The present study includes 42 patients with a mean age of 59.45 ± 8.36 years (range 47–78 years) who were histologically diagnosed with hypopharyngeal carcinoma, and 8 of vocal cord polyp patients, with a mean age of 55.68 ± 8.27 years (range, 48–75 years), that served as age and gender-matched controls. The staging of the tumor was determined in accordance with the American Joint Committee on Cancer (AJCC), Tumor-Node Metastasis (TNM) classification. None of the patients had received any chemotherapy, radiation therapy or immunotherapy within 2 months prior to surgery. Patients with any other chronic diseases such as tuberculosis, diabetes, autoimmune diseases or other malignant tumors were excluded. Cancer tissues and pericancerous tissues were identified by stereoscopy and quick frozen sectioning. Two pieces of tissues were collected and snap-frozen for RNA extraction and protein preparation. Similarly, normal controls who had suffered fever or viral infection in the past 1 week, pregnancy, or recent accident were also excluded from the study. The protocol was approved by the Ethics Committee of Institute of Basic Medicine, Shandong Academy of Medical Sciences, and all participants signed informed consent. Representative samples of tumors and of normal control tissues were collected at surgery, snap-frozen immediately in Eppendorf tubes and stored at −80 °C to avoid RNA and protein degradation until sectioning for RT-PCR and western blot analysis.

The total RNA extraction kit was purchased from Transgen Biotech Company (Beijing, China). M-MLV reverse transcriptase and Taq DNA polymerase were purchased from Promega Corporation (Madison, WI, USA). PCR primers for the detection of MMP-2, MMP-9 and β actin mRNA were designed using the OLIGO Primer Analysis Software, version 5.0 (NBA, Software and Research Services for Tomorrow's Discoveries, National Biosciences, Plymouth, MN, USA). The PCR oligomers were synthesized by a DNA/RNA synthesizer (Applied Biosystems) at BioSune biological technology corporation, Shanghai, China. Primer sequences are listed in Table 1. Rabbit anti-human MMP-2 monoclonal antibody and rabbit anti-human MMP-9 polyclonal antibody were purchased from Abcam Company (Cambridge, UK). Rabbit anti-actin polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase-labeled goat anti-rabbit IgG were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).

The RT-PCR method was described briefly. RNA was extracted from tissues using the guanidine thiocyanate phenol-chloroform method. The quality of the RNA yield was assessed by electrophoresis on a 1.5% agarose gel in 0.5 moL Tris/Borate/EDTA buffer. The optical density of the RNA samples was measured and samples exhibiting an A260‒A280 ratio of 1.8–2.0 were used to obtain cDNA. RT-PCR was performed using a RNA PCR kit (Perkin-Elmer, Norwalk, CT, USA). The Relative Intensity (RI) of each band was determined according to the following equation: RI = the sum density of target gene/the sum density of β-actin. To exclude the possibility of contamination, reactions containing RT-PCR reagents including cytokine PCR primers without sample RNA were used as the negative control groups.

SDS-PAGE and immunoblotting were performed according to standard techniques. Briefly, the prepared tissues (100 mg) were lysed at 4 °c for 30 min in lysis buffer (Beijing Leagene Biotech. Co, Ltd, Beijing, China). The lysates were centrifuged at 15,000 rpm for 20 min at 4 °c to remove nuclei and undisrupted tissues. Protein concentration was determined using Bio-Rad protein assay solution (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as the standard.13 The protein samples were boiled for 10 min and loaded onto a 10% SDS-PAGE gel followed by electrophoresis for 2 h. The proteins were electrophoretically transferred onto a 0.22 μm nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% skim milk at room temperature for 1 h, and immunoblotted with monoclonal rabbit anti-human MMP-2, polyclonal rabbit anti-human MMP-9 and β-actin primary antibodies. After the membrane was washed three times at 5 min intervals in PBS-T, the membrane was subsequently incubated with goat anti-rabbit IgG-HRP diluted to 1:2000 for 1 h at room temperature. After the membrane was washed three times at 5 min intervals in PBS-T, and the immunoblots were then visualized using a LAS4000 Chemiluminescence Imager (Fijifilm, Tokyo, Japan) with associated software. For presentation, immunoblots were opened in PhotoShop CS2 (Adobe Systems, Mountain View, CA, USA).

