The Relationship Between RIPK4Expression And The Clinical Characteristics And Biological Properties Of TSCC

ObjectiveOral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. OSCC normally occurs in bucca, gingiva, palate and maxillary sinus, especially in tongue. Tongue squamous cell carcinoma (TSCC) has poorer5-year survival rates (48.0%-70.9%) and the lower rate of local tumor control.However, the mechanisms involved in the tumorigenesis of TSCC have not been well characterized yet.The RIPK4(receptor-interacting serine/threonine kinase protein4) was first described as an unidentified protein found to interact with protein kinase C (PKC)(31Pectin yeast two-hybrid assays. Due to the abnormal epidermal differentiation, REPK4deficiency in mice results in prenatal lethality. The epidermis of RIPK4-/-mice is thickened with expanded spinous and granular layers, and contains an outermost layer of parakeratotic cells instead of enucleated squamous cells. RIPK4autonomously functions in a cell within the keratinocyte lineage. RIPK4is an ankyrin repeat-containing kinase essential for keratinocyte differentiation and cuaneous inflammation. The goal of the present study was to assess the relationship between RIPK4expression and the clinical characters and biological properties of TSCC.MethodsFor this study we obtained109samples of which36samples from paracancer normal tissue and73samples of surgically excised TSCC from patients who were treated at the First Affiliated Hospital of Zhejiang University during January2006to December2012. Of these,32patients had highly differentiated tumors (according to WHO),23had moderately differentiated tumors, and18had poorly differentiated tumors. Among these patients20had lymph node metastasis. None of these patients underwent radiotherapy, chemotherapy, or any other interventional palliative or therapeutic measure prior to operation, and all with the local ethics committee approval. All specimens were obtained from the central region of the tumor and pulverized in liquid nitrogen in a cold mortar, or fixed in10%formalin and embedded in paraffin. Clinical characteristics of the patients were recorded, including age, gender, education background, alcohol consumption, smoking habit, diabetes, hypertension, and lymph node metastasis. Multivariate logistic regression analysis was used to investigate the association between RIPK4and clinical characteristics mentioned above. After the human tongue squamous cell carcinoma cell lines TCA-8113transfected with pRIPK4or pRIPK4-shRNA plasmid, mRNA expression levels were detected through fluorescence quantitative PCR.Western Blot was used to detect RIPK4protein expression. Scratch wound healing assay and invasion assay were used to find out if changes of RIPK4expression were related to cell migration and invasion. ResultsIt was showed that nomal RIPK4expression level were found both in paracancerous normal tissue and normal tongue epithelial cells. RIPK4expression level in well differentiated tumor was high, while the expression level of RIPK4decreased obviously in low differentiation cancer. The average score of RIPK4in well differentiated tongue squamous cell carcinoma was2,1.2in the middle differentiation tongue squamous cell carcinoma, and0.54in the poorly differentiated tongue squamous cell carcinoma. The RIPK4mRNA levels in paracancerous normal tissue were higher than any degree of TSCC. This indicated that there was a significant correlation between the RIPK4levels and the TSCC degree of differentiation. RIPK4levels increased with age (r=0.66,P=0.04), from a mean of1.13in patients aged31to55years old,to1.65in patients aged56-69years old (r=0.03, P<0.01). RIPK4level had a significant association with gender. Men had higher RIPK4levels (P=0.03, chi square test). The education background, smoking, drinking, diabetes, hypertension and lymph node metastasis had no impact on RIPK4levels. We transfected pRIPK4, pNC-shRNA, or pRIPK4-shRNA to TCA-8113cells. The transfection rates of the four different groups were all above60%, and showed no statistical significance. The RIPK4mRNA expression, and the migration and invasion of TCA-8113were measured and found that down-regulation of RIPK4could significantly increase its ability of migration and invasion. These results indicated that RIPK4played an important role in migration and invasion in tongue squamous cell carcinoma cell lines. Confocal laser scanning image displayed RIPK4expression level of TCA8113cells and human tongue squamous carcinoma cells were found in the cytoplasm which decreased obviously contrasted with the expression of RIPK4in NOK cells and normal tongue tissue. Conclusionsl.The lower the level of tongue squamous cell carcinoma is, the lower the RIPK4level is.2.RIPK4is positively related with age and men have a higher expression lever3.The dicreasing of RIPK4can strengthen the migration and invasion ability of the tongue squamous carcinoma epithelial cell TCA-8113.