Expression Profiling And Identification Of Genes Associated With Lung Squamous Cell Carcinoma

Lung cancer is the leading cause of cancer death in the world. Non-small-cell lungcancers compose 80％of all lung carcinomas with squamous cell carcinomas (SCC)representing the majority of these tumors. Over the past decades, much has been knownabout the molecular changes in lung cancer; however, the molecular mechanism underlyingthis malignant progression is still poorly understood.To explore the genes related to lung carcinogenesis, six cDNA libraries, consisting ofgenes preferentially up- and downregulated genes in human lung cancer (An Q et al., 2003;Sun W et al., 2004; Liu Y et al., 2007), were previously established in our lab. In this study,to identify those differentially genes among the libraries, which might play important roles inlung carcinogenesis, cDNA microarrays containing 1,642 gene clones identified in thelibraries were fabricated. These cDNA microarrays were then employed for comparison ofexpression profiles of human lung SCC with 27 fresh tumor tissues and their matchedadjacent normal lungs. The analysis revealed 32 upregulated genes and 19 downregulatedgenes in lung SCC tumor tissue, with 92％(29/32) and 79％(15/19) in agreement with thoseresults found in the libraries. Using semi-quantitative reverse transcription-PCR (RT-PCR), 8out of the 32 upregulated genes were substantially examined in the 27 tumor pairs used forcDNA microarray analysis. Differential expression was confirmed in seven of theses eightgenes, including CRKL, SNRPG, IGFBP5, SQLE, RAP2B, CLDN1, and TBL1XR1. Theelevated mRNA expression of RAP2B, CLDN1 and TBL1XR1, three genes located onchromosome 3q, were further validated in 64.3％(18/28), 82.1％(23/28), and 75.0％(21/28)of lung SCC tumor tissues, respectively, by quantitative real-time RT-PCR analysis.Moreover, western blot analysis showed that the protein expression of CLDN1 and TBL1XR1were also upregulated in 85％(17/20) and 53.3％(8/15) of lung SCC tumour samples, as wellas in lung cancer cell lines and in one human immortalized bronchial epithelial cell line.To identify additionally differentially expressed genes in human lung cancer, cDNAmicroarrays containing 15K genes, were further applied to analyze 11 lung SCC tumorsamples, with normal lung samples as a reference. This analysis resulted in the identificationof 380 upregulated genes and 372 downregulated genes in the tumor tissue. Gene Ontologyanalysis was subsequently performed on these differentially expressed genes. The analysisrevealed the predominant overrepresented functional classes related to the development ofcancer, including those involved in cell proliferation, cell cycle, cell differentiation, cell adhesion, DNA damage and repair, cell signaling, organ development, blood vesseldevelopment, and immune response functions (P＜0.05). Further, nine upregulated genes incell cycle-associated functional class, including BUB1, MAD2L1, KIF11, ORC6L, CDC2,CDKN3, CKS2, MYBL2, and CHEK1, were examined in 24 lung SCC tumor tissues andcorresponding normal tissues, by quantitative real-time RT-PCR. Differential expression ofthese nine genes was in accordance with that found by cDNA microarray analysis. Amongthem, overexpression of ORC6L or CKS2 was associated with lymph node metastasis of lungSCC, and overexpression of BUB1 or ORC6L with tobacco smoking.Furthermore, Agilent Human Oligonucleotide Microarrays (22K genes/microarray) wereused to delineate the expression profiles of 30 lung squamous cell carcinomas, with theircorresponding normal lungs as the controls. Preliminary data revealed 129 genes showingsignificant differences in the lung SCC tumor groups with and without lymph node metastasisNotably, among these genes, an index based on the top 44 genes showed clear stratification ofthe lung SCC patients with or without lymph node metastasis.The differentially expressed genes reported in this study will provide a valuableresource for understanding the pathogenesis of lung SCCs and for discovery of noveldiagnostic or therapeutic targets. Further studies with Agilent 22K microarrays will lead tothe discovery of the predictive gene expression signature to identify patients with early-stagehigh-risk lung SCC who might benefit from adjuvant therapy following surgery.