Synergistic Effects of Targeting Survivin and CDK 1 on Nasopharyngeal Carcinoma in Vitro and in Vivo

Background: To explore the impact of pU6-based tandem survivin and CDK1-specific short hairpin RNA on the biological behaviors of CNE-2 nasopharyngeal carcinoma cells in vitro and in vivo. Patients and Methods: The vectors of pU6-survivinshRNA, pU6-CDK1shRNA and pU6-survivinshRNA-CDK1shRNA were constructed and transfected into CNE-2 cells with Lipofectamine TM 2000, respectively. The mRNAs and proteins of CDK1 and survivin were determined by RT-PCR and Western blotting, accordingly. MTT assay was employed to evaluate the proliferation of CNE-2 cells, and flow cytometry was performed to determine the apoptosis of CNE-2 cells. The effects of interfering survivin and CDK1 on tumorigenesis were evaluated by tumor xenografts experiments. Results: Effective plasmids were successfully constructed knocking down survivin and/or CDK1. The proliferation inhibition of CNE-2 cells by pU6-survivinshRNA-CDK1shRNA (32.5%) was higher than that of by pU6-survivinshRNA (25.6%) and pU6-CDK1shRNA (15.6%), and apoptosis in CNE-2 cells simultaneously interfering survivin and CDK1 (15.2%) dramatically increased when compared to those of interfering survivin (5.4%) or CDK1 (4.7%) alone. Furthermore, simultaneously interfering survivin and CDK1 is more effective than interfering alone component in inhibiting tumor growth of fBalb/C nude mice xenografted with CNE-2 cells. Conclusion: The results altogether indicate that interfering survivin and CDK1simutaneously can produce synergistic effects of anti-nasopharyngeal carcinoma, which could be a potential therapeutic method.


Introduction
Nasopharyngeal carcinoma (NPC) is one of the most popular cancers in southern China [1].Though NPC is sensitive to radiotherapy, the outcome of the treatment is depressing.The five year survival rate is only 50% -60% due to factors below: most NPC organization cells is undifferentiated with high malignancy; the NPC anatomical location impedes radical resection and the remaining NPC cells serve as seeds for future recurrence and metastasis in future; NPC cells are insensitive to chemotherapy; NPC cells develop radio resistance during radiotherapy.Therefore, it is essential to develop a new therapeutic technology to improve the unsatisfactory outcome of NPC treatment.With the development of technology of gene recombination and transgenosis, RNA interference technology has become a powerful tool of knocking out targeted gene expression, which offers an optimistic treatment for various cancers.
Multiple up-regulated genes are involved in tumorogenesis and metastasis, so the treatment of targeting a single gene is generally unsatisfactory.Therefore, researchers attempt to solve the problem through two ways: targeting th enodal protein or targeting multiple proteins simultaneously.Survivin, a member of the inhibitor of apoptosis (IAP) family, is closely associated with many physiological and pathological processes, including interaction with multiple regulatory factors, modifiers and cellular networks [2] [3] [4] [5].Survivin is highly expressed in almost all cancers, including NPC.However, expression of survivin is undetectable or rather low in almost all normal mature cells, which makes survivin an ideal target for various cancers [3] [4] [5] [6].CDK1, an important member of controlling mitosis, plays a key role in G1/S and G2/M phase transitions of eukaryotic cell cycle [7] [8].Moreover, CDK1 is also the enzyme for catalyzing the phosphorylation of 34 survivin threonine residue site [9].Overexpression of CDK1 in cancers induces cell proliferation and chromosomal instability, which also makes CDK1 a potential therapeutic target [10] [11] [12].Short hairpin RNAs (shRNA) is efficient in silencing specific gene in mammalian cell [13].
Therefore, the study is aimed at observing impacts of knocking out survivin and CDK1 simultaneously on nasopharyngeal carcinoma by constructing pU6-survivin shRNA -CDK1 shRNA , and evaluating the potential value of survivin and CDK1 in tumor therapy.

