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Correlation of nm 23 expression with pharyngeal cancer and patient prognoses

The present study investigated the expression of non-metastasis 23 (nm 23) in laryngeal cancer tissues and analyzed its correlation with the prognoses of laryngeal cancer patients. A total of 122 laryngeal cancer patients who were admitted to The Affiliated Hospital of Jining Medical College from June 2009 to June 2012, and 30 normal laryngeal mucosal tissues were selected as the control group. Immunohistochemical staining method was used to test the expression of nm 23 in tissues. Quantitative-polymerase chain reaction (q-PCR) and western blotting were conducted to test the expression of nm 23 in tissues at the gene and protein levels, respectively. Moreover, the prognoses of patients were analyzed. The positive expression rate (90.00%) of nm 23 in the normal laryngeal mucosal tissues was markedly higher than that in laryngeal cancer tissues (56.56%) (p<0.05). The expression of nm 23 proteins was correlated with the clinical staging of laryngeal cancer and the metastasis of lymph nodes (p<0.05). The expression of both nm 23 genes and proteins in the laryngeal cancer tissues were significantly lower than those in the normal laryngeal mucosal tissues (p<0.05). The survival rate of the positive nm 23 expression was substantially higher than that of the negative expression with a statistically significant difference (p<0.01). In conclusion, the expression of nm 23 proteins plays an important role in the development and metastasis of laryngeal cancer and may be taken as one of the indicators to evaluate the prognoses of such patients.


Introduction
Pharyngeal cancer, the most prevalent malignant tumor in head and neck, tends to attack laryngeal mucous membranes (1). Currently, the pathogenesis of pharyngeal cancer is yet to be identified. In the 1990s, scholars initially segregated and identified non-metastasis 23 (nm 23) and later it was verified that the expression of nm 23 is closely correlated with the metastasis suppression in patients (2). Studies have shown that the expression product of nm 23 can directly affect the signal transduction system in cells and furthermore, have effects on the microfilaments and microtubules involved in biological activities of cells. In addition, it inhibits metastasis of tumor cells (3,4). To date, although many scholars have reported the mechanism of nm 23 expression to inhibit metastasis of tumor cells, the study on the relation between nm 23 and prognoses of pharyngeal cancer patients remain controversial, and no agreement exists in the academic circle. In the present study, pharyngeal cancer patients diagnosed and treated from 2009 to 2012 were retrospectively analyzed. Immunohistochemical staining, quantitative-polymerase chain reaction (q-PCR) and western blotting were used to analyze the expression of nm 23 in different tissues. Besides, such patients were followed up for 5 years. Main reagents. Rabbit anti-human nm 23 (Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China), goat anti-rabbit secondary antibody and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal reference antibody (Becton Dickinson; BD Biosciences, San Jose, CA, USA), diaminobenzidine (DAB) developer and citrate buffer powder (Xinyu Biotech, Shanghai, China), immunohistochemistry kit (DASF Bio, Nanjing, China), reverse transcription (RT) kit (Boyi Biotech, Shanghai, China), real-time quantitative fluorescent PCR kit (Vipotion Biotechnology Co., Ltd., Guangzhou, China), TRIGene reagent (GenStar BioSolutions Co., Ltd., Beijing, China), RT kit (Takera, Dalian, China), total protein extraction kit (KeyGen Biotech Co., Ltd., Nanjing, China) and bicinchoninic acid (BCA) protein assay kit (Nanjing Senbeijia Biotechnology Co., Ltd., Nanjing, China).