To determine the levels of MMP-2 and MMP-9 in hypopharyngeal carcinoma, data analysis was performed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Data were presented as the mean ± standard deviation. The paired samples t-test was used to compare differences between hypopharyngeal carcinoma and pericarcinoma tissues. One-way analysis of variance was used to compare the differences between groups at different clinical stages, lymph node metastasis and tumor pathological stage. p < 0.05 was considered to indicate a statistically significant difference.

Table 2 shows the descriptive characteristics of the study subjects which include 42 hypopharyngeal carcinoma patients with an average age of 59.45 ± 8.36 years (range 47–78 years), and 8 vocal cord polyp patients with an average age of 55.68 ± 8.27 years (range 48–75 years). Out of total 42 hypopharyngeal carcinoma patients, 38 (90.48%) were male and only 4 (9.52%) were female, a male to female ratio up to 9.5. In accordance with TNM classification, the majority of patients presented with large tumors (T3 + T4; 64.28%) and lymph node involvement (N+; 66.67%). The majority of patients exhibited advanced stage disease (Stage III + IV, 80.95%), while 19.05% exhibited early stage cancer (Stage I + II). All the patients were pathologically diagnosed with squamous cell carcinoma (Fig. 1). Histologically, 71.43% of patients exhibited poorly- or moderately-differentiated tumors.

Figure 1.

Pathological results from hypopharyngeal carcinoma patients (H&E, the scale bar is set at 400 μm). A, Pericarcinoma tissue; B, Carcinoma tissue.

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Total RNA of 84 fresh tissues obtained from hypopharyngeal carcinoma patients and 8 vocal cord polyp tissues were prepared. The mRNA expression profiles from the aforementioned tissues were analyzed by RT-PCR. To exclude the possibility of carry-over contamination, reactions containing all RT-PCR reagents including cytokine PCR primers without sample RNA were used as negative controls. No contamination was detected. The mRNA expressions of MMP-2 and MMP-9 in tissues were analyzed. The expressing capacity (measured as Relative Intensity [RI] to the ratio of β-actin) of patients for MMP-2 and MMP-9 was higher in hypopharygeal carcinoma tissues than in pericarcinoma tissues. There was no MMP-2 and MMP-9 mRNA expression in polyp of vocal cord tissues (Fig. 2). In all 42 patients, 37 cancer tissues (88.1%) and 34 pericarcinoma tissues (81.0%) expressed MMP-2 mRNA (RI 0.509 ± 0.319 and 0.353 ± 0.268, respectively; t = 2.529, p = 0.015); and 35 cancer tissues (83.3%) and 30 pericarcinoma tissues (71.4%) expressed MMP-9 mRNA (RI 0.461 ± 0.249, and 0.218 ± 0.208, respectively; t = 4.781, p < 0.001). Cancer tissues exhibited a higher level mRNA expression of MMP-2 and MMP-9 than pericarcinoma tissues, and this difference was statistically significant.

Figure 2.

The mRNA expression of MMP-2 and MMP-9 in tumor tissues, pericarcinoma tissues and polypoid control tissues obtained from hypopharyngeal carcinoma patients and polyp of vocal cords patients. (A) Representative mRNA expression of MMP-2 and MMP-9 in tumor tissues and polypoid control tissues. Cancer tissues exhibited stronger mRNA expression of MMP-2 and MMP-9, but there was almost no expression in polypoid control tissues (lane 1, 1 kb DNA ladder; lane 2, carcinoma tissue; lane 3, polypoid control tissue). (B) Representative mRNA expression of MMP-2 and MMP-9 in tumor tissues and pericarcinoma tissues. Cancer tissues exhibited a higher level mRNA expression of MMP-2 and MMP-9 than pericarcinoma tissues (lane 1, 1 kb DNA ladder; lane 2, carcinoma tissue; lane 3, pericarcinoma tissue). (C) Relative intensity of MMP-2 and MMP-9 mRNA expression in carcinoma tissues and pericarcinoma tissues. Carcinoma tissues exhibited higher levels of MMP-2 and MMP-9 mRNA expression than pericarcinoma tissues. (*p < 0.05 and **p < 0.01).

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MMP-2 and MMP-9 mRNA expressions were enhanced with the increase of the tumor clinical Stages (MMP-2: F = 4.003, p = 0.026, MMP-9: F = 5.501, p = 0.008). Especially in Stage Ⅳ was obviously higher than that in Ⅰ+Ⅱ (Fig. 3). Their expression intensity were associated with the metastasis of lymph nodes (N staging) and increased with the degree of lymphatic metastasis (MMP-2: F = 4.584, p = 0.016; MMP-9: F = 4.643, p = 0.006) (Fig. 4). The expression of MMP-2 and MMP-9 in the cancer tissues is enhanced with the increase of tumor pathological stage (MMP-2: F = 3.884, p = 0.029, MMP-9: F = 3.783, p = 0.032) (Fig. 5).

