The effect of NMRAL1 protein on HTATIP2 and investigation as A biomarker in head and neck cancer

Head and neck squamous cell carcinoma is the most common head and neck cancer type. Cancer cells need NADPH to reduce increased oxidative stress and preserve redox balance. NADPH performs by interacting with proteins such as Htatip2 and Nmral1. Purpose of the study is to determine altering expression level and cellular location of Htatip2 in absence of Nmral1. Furthermore, to explore the expression levels of c-MYC and cyclin D1, which have been shown to interact with Htatip2, in absence of Nmral1. HTATIP2, c-MYC and CCND1 gene expression levels were examined by silencing the NMRAL1 gene with siRNA in cancerous and normal cell lines. The cellular localization of Nmral1 and Htatip2 was determined by immunocytochemistry assay. Also, mRNA and protein expressions in tumor and normal tissues of 50 patients with larynx cancer were examined. The NMRAL1 gene was silenced 62% in head and neck normal cell line and %74 in cancer cell line. In normal cell line, HTATIP2 mRNA expression was decreased while c-MYC and CCND1 did not change after silencing. After NMRAL1 silencing, the amount and location of Htatip2 protein did not change in the cancer cell line but it was concentrated in the cytoplasm in the normal cell line. Low expression of NMRAL1 was decreased cell viability by approximately 60% in both cell lines. NMRAL1 (p=0.001) and c-MYC (p<0.001) mRNA expression was decreased in laryngeal tumor tissues. c-MYC and CCND1 expression was decreased in 85.2% and 65.9% of patients with increased HTATIP2 expression, respectively. c-MYC expression was also decreased in 85.7% of patients with decreased NMRAL1 mRNA expression. As a result, absence of the NMRAL1 gene in normal head and neck cell line, the Htatip2 protein is more concentrated in the cytoplasm. This suggests that cytoplasmic functions of Htatip2 might be increased. HTATIP2 has a suppressive effect on c-MYC and CCND1 gene expression in laryngeal cancer. NMRAL1 gene has a positive correlation with c-MYC but contrarily with CCND1. Further studies are needed to determine this predicted association. Key Words: Head and neck cancer, NMRAL1, HTATIP2, siRNA, Western blot