Methods
Immunohistochemical staining. Paraffin-embedded sections of two kinds of tissues were obtained, and after deparaffinating and hydration, phosphate-buffered saline (PBS) was used to wash the sections. In order to weaken the non-specific background staining caused by endogenous peroxidase, the foregoing sections were added with the blocking solution to block for 20 min and then, sealed by 10% serum for 10 min at room temperature. Afterwards, the primary antibody solution (diluted by 1:50), containing matrix metallopeptidase-9 monoclonal antibody (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1), was added for the overnight incubation at 4˚C. Again, PBS was used to wash the resulting product and then, the secondary antibody solution (diluted by 1:50) was added for incubation at room temperature. The incubating products were washed using PBS 30 min later. After that, the sections were added with streptomycin avidin-peroxidase (SP) solution for 0.5 h of incubation at room temperature. After incubation, the foregoing sections were washed by PBS. Then, the sections were added with DAB developer for color development, washed by distilled water, counterstained and sealed for observation.
Expression of nm 23 messenger ribose nucleic acid (mRNA) determined via q-PCR. The primers were synthesized by Huamei Ruikang (Beijing) International Biotech Research Institute with the sequences shown in Table I. The total RNA in tissues was extracted according to the instructions of the total RNA extraction kit. The complementary deoxyribose nucleic acid (cDNA) was synthesized using reverse transcription kit.
PCR system (25 µl) contained: 12.5 µl SYBR Premix Ex Taq™ II, 1 µl forward primer, 1 µl reverse primer, 2 µl cDNA and 8.5 µl dH 2 O. Reaction condition: Pre-degeneration for 3 min at 94˚C, degeneration for 20 sec at 94˚C, annealing for 20 sec at 58˚C, and extension for 30 sec at 72˚C, 40 cycles in total. GAPDH was taken as the internal reference. Real-time PCR instrument automatically calculated and displayed the relative mRNA expression level of nm 23.
Expression of nm 23 proteins tested by western blotting. The total protein in the tissues was extracted according to the instructions of the total protein extraction kit, and BCA protein assay was performed to determine the concentration of the extracted proteins. Then, they were stored at -70˚C for standby use. Gels at different concentrations were prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-page). The corresponding positions of two proteins in gels were roughly confirmed based on markers and then, the proteins were transferred onto membranes for 35 min and sealed by 5% skim milk powder at 37˚C. The proteins were added with the rabbit anti-human nm 23 and GAPDH polyclonal antibodies (diluted by 1:1,000; cat. no. 3345; Cell Signaling Technology, Danvers, MA, USA) 90 min later and incubated at 4˚C overnight. The next day, the incubating products were added with Tris-buffered saline with Tween-20 (TBST) and placed on a shaking incubator to shake and wash 3 times (15 min each time). After that, the goat anti-rabbit secondary polyclonal antibody solution (diluted by 1:1,000; cat. no. 7074; Cell Signaling Technology) was added for 1 h of incubation at 37˚C. When the incubation ended, TBST solution was added, and the sequent operations were performed as above. Afterwards, in a dark room, the enhanced chemiluminescent (ECL) fluid (Millipore Sigma, Burlington, MA, USA) was added to develop color, followed by exposure, development and fixation. Finally, ChemiDoc™ MP Imaging System was used to scan, and the professional image processing program of Image J was adopted to analyze images and record the absorbance value, with β-actin as the internal reference.
Statistical analysis. Statistical Product and Service Solutions (SPSS) 17.0, a professional statistics software (Beijing Xinmei Jiahong Technology Co., Ltd., Beijing, China), was used to analyze the data of the present study. The measurement data were expressed as mean ± standard deviation, and the one-way analysis of variance was used for inter-group comparisons and the post hoc test was Least Significant Difference test. GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was adopted for the survival analysis. α=0.05 was taken as the statistical verification standard.

Results
Immumohistochemical staining. The immunohistochemical staining results showed that the positive expression rate of nm 23 in pharyngeal cancer tissues was 56.56% (69/122), while that in the normal laryngeal mucosal tissues was 90.00% (27/30). Differences between the two groups were statistically significant (p<0.05) ( Fig. 1 and Table II).

Relationship between nm 23 expression and clinical features of pharyngeal cancer.
The comparison of the nm 23 expression in pharyngeal cancer tissues with different pathological grades showed no obvious difference, without statistical significance (p>0.05). In the the clinical staging, the expression rate (86.96%) in stage I-II cases was significantly higher than that (48.48%) in stage III-IV cases, with a statistically significant difference (p<0.05). Similarly, that (75.00%) in cases without lymphatic metastasis was also obviously higher than that (37.78%) in cases with lymphatic metastasis, and the difference presented the statistical significance (p<0.05) (Table III).
q-PCR results. The relative expression level of nm 23 mRNA in tissues was tested via q-PCR, and the results showed that the expression level of nm 23 mRNA in the normal pharyngeal mucosal tissues was significantly higher than that in Table II   the pharyngeal cancer tissues, with a statistically significant difference (p<0.05) (Fig. 2).
Western blotting. Western blotting was conducted to detect the expression of nm 23 proteins. According to the result, the nm 23 proteins were significantly highly expressed in the normal pharyngeal mucosal tissues, and the difference from that in the pharyngeal cancer tissues was statistically significant (p<0.05) (Figs. 3 and 4).
Analysis of prognosis. The survival rate of patients with nm 23 protein positively expressed was higher than that of patients with the nm 23 protein negatively expressed. Especially in terms of 3-year survival rate, the survival rate of patients with the negative nm 23 protein expression was decreased to 12.96%, while that of patients with the positive expression reached up to 82.35% and began slowly decreasing. The difference between the two groups of patients presented a markedly statistical significance (p<0.01) (Fig. 5).