Generation of shRNA pU6-M4 Plasmids
Genbank accession number (survivinNM_001012271) and (CDK1 NM_001170406) were used for this study.Scramblesh RNA sequence without significant homology to mouse and human gene sequences, was used as negative control to validate the specific effects.Terminator code (TTTTT), restriction sites of BamHI (G^ATCC) and HindIII (A^AGCCT) were added to where to form Bam-HI-sense-loop-anti-sense-terminator-HindIII. A series of plasmids containing shRNA specific to Survivin and CDK1gene, either alone or in combination, were cloned into pU6-M4 vector by ligating the BamHI/HindIII-digested shRNA fragment to the vector after digestion by the same restriction endonucleases as described previously.The diagram of constructed pU6-survivin shRNA -CDK1 shRNA was as shown in Figure 1.

Reverse-Transcription PCR and qPCR
To evaluate the interference efficiency of constructed plasmids, the seven groups Journal of Cancer Therapy The 5' primer of survivin gene was 5'-TCAAGGACCACCGCATCTCTA-3', and the 3' primer was 5'-TGAAGCAGAAGAAACACTGGGC-3'.The 5' primer of CDK1 was 5'-CCTAGCATCCCATGTCAAAAACTTGG-3' and the 3' primer was 5'-TGATTCAGTGCCATTTTGCCAGA-3'.The 5' primer of β-actin that acted as internal reference was 5'-GTGGTGGTGAAGCTGTAGCC-3', and the 3' primer was 5'-GAGACCTTCAACACCC-3'.RT-PCR was performed with the following parameters: predegeneration at 95˚C for 2 min, synthesis of cDNA with SuperScript TM one-step RT-PCR kit on a PCR cycler by heating 60˚C for 1 min, followed by 40 cycles of amplification (denaturation at 95˚C for 15 s, anealing at 55˚C for 15 sec, extending at 72˚C for 45 s), and a final extension step at 72˚C for 5 min.The PCR cycle parameters were as following: predegeneration at 95˚C for 30 sec; 40 cycles of amplification (denaturation at 95˚C for 5 sec, and annealing at 60˚C for 20 sec).

Western Blot
loading buffer.Lysates from CNE-2 cells were added with protease inhibitorcocktail (Roche) and 1 mM PMSF (Calbiochem).The supernatant was acquired with centrifuging at 13,000× g for 10 min.The lysates were denatured and separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to PVDF membranes (Millipore, Bedford, MA, USA).5% nonfast milk in Tris bufferedsaline (TBS) buffer with 0.05% Tween-20 was used to block at room temperature for 1 hour.Then the primary rabbit antibodies of anti-human CDK1 (1:1000) and survivin (1:1000) were added and incubated at 4˚C overnight.After five times of rinsing with TBST buffer, secondary goat anti-rabbit horseradish peroxidase conjugated antibody was added and incubated at room temperature for 2 h.Bands were visualized by ECL plus western blot detection reagent (GE, USA).The house-keeping gene β-actin was used as control and detected by using rabbit anti-β-actin antibody (1:4000, Abmart, China) plus HRP conjugated goat anti-rabbit secondary antibody (1:5000, Sigma).

Evaluation of Cell Growth Inhibition Rate (IR) by MTT Assay
CNE-2 cells were seeded into 96-well plates at a density of about 3 × 10 5 /well.CNE-2 cells were classed into five groups: transfection with pU6-survivin shRNA ; transfection with pU6-CDK1 shRNA ; transfection with pU6-survivin shRNA -CDK1 shRNA ; transfection with pU6-survivin shRNA -CDK1 shRNA ; Control without adding anything.CNE-2 cells were transfected with 5 μg plasmids and 5 μl of Lipofectamine TM 2000 followed by incubating for 48 h.Then, 15 μl of MTT (5 mg/ml) was added and cultured 4 h, followed by adding 150 μl DMSO to each well.OD value of each well was analyzed at wavelength of 490 nm after shaking for 10 min, and the cell growth inhibition rate (IR) was calculated with the following formula: IR = (Acontrol group − A experimental group)/A control group × 100%.Each Experiment was repeated thrice.

Evaluation of CNE Cell Apoptosis by Flow Cytometry
The CNE-2 cells were categorized into the following five groups: transfection with pU6-survivin shRNA ; transfection with pU6-CDK1 shRNA ; transfection with-pU6-survivin shRNA -CDK1 shRNA ; transfection with pU6-survivin shRNA NC-CDK1 shRNA NC; Mock transfection without adding anything.CNE-2 cells were harvested after 48 hour of post-transfection and digested with 0.25% trypsin to produce single-cell suspension, cells were adjusted to 1 × 10 6 /ml and then fixed with 70% cold ethanol at 4˚C overnight.CNE-2 cells were further treated with Annexin V and PI and incubated for 30 min at room temperature in the darkness.The percentage of CNE-2 apoptosis was determined by Flow cytometry (Flow cytometry cycle detection kit, FACSort flow cytometry, B & D companies; USA) operated by a specialist.

