC-3 cells were divided into three groups and transfected with pPSMAe/p-shNS-poly(A) (the silencer group), empty pPSMAe/p (the vector group) or PBS (the control group). RT–PCR and Western blot results showed that compared with the NS mRNA and protein levels in the vector and control groups, the levels in the silencer group were reduced by ∼60% in LNCaP cells (P<0.01) (Figure 4a). In contrast, in PC-3 cells, the NS mRNA and protein levels were similar in the vector, control and silencer groups (P>0.05) (Figure 4a).
3.4. RNAi-mediated NS knockdown reduces cell proliferation rate in LNCaP cells
LNCaP and PC-3 cells were transfected in three groups as described above. The cell counting analysis showed that in LNCaP cells, while no proliferation difference was observed between the vector group and the control group (P>0.05), the cell proliferation rate of the silencer group was significantly lower than that of the vector and control groups at 72, 96, 120 and 144 h after inoculation (P<0.05) (Figure 4b).
In contrast, in PC-3 cells, the proliferation rate of the silencer group was not significantly lower than that of the vector and control groups at 36, 48, 60 and 72 h after inoculation (P>0.05) (data not shown).
3.5. RNAi-mediated NS knockdown reduces the percentage of S-stage cells and increases the percentage of G1-stage cells in LNCaP cells
LNCaP and PC-3 cells were transfected in three groups as described above and cell-cycle stage percentages were analysed (Figure 4c). FCM results showed that in LNCaP cells, the percentage of S-stage cells of the silencer group (14.17±0.32%) was significantly lower than that of the vector group (19.65±0.64%) and the control group (20.61±0.80%) (P<0.01). On the contrary, the percentage of G1-stage cells of the silencer group (74.69±1.01%) was significantly higher than that of the vector group (71.06±0.38%) and the control group (70.17±0.32%) (P<0.01) (Figure 4d).
In contrast, in PC-3 cells, the percentages of S-stage cells and G1-stage cells remained similar among the vector, control and silencer groups (P>0.05; S1 stage: 29.47±0.47, 29.86±0.36 and 28.86±1.13%, respectively; G1 stage: 62.56±0.62, 61.71±1.07 and 63.34±0.81%, respectively) (Figure 4d).
3.6. RNAi-mediated NS knockdown increases the early apoptosis ratio in LNCaP cells
LNCaP and PC-3 cells were transfected in three groups as described above and early apoptosis ratio was analysed. Cells were dyed according to the instructions of Annexin V-EGFP cell apoptosis detection kit. The percentage of early apoptosis was determined by FCM. The results showed that in LNCaP cells, the early apoptosis ratio in the silencer group (5.50±0.79%) was significantly higher than that in the vector group (2.15±0.24%) and the control group (1.30±0.37%) (P<0.01) (Figure 4e).
PC-3 cells displayed a lower early apoptosis ratio. This ratio remained similar between the silencer, vector and control groups (P>0.05; 1.03±0.32, 1.01±0.38% and 0.93±0.43%, respectively) (Figure 4e).
3.7. RNAi-mediated NS knockdown reduces LNCaP xenograft tumour growth in vivo
To generate LNCaP tumour xenografts, 2×107 cells in 100 μl of PBS were inoculated subcutaneously into the right flank of male BALB/c nude mice at 6 weeks.
There were visible tumours in all 12 nude mice 7 days after inoculation, showing a tumour formation rate of 100%. The tumour diameter reached 0.6 cm 10 days after inoculation and no mouse died. No swelling or ulceration was observed in the lumps. Mice were divided into three groups (the silencer, vector and control groups) as described in the Materials and Methods. Initially, no difference in the tumour volume was observed between these three groups (P>0.05; 0.095±0.003, 0.099±0.006 and 0.098±0.005 cm3 respectively).