Discussion
Since nm 23 was discovered in the 1990s, scholars all over the world have made a large number of systematic studies on the effects of nm 23 on various cancers (5,6). Pharyngeal cancer patients often die of metastasis, and the metastasis of tumor is a very complex process, starting from the primary tumor cells infiltrating into the surrounding cytoplasmic matrix (7,8).
Currently, it is known that non-metastasis genes can participate in several steps of tumor cell metastasis process through various pathways and serve as one of natural defenders against the tumor metastasis in the body (9,10). Pathological studies have shown that the lymphatic metastasis is one of the earliest incidents during metastasis and diffusion of most solid tumors in organic bodies and also one way of tumor metastasis (11,12). As a metabolic suppressor gene, nm 23 has been verified through many in vitro and in vivo experiments. Marioni et al (13) found that nm 23 can significantly suppress the cancerization of melanoma cells, such as inhibiting cell activity, tumor cell growth and distal infiltration and metastasis. Fang et al (14) reported according to the clinical examinations that the expression of nm 23 in the patients is significantly negatively correlated with the invasion and metastasis of tumor cells and has a marked effect on the prognoses of patients. According to the findings of He et al (15), the nm 23 mRNA expression level is significantly decreased in highly metastatic breast cancer tissues, but Han et al (16) studied the expression of nm 23 in thyroid tumors with the same technical method, and the results showed that although nm 23 in coding region is not mutated, there is no obvious negative correlation between the expression level of nm 23 and metastasis in thyroid tumor tissues. Therefore, it was inferred that the effects of nm 23 in different cancer tissues may vary to some degree. However, the effect of the nm 23 expression on pharyngeal cancer tissues remains controversial. It was found by Tong et al (17) that the lowly expressed nm 23 is closely correlated with the metastasis of pharyngeal cancer, while Fu et al (18) reported that the expression of nm 23 protein is not significantly correlated with the growth position and pathological grading of laryngeal cancer, but negatively correlated with lymphatic metastasis and clinical staging. On the contrary, some scholars held that the expression of nm 23    is positively correlated with lymphatic metastasis, but has no relation with clinical staging (19).
The retrospective analysis results of this study showed that the positive expression rate (90.00%) of nm 23 in the normal pharyngeal mucosal tissues was significantly higher than that (56.56%) in the pharyngeal cancer tissues, with a statistically significant difference (p<0.05). The positive expression percentage (86.96%) of nm 23 in stage I-II cases was markedly higher than that (48.48%) in stage III-IV cases, with a statistically significant difference (p<0.05). Similarly, that (75.00%) in cases without lymphatic metastasis was also obviously higher than that (37.78%) in cases with lymphatic metastasis, and there was a statistically significant difference (p<0.05). This indicates that the expression of nm 23 proteins is significantly correlated with clinical staging of pharyngeal cancer and lymphatic metastasis. At the same time, it was indicated that at the gene and protein levels, the expression of nm 23 in the pharyngeal cancer tissues was significantly lower than that in the normal pharyngeal mucosal tissues (p<0.05), which is consistent with the result of the study by Yang et al (20). According to the postoperative 5-year prognosis follow-up results of pharyngeal cancer patients, the survival rate of patients with nm 23 positive expression was significantly higher than that of patients with negative expression, and the difference showed a statistical significance (p<0.01). Especially in terms of three-year survival rate, the survival rate of patients with nm 23 proteins negatively expressed was decreased to 12.96%, while that of patients with nm 23 proteins positively expressed reached up to 82.35%, indicating that the higher the positive expression of nm 23 is, the better the prognoses of pharyngeal cancer patients are.
In conclusion, the expression of nm 23 protein plays an important role in the occurrence, development and metastasis of pharyngeal cancer and may be regarded as one of the indicators to evaluate the prognoses of such patients.