The pU6-SurvivinshRNA-CDK1shRNA Was More Effective in Inhibiting Tumor Growth in Nude Mice Received CNE-2 Xenograft
The tumor growth speeds of the pU6-survivin shRNA NC-CDK1 shRNA NC group and mock transfection group were the fastest, and both were higher than those of the other three groups.As shown in Figure 4, the tumor volume of CNE-2 xenografts in nude mice treated with pU6-survivin shRNA -CDK1 shRNA at a dose of

Discussions
Uncontrolled cell proliferation and abnormal cell death can lead to cancer.
Therefore, therapies against cancer are to restore balance through targeting cancerous cells by blocking cellular growth or enhancing death.Expression of Survivin in cancer and their correlation with cell proliferation is well documented for various cancers, and silencing survivin has been proved to be efficient in treating cancers.Though knocking out survivin alone manifested antitumor effects both in vitro and in vivo, its efficiency didn't meet clinical demands, therefore, run on sentence some studies tried to combine silencing survivin and other treatments.In this study, we proved that the antitumor effects of targeting survivin and CDK1 perform significant better than either component alone.
In our nasopharyngeal carcinoma xenografts experiments, mice were administrated with the three kinds of plasimids, i.e. pU6-survivin shRNA -CDK1 shRNA , pU6-survivin shRNA and pU6-CDK1 shRNA , They all manifested obvious inhibiting growth of CNE-2 xenografts.Meanwhile, pU6-survivin shRNA -CDK1 shRNA was superior to pU6-survivin shRNA and pU6-CDK1 shRNA in decreasing tumor volume, and the synergistic effects of targeting survivin and CDK1 were apparent (Figure 3).Our results also manifested that, compared with the single gene suppression, co-interfering survivin and CDK1 was more effective in reducing expressions of   [6].CDK1 is a key modulator involving the initiation and transition process in mitosis.CDK1 is also the enzyme for activating phosphorylation of multiples proteins, including survivin.Silencing CDK1 leads to inhibiting dephosphorylation of survivin (T34A) into Phosphorylation 34 site of survivin (T34E) [9].It has been proved that interfering phosphorylation of survivin (T34A) will lower the level of survivin and promote apoptosis [14], which may be due to the incapability of survivin (T34A) combining with acceleration degradation of surviving by ubiquitin [15].Therefore, silencing CDK1 and surviving simultaneously will generate synergistic effect in inhibiting growth and inducing apoptosis of tumor cells.
The combined targeting of survivin and CDK1 plasmids is superior to targeting of survivin and CDK1 alone in vitro and in vivo, the suppression of tumor growth was still incomplete.Many factors, including efficiency of transfection, expression levels and stability, result in incomplete inhibition of tumor growth.Moreover, no differential attack of constructed vectors, which shunts some plasmids to non-tumor tissue, also contributes to unsatisfactory antitumor effect.Therefore, it is necessary to solve the problem of designing efficacious in vivo delivery systems to transport designed vectors into tumor.
In brief, our results suggest that targeting survivin and CDK1 that based on constructed pU6-survivin shRNA -CDK1 shRNA represents a more promising therapeutic approach for treating NPC than targeting survivin or CDK1 singly.The synergistic effects of silencing survivin and CDK1 suggest that targeting the two genes should produce robust efficacy against a variety of tumors.

Figure 2 .
Figure 2. mRNA and protein levels of survivin and CDK1 in CNE-2 cells transfected with various recombinant plasmids.(a) RT-PCR analysis of survivin mRNA in CNE-2 cells transfected with pU6-survivin shRNA ; (b) RT-PCR analysis of CDK1mRNA in CNE-2 cells transfected with pU6-CDK1 shRNA ; (c) relative mRNA levels of survivin and CDK1 in CNE-2 cells transfected with different plasmids revealed by qPCR; (d) levels of survivin and CDK1 protein in CNE-2 cells transfected with different plasmids revealed by western blot.

Figure 4 .
Figure 4. Growth curves of CNE-2 treated with different components.

Table 1 .
The growth inhibition rate of CNE-2post-interference of 24 h.