Mice were intratumourally injected with pPSMAe/p-shNS-poly(A) and liposome (the silencer group); pPSMAe/p and liposome (the vector group); or PBS and liposome (the control group). Tumour volume was recorded and the growth curves are shown in Figure 5(a). Our results indicated that compared with the tumour growth rate of the vector and control groups, the rate of the silencer group was significantly decreased after they were treated with cell-selective NS RNAi (Figures 5a and 5b). In addition, the silencer group also showed the lowest final tumour weight among the three groups (Figure 5c; P<0.01; the silencer group: 0.49±0.14 g, the vector group: 1.57±0.124 g, and the control group: 1.65±0.18 g). In conclusion, tumour growth curves and final tumour weight showed that LNCaP cell proliferation in vivo was significantly reduced after RNAi-mediated NS down-regulation.
3.8. RNAi-mediated NS knockdown in LNCaP xenograft tumour
Finally, to confirm NS knockdown in the LNCaP xenograft tumour, IHC staining was done (Figure 5d). NS protein expression was high in the nucleus and low in the cytoplasm in LNCaP xenograft tumour tissues. Compared with vector and control group, the NS-positive staining of the silencer group was significantly lower. The positive rate of silencer group, vector group and control group was 34.0±2.9, 70.3±3.5 and 74.0±4.2%, respectively (Figures 5d and 5e; P<0.01). No significant difference in the NS-positive rate was observed between the vector group and the control group (Figures 5d and 5e; P>0.05). Furthermore, the staining intensity was also decreased in the silencer group. The PBS control group showed negative staining (−) (1.1±0.1%).
Cell-specific promoters belong to the RNA Pol II promoters and enable targeted gene expression to avoid causing side effects in other tissues and organs. Their termination sequence poly(A) not only directs 3′ end processing but also controls the extent and speed of transcription in vivo. Transcription suspension occurs before final termination when RNA Pol II encounters the poly(A) sequence. The G/C-rich region of poly(A), which is required in transcription termination, does not participate in poly(A)-dependent transcription suspension (Hattori and Maitani, 2006). The only region that determines transcription suspension is the AATAAA hexamer in the poly(A) signal and one or two such hexamers are sufficient to slow down or stop the transcription of RNA Pol II. This hexamer may play an independent role in the 3′ end processing and transcription termination. Polymerases that slow down or pause after encountering this hexamer may restore their speed if the transcription termination signal is absent (Nag et al., 2006). Other termination sequences also exist. Song et al. (2004) showed that the CMV promoter, which is a RNA Pol II promoter, combined with the termination sequence AATAAA hexamer or the U6 promoter combined with poly(U) termination sequence can both drive the transcription of JNK (c-Jun N-terminal kinase) shRNA at similar efficiency. Furthermore, when the AATAAA hexamer was replaced by poly(U), the CMV promoter could still drive the shRNA transcription and knock down JNK effectively (Song et al., 2004).
We established the cell-specific promoter PSMAe/p in combination with the termination sequence poly(A) as a specific and efficient system to drive shRNA transcription in PSMA-expressing prostate cancer LNCaP cells. We confirmed by PCR that PSMA was specifically expressed in LNCaP cells, but not in PC-3, EJ and HEK-293 cells. After constructing three EGFP-specific shRNA expression vectors: pPSMAe/p-shEGFP-poly(A), pPSMAe/p-shEGFP-minipoly(A) and pPSMAe/p-shEGFP-poly(U), we showed that while EGFP expression was not affected in PC-3, EJ and HEK-293 cells (Figures 2 and 3), EGFP expression was significantly reduced in LNCaP cells in the interference group compared with the empty vector group (Figures 2 and 3). These results confirmed that PSMAe/p specifically drives transcription in LNCaP cells, but not in the other cells. Our results are consistent with Zeng et al. (2005), who showed that, when driven by PSMAe/p, EGFP is only expressed in PSMA-expressing LNCaP cells, but not in PC-3 and DU145 cells. Furthermore, pPSMAe/p-shEGFP-poly(A) induces the greatest EGFP knockdown followed by pPSMAe/p-shEGFP-minipoly(A), whereas pPSMAe/p-shEGFP-poly(U) shows the lowest silencing effects on EGFP (Figures 2 and 3). These results indicate that the simplified poly(A) termination signal shows the highest transcription–termination efficiency in the PSMAe/p-driven shRNA expression vector. The termination efficiency of its core structure, the AATAAA hexamer (minipoly(A) signal), is lower probably due to the lack of GC-rich region, which is consistent with Nag et al. (2006). In contrast with Song et al. (2004), poly(U) signal shows the lowest transcription–termination efficiency. The reason for this discrepancy may be that the transcription–termination ability of poly(U) differs for different RNA Pol II promoters.
