Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer, affecting ~500,000 individuals worldwide annually. of and mRNA were statistically correlated with a decrease in disease-free survival (DFS) (log-rank test, P 0.001). The results of the multivariate logistic regression analysis revealed that high expression levels of the and gene pair were associated with a high odds ratio for recurrence of 14.62 (95% confidence interval: 2.77C77.26; P=0.002). Therefore, the upregulation of and mRNA may play a critical role in the progression of HNSCC and provide useful information as a prognostic predictor for HNSCC patients. and gene is localized to 6p11C12 and mRNA is present in a number of tissues, including the heart, stomach, kidney, skeletal order GW3965 HCl muscle and placenta, whereas type XXI collagen is an extracellular matrix component of blood vessel walls (7). The gene encodes the chain of collagen XXI, which is a member of the fibril-associated collagens with interrupted triple helices (FACIT) collagen family. Similar to other members of the FACIT order GW3965 HCl collagen family, collagen XXI, which localizes to tissues containing type I collagen, may play a role in maintaining the integrity of the extracellular matrix (8). The gene positioned on human chromosome 8q24.2 encodes a collagen that structurally belongs to the FACIT protein family. Collagen XXII is a novel gene product, which is a specific extracellular matrix protein present only at the tissue junctions of muscles, tendons, the heart, articular cartilage and skin. Collagen XXII is deposited in the basement membrane zone of the myotendinous junction (9). and tumor progression was order GW3965 HCl previously described for several types of cancer (10,11). and in HNSCC tissues (typical SCC specimens) and normal mucosal tissues from the same individuals, in order to determine the correlation between their expression and disease progression. Materials and methods Tumor specimens and patients Patients diagnosed with HNSCC (n=70) who were treated at the Department of Otolaryngology/Head and Neck Surgery, Hamamatsu University School of Medicine (Shizuoka, China), were included in this study. HNSCC tumor specimens were obtained from the 70 patients during surgery. Clinical information, including age, gender, tumor site, smoking status, alcohol consumption, tumor size, lymph node status and tumor stage were obtained from the clinical records. The mean patient age was 65.0 years (range, 37C85 years) and the male:female ratio was 55:15. The primary tumors were located in the oral cavity (n=24), pharynx (n=19), larynx (n=15) and paranasal sinuses (n=12). Matched order GW3965 HCl pairs of head and neck tumors and adjacent normal mucosal tissues were obtained from the surgical specimens of 44 patients for initial expression screening. All patients provided written informed consent under a protocol approved by the Institutional Review Board of the Hamamatsu University School of Medicine. RNA extraction and quantitative polymerase chain reaction (qPCR) Frozen tissue specimens were stored at ?80C until RNA extraction. Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Rabbit Polyclonal to CNNM2 Germany) and treated with RNase-Free DNase (Qiagen). cDNA was generated from DNase-treated total RNA using random primers (Invitrogen Life Technologies, Carlsbad, CA, USA) with Superscript II reverse transcriptase (Invitrogen Life Technologies). The primer sequences and PCR conditions are provided in Table I. All the qPCR reactions were performed with the Thermal Cycler Dice? Real Time Program TP800 (Takara, Tokyo, Japan). For every PCR evaluation, 2 l of diluted cDNA, 12.5 l of SYBR? Premix Former mate Taq? Perfect REAL-TIME (Takara) and 0.5 l from the primers had been put into a final level of 25 l. The thermal cycler circumstances had been the following: a short denaturation stage at 95C for 10 sec, accompanied by 45 cycles of denaturation at 95C for 5 sec and annealing/expansion at 60C for 30 sec (two-step response). Evaluation was performed with Thermal Cycler Dice REAL-TIME System TP800 software program, edition 1.03A (Takara) based on the producers instructions. For evaluations between examples, the mRNA manifestation of the prospective genes was normalized to mRNA manifestation. Desk I Primers for quantitative polymerase string reaction..