Figure 3.

The mRNA expression of MMP-2 and MMP-9 in tumor tissues of different clinical stages obtained from hypopharyngeal carcinoma patients. (A) Representative mRNA expression of MMP-2 and MMP-9 in tumor tissues of different clinical stages. Expression of MMP-2 and MMP-9 was elevated with increasing clinical stage (lane 1, 1 kb DNA ladder; lane 2, Stage I + II patient; lane 3, Stage III patient; lane 4, stage IV patient). (B) Relative intensity of MMP-2 and MMP-9 mRNA expression in tumor tissues of different clinical stages. Advanced stage patients exhibited higher levels of MMP-2 and MMP-9 mRNA expression than early stage patients (*p < 0.05 and **p < 0.01).

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Figure 4.

The mRNA expression of MMP-2 and MMP-9 in tumor tissues of different N stages obtained from hypopharyngeal carcinoma patients. (A) Representative mRNA expression of MMP-2 and MMP-9 in tumor tissues of different N stages. Expression of MMP-2 and MMP-9 was enhanced with increasing N stage (lane 1, 1 kb DNA ladder; lane 2, stage N0 patient; lane 3, stage N1 patient; lane 4, stage N2 patient); (B) Relative intensity of MMP-2 and MMP-9 mRNA expression in tumor tissues of different N stages. Lymphatic metastasis patients exhibited higher levels of MMP-2 and MMP-9 mRNA expression than stage N0 patients (*p < 0.05 and **p < 0.01).

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Figure 5.

The mRNA expression of MMP-2 and MMP-9 in tumor tissues of different pathological stages obtained from hypopharyngeal carcinoma patients. (A) Representative mRNA expression of MMP-2 and MMP-9 in tumor tissues of different pathological stages. Expression of MMP-2 and MMP-9 was declined with the degree of differentiation becomeing better (lane 1, 1 kb DNA ladder; lane 2, well differentiated patient; lane 3, moderately differentiated patient; lane 4, poorly differentiated patient); (B) Relative intensity of MMP-2 and MMP-9 mRNA expression in tumor tissues of different pathological stages. Poorly differentiated patients exhibited higher levels of MMP-2 and MMP-9 mRNA expression than well differentiated patients (*p < 0.05 and **p < 0.01).

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The protein expressions of MMP-2 and MMP-9 were analyzed by western blot analysis. The results showed that the protein expression levels of MMP-2 and MMP-9 was consistent with the mRNA expression in hypopharyngeal carcinoma and pericarcinoma tissues. The protein expression of MMP-2 and MMP-9 in cancer tissues was higher than that in pericarcinoma tissues. There was no protein expression in polyp of vocal cord tissues (control tissues). The protein expression level of MMP-2 and MMP-9 was significantly enhanced with the increase of the tumor clinical stages and the metastasis of lymph nodes (N staging) (Fig. 6).

Figure 6.

Protein expressions of MMP-2 and MMP-9 in tumor tissues and pericarcinoma tissues obtained from hypopharyngeal carcinoma patients. (A) Representative protein expression of MMP-2 and MMP-9 in tumor tissues and polypoid control tissues. Cancer tissues exhibited stronger protein expression of MMP-2 and MMP-9, but there was almost no expression in polypoid control tissues (lane 1, tumor tissue; lane 2, polypoid control tissue). (B) Representative protein expression of MMP-2 and MMP-9 in tumor tissues and pericarcinoma tissues. MMP-2 and MMP-9 protein expression were higher in tumor tissues than that in percarcinoma tissues (lane 1, tumor tissue; lane 2, pericarcinoma tissue). (C) Representative protein expression of MMP-2 nd MMP-9 in tumor tissues of different clinical stages. Expression of MMP-2 and MMP-9 protein was elevated with increasing clinical stage (lane 1, Stage I + II patient; lane 2, Stage III patient; lane 3, Stage IV patient). (D) Representative protein expression of MMP-2 and MMP-9 in tumor tissues of different N stages. Expression of MMP-2 and MMP-9 protein was enhanced with increasing N stage (lane 1, stage N0 patient; lane 2, stage N1 patient; lane 3, stage N2 patient).

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