NS gene, an important endogenous functional gene of prostate cancer, was to test the antitumour effects of pPSMA-shNS-poly(A)-mediated knockdown in vitro and in vivo. Our in vitro studies showed that the transfection of pPSMA-shNS-poly(A) into LNCaP cells decreases NS expression, arrests cell cycle at G1, decreases cell proliferation and increases early apoptosis (Figure 4). In contrast, these effects were not observed in PC-3 cells transfected with pPSMA-shNS-poly(A) (Figure 5). To observe the antitumour effects in vivo, we established the ectopic xenograft model using LNCaP cells in nude mice and showed that intratumoural injection of pPSMA-shNS-poly(A) reduces NS expression and slows down the growth of LNCaP xenograft tumours (Figure 5).
In summary, PSMAe/p-poly(A) combination specifically drives the transcription of shRNA in LNCaP cells and causes cell-selective silencing in vitro and in vivo. The reason that PSMAe/p in the vector only works in the LNCaP cells may be as follows. The major regulatory elements that start gene transcription include two parts: cis-regulatory elements and trans-regulatory factors. Only a right combination of cis-, trans-elements, and the RNA Pol II can form the transcription initiation complex and start transcription. One potential mechanism for the cell-selectivity of the PSMA promoter may be that the trans-acting factors that can interact with the cis-elements of PSMA promoter may exist only in LNCaP cells but not in PC-3, EJ and HEK-293 cells.
The strategy of gene silencing was effective in prostate cancer LNCaP cells because PSMA was strongly expressed in LNCaP cells. However, PSMA was not expressed in PC-3 cells, though PC-3 cell line was also derived from prostate cancer. That is to say, our method of gene silencing can be effective in prostate cancer as long as the cancer cells express PSMA. Meanwhile, PSMA shows high levels of prostate tissue-specific expression in more than 98% of patients and almost all patients with prostate cancer metastasis. Furthermore, its expression is actually strengthened after the treatment of hormone deprivation. Therefore we believe that our method of gene silencing can be effective in prostate cancer, including in advanced stage patients.
Our study demonstrated that PSMAe/p–poly(A) combination is a promising delivery system for targeted RNAi gene therapy of prostate cancer. We showed one effective antitumour strategy by targeting NS protein, an important target in prostate cancer, with PSMAe/p-shNS-poly(A). These results not only provide important experimental evidence for cell-specific RNAi-based therapy for prostate cancer but also serve as an important step for developing new strategies to treat prostate cancer, especially castration-resistant prostate cancer.
Ranlu Liu carried out the molecular studies, participated in the sequence alignment and drafted the paper. Jiantao Sun participated in the design of the study and performed the statistical analysis. Zhihong Zhang participated in the design and co-ordination of the study and helped to draft the paper. Yong Xu participated in the design and co-ordination of the study.
This swork was supported by the
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Received 30 December 2011/24 April 2012; accepted 21 May 2012
Published as Cell Biology International Immediate Publication 21 May 2012, doi:10.1042/CBI20110662
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 2 pPSMAe/p-shEGFP-poly(A) induces greatest EGFP silencing in LNCaP cells: microscopy and FCM analyses
Figure 3 pPSMAe/p-shEGFP-poly(A) induces greatest EGFP silencing in LNCaP cells: RT–PCR and Western blot analyses
Figure 4 pPSMAe/p-shNS-poly(A)-mediated NS knockdown reduces cell proliferation, arrests cell cycle at G1 and increases early apoptosis