PD2-2-2: Identification of differentially expressed genes in the course of lung adenocarcinoma development.

Abstract

In 1999, WHO defined atypical adenomatous hyperplasia (AAH) as preinvasive lesion of adenocarcinoma. Recently, we developed two immortalized cell lines of AAH (PL16T) and its counterpart, bronchial epithelium (PL16B) by transfection of SV40 large T antigen (Shimada A et al, Cancer Sci. 96:668-675,2005). Using the two cell lines, differentially expressed genes in AAH cells were screened by cDNA microarray analysis. Total RNA was extracted from PL16T and PL16B, respectively, and examined using cDNA microarray (CodeLink Human Whole Genome Bioarray, GE Health Care, USA) To confirm the differences in expression levels of genes selected by cDNA microarray analysis, quantitative real-time PCR (qRT-PCR) assay were carried out using PL16T and PL16B. Then, the expression differences of the selected genes were further examined using total RNA extracted from 24 small sized adenocarcinomas (less than 2 cm in diameter) by qRT-PCR assay. The 24 small sized adenocarcinomas contained 7 in situ adenocarcinomas (Bronchioloalveolar carcinoma (BAC); type A and B), 14 early invasive adenocarcinomas (mixed adenocarcinoma with BAC component; type C) and 3 poorly differentiated adenocarcinomas. Totally, the expression level of 30298 genes was compared between PL16T and PL16B and fourteen genes were expressed 7 times or more in PL16T than PL16B. Among the 14 genes, we succeeded to compare the expression differences of 7 genes between the two cell lines by qRT-PCR and selected two genes that were expressed 10 times more in PL16T thanPL16B. The two genes are Sushi domain containing 2 (SUSD2) and Neuromedin U (NUM). About the two genes, we examined their expressions using 24 small sized adenocarcinomas by qRT-PCR. SUSD2 was expressed significantly more in situ adenocarcinomas than normal lung tissue (p=0.022), but decreased in early invasive adenocarcinoma (p≤0.01). On the other hand, NMU expression was gradually increased according to the malignant progression from in situ carcinoma to early invasive adenocarcinoma, It was expressed significantly more in early invasive adenocarcinoma than normal lung tissue (p=0.043). Using immortalized AAH and normal cell lines and cDNA microarray, we detected two specific genes (SUSD2 and NMU) that express differentially between early adenocarcinoma and normal tissue. The expression pattern of SUSD2 and NMU in early stage adenocarcinoma was different. SUSD2 expression is thought to be related to carcinogenesis, but NMU expression may be associated with malignant progression.