Functional Analysis of Nrf1 Gene inNasopharyngeal Carcinoma

• Main Authors: Ting-Ying Chen, 陳亭螢
• Other Authors: Chin-Tarng Lin
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/efz7w2

碩士 === 國立臺灣大學 === 病理學研究所 === 106 === Nasopharyngeal carcinoma ( NPC ) is arising from nasopharyngeal epithelium. Endemic regions of NPC are shown in southern China, southeast Asia, northern Africa and Taiwan. The specific etiology is not clear now, but some evidences show that NPC is related to environmental factors, heredity, and Epstein-Barr virus infection . The purpose of this research was to find out the genes associated with NPC pathogenesis. Previously we compared the mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells by cDNA microarray analysis, and found that 11 genes showed significantly increased or decreased expression. Then we used quantitative RT-PCR for further confirmation, and found that FGFR1 gene and SPARK gene were significantly decreased in NPC cell lines. In addition, we also found that the transcription factor NRF1 ( Nuclear respiratory factor 1 ) could regulate the FGFR1 gene and SPARK gene expression. When we compared NRF1 gene expression between NPC cell lines and normal nasal mucosal epithelial cells by quantitative RT-PCR, Western blotting and immunocytochemistry, we found that NRF1 gene was significantly increased expression in NPC cell lines. In order to understand the role of NRF1 expression in nasopharyngeal carcinoma, we used CRISPR/Cas9 to knockout NRF1 gene in NPC TW01 cell line which was established from our lab. After knockout of NRF1, we used Western blotting to confirm the NRF1 expression in NPC cells, and found that the NRF1 protein was significantly decreased. Then we produced 38 single stable cell lines, and chose the best gene knockout clone for the following experiments. We found that NRF1 knockout could decrease cell proliferation, migration, and invasion activities of NPC cells. When we added hydrogen peroxide into medium to increase oxidative stress, we found that NPC cells which expressed lower NRF1 had worse viability. As for metabolism, we found that both of mitochondrial respiration ability and glycolysis ability were downregulated in knockout of NRF1 cells. The in vivo animal experiment by injection of NRF1 knockout cells also revealed that the tumors growth was decreased, which confirmed the results of in vitro assay. According to in vitro assay and xenograft experiment, we suggest that NRF1 plays a role as an oncogene in nasopharyngeal carcinoma pathogenesis.