Utilizing RNA interference technique to investigate effect of aryl hydrocarbon receptor on expression of cancer-related genes and cell growth in human lung adenocarcinoma

• Main Authors: Po-Hung, 陳柏宏
• Other Authors: 蔡菁華
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/75590759949182187966

碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 94 === Malignant neoplasms have been the leading cause of deaths in Taiwan since 1982. In 2004, lung cancer is the first and second leading cause of cancer death among females and males in Taiwan, respectively. Epidemiological studies indicated that exposure to environmental carcinogens (such as dioxins ) might be one of etiologies for human lung cancer, and increase the risk of lung cancer. 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is ligand of aryl hydrocarbon receptor (AhR). Liganded AhR up-regulates expression of cytochrome P450 1 (CYP1) enzymes in target cells. Our previous studies indicated that AhR expression was increased in human lung adenocarcinomas (AD) tissues and cell lines. It suggested that AhR might play an important role in the development of lung adenocarcinomas. There are two objectives in the present study: 1) to evaluate the physiologic role of AhR in lung AD cells with the RNAi technique, 2) to study effect of AhR expression on constitutive expression of CYP1A1 and CYP1B1 in lung AD cells. We created a DNA vector containing human HU6 promoter followed by the AhR siRNA sequence. This construct was transfected into lung AD cell lines H1355 and G418 resistant stable clones were selected. The quantitative real-time PCR and Western immuno-blotting analysis showed that AhR mRNA and protein levels in these stable clones were respectively inhibited up to 80% and 70%. We found that AhR expression affected CYP1B1 mRNA and protein expressions, but not CYP1A1. Utilizing MTT assay, anchorage independent colony formation assay and flow cytometry for measuring intracellular ROS, we found that cells with AhR high expression formed more colonies in soft agar and generated more intracellular ROS than those with low AhR expression. Therefore, AhR might promote cancer cell growth. On the other hand, we found that TSP1 and MTS1 (S100A4) expressions were correlated with AhR by Human cancer pathway microarray assay. Utilizing Transcription Factor Protein / DNA array, we found that activity of many transcriptional factors for DNA binding was modified by AhR. For example, estrogen receptor (ER), nuclear factor 1 (NF-1), POU domain, class 2, transcription factor 1 (Oct-1) et al. were correlated with AhR, and progesterone receptor (PR) was inversely correlated with AhR expression. Furthermore, we also found that AhR expression affected some proteins expression. 14-3-3 gamma, Galectin-1 and Hsp27 expressions were inversely correlated with AhR expression, and GRB10 expression was correlated with AhR expression. Taken together, our results suggested that AhR might play an important role in the tumorigenesis.