Expression of the Pokemon Proto-oncogene in Nasopharyngeal Carcinoma Cell Lines and Tissues

The proto-oncogene Pokemon is a POZ and Krüppel (POK) erythroid myeloid ontogene, a member of the POK family. Because of containing broad complex, tramtrack, and bric-a-brac/poxvirus and zinc finger (BTB/POZ) domain, Pokemon protein has several important structures, such as zinc finger and actin-binding repeats (Albagli et al., 1995; Collins et al., 2001). It is also called FBI-1 (Pessler et al., 1997), OCZF (Kukita et al., 1999), LRF (Davies et al., 1999), Zbtb7A (Kelly and Daniel, 2006). Pokemon can specially suppress a regulation pathway between ARF and p53, as a result, tumorigenesis will be promoted with down-regulated ARF (Maeda et al., 2005). Pokemon i s aber ran t ly overexpressed in nasopharyngeal carcinoma (NPC) tissue by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) (Gao et al., 2009). NPC incidence and mortality trends have been high in southern China (Wei et al., 2010; Wei et al., 2010; Huang et al., 2012). It is necessary to study further the connection between Pokemon and tumorigenesis of NPC. According to the World Health Organization (WHO) classification system (1978), non-keratinising carcinoma and undifferentiated carcinoma (types II and III) can be considered together as undifferentiated carcinomas of the nasopharyngeal type (UCNT) (Tao

P o k e m o n i s a b e r r a n t l y o v e r e x p r e s s e d i n nasopharyngeal carcinoma (NPC) tissue by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) (Gao et al., 2009).NPC incidence and mortality trends have been high in southern China (Wei et al., 2010;Wei et al., 2010;Huang et al., 2012).It is necessary to study further the connection between Pokemon and tumorigenesis of NPC.According to the World Health Organization (WHO) classification system (1978), non-keratinising carcinoma and undifferentiated carcinoma (types II and III) can be considered together as undifferentiated carcinomas of the nasopharyngeal type (UCNT) (Tao

Patient selection
After obtaining patient approval, 10 chronic rhinitis and 10 NPC fresh tissues were obtained by biopsy.The tissues were confirmed by pathology from the formalinfixed paraffin wax-embedded samples.The pathological type of NPC was UCNT.This study was approved by the ethics committee of The People's Hospital of Guangxi Zhuang Autonomous Region.

RNA extraction and quality test
Total RNA of the cell lines was extracted by TRIZOL (Invitrogen, Carlsbad, USA).RNA amount was detected by Nanodrop 2000 (Thermo Fisher Scientific Inc, Waltham, USA).4μl total RNA was analyzed by 1.2% agarose gel electrophoresis with 0.6 mol/L formaldehyde (Robert, 2008).The gel was photographed by Bio Imaging System Gene Genius (Syngene, Cambridge, United Kingdom).An equal amount (4μg) of total RNA was synthesized as a first-strand cDNA using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Pittsburgh, USA).
Purified Pokemon PCR products were analyzed by the 3730 automatic DNA sequencer (ABI, Carlsbad, USA).

Statistical analysis
Data analysis of Real-time PCR and western-blot was performed by Sigma Plot 8.0.Statistical comparisons amount the data were performed by t test (SPSS.13.0).P values less than 0.05 was considered to be statistically significant.

Quality test of total RNA from the cell lines
Total RNA of the cell lines was not degraded and not polluted by DNA or protein (Table 1, Figure 1).A260/ A280 of all RNA value was between 1.90 and 2.00.

RT-PCR and sequencing of the cell lines
The electrophoretic bands indicated that CNE3   Pokemon band was weakest (Figure 2A).The gray value ratios of Pokemon and GAPDH indicated: CNE1>CNE2>NP69 >C666-1>CNE3 (Figure 2B).PCR sequences of the cell lines were matched with the Pokemon, according to the NCBI blast database (Figure 2C).

Western-blot of chronic rhinitis and UCNT
Subsequently, 3 chronic rhinitis and 3 NPC biopsy specimens were selected randomly in 10 chronic rhinitis and 10 NPC biopsy specimens.As a result, the protein bands indicated that Pokemon protein expression was commonly high in UCNT (T6, T24 and T32).however, Pokemon was not expressed in chronic rhinitis (T4 and T8), there was a little protein expression in T9 (Figure 4A).The Pokemon protein expression level of UCNT was further higher than Pokemon protein expression of chronic rhinitis (Figure 4B).

Discussion
Our group firstly used the NPC cell lines from different tissue types to study Pokemon mRNA and protein expression.CNE1 came from a well differentiated squamous carcinoma (Department of Virology, 1978).CNE2 came from a poorly differentiated squamous carcinoma (Gu et al., 1983).CNE3 came from an UCNT with primitive adenoid structure, then transformed to a poorly differentiated adenocar-cinoma (Liu et al., 2013).C666-1 came from an undifferentiated cell lines (Cheung et al., 1999).The results showed that different Pokemon protein level was expressed in different cell lines.Pokemon was highest in C666-1 and lowest in CNE3, there was no significant difference between well differentiated and poorly differentiated squamous carcinoma.Furthermore, immortalized nasopharyngeal epithelial cell NP69 was used as a normal control because of its non-tumorigenic characteristic (Tsao et al., 2002).As a result, Pokemon protein expression of the four NPC cell lines was higher than NP69 Pokemon protein.Selecting UCNT and chronic rhinitis biopsy specimens separately as the experimental group and control group, we found that Pokemon protein of NPC tissues was obviously higher than Pokemon of control group.The result was in conformity with IHC result of NPC tissues, which was highly expressed in 86 NPC tissues (77.9%) and higher than expression in 30 nasopharyngitis tissues (23.3%) (Gao et al., 2009).The different results illustrate that the regulation mechanisms including Pokemon exist possibly some difference through comparing Pokemon mRNA and protein expression levels in the cell lines.Pokemon mRNA expression level of CNE1 was highest, C666-1 result was only higher than CNE3 result, NP69 result was surprisingly higher than C666-1 result.However, Pokemon protein expression level of C666-1 was highest, NP69 result was lowest.It is well known that transcription of Pokemon is repressed in some degree by directly binding competition of Sp1 and p53, and interacted by corepressors such as mSin3A, NCoR, SMRT (Choi et al., 2009).Therefore, NP69 Pokemon protein expression being in the maximal degree illustrates conversely that Pokemon protein probably plays an important role in the carcinogenesis of NPC through repressing tumor suppressor genes and promoting oncogenes.The speculation is consistent with some studies which Pokemon participates in various regulation mechanisms (Lin et al., 2012;Yang et al., 2012;Jin et al., 2013;Zhang et al., 2013;Zhu et al., 2013).
In view of continuous presence of Epstein-Barr Virus (EBV) in C666-1 (Cheung et al., 1999;Liu et al., 2013), the EBV oncogenes probably participate in the regulation mechanisms of Pokemon, even promote Pokemon protein expression and in coordination with elevating tumorigenicity of EBV products, which play an pathogenic role in the development of NPC (Yip et al., 2010;Yoshizaki et al., 2013).According to WHO classification system standard (2005), the pathological type of C666-1 is UCNT.Because of close relationship between UCNT and EBV (Klein et al., 1974;Andersson-Anvret et al., 1979;Wei et al., 1996;Han et al., 2012), the study data will be useful for clinical diagnosis and therapy of UCNT if C666-1 is used for further study.
In a summary, the study confirms further the association between Pokemon and NPC.Pokemon probably will become a newly molecular target therapy of UCNT.Basing on the study, we are observing the changes of biological characteristics of the NPC cell lines after interrupting Pokemon mRNA expression of by short hair RNA (shRNA) (data not shown).

(CNE1
Figure 3. Real-time PCR and Western-blot of the Cell Lines.(A) The data of NP69 was used as a control, delta-delta Ct calculated mRNA expression level of Pokemon in other cell lines.(B) The bands represented the proteins expression level of Pokemon and β-actin of the cell lines.(C) The bar charts represented ratios of Pokemon and β-actin protein.All data represented the mean of three independent experiments ± SD.Statistical significance was tested using Independent-sample t test with *P<0.05 and **P<0.01

Figure 4 .
Figure 4. Western-blot of Chronic Rhinitis and UCNT.(A) The bands represented the proteins expression level of Pokemon and β-actin of chronic rhinitis and UCNT.(B) The data of chronic rhinitis T9 was used as a control.The bar charts represented ratios of Pokemon and β-actin protein.All data represented the mean of three independent experiments ± SD.Statistical significance was tested using Independent-sample t test with *P<0.05 and **P<0.01