Elsevier

Lung Cancer

Volume 56, Issue 3, June 2007, Pages 307-317
Lung Cancer

Identification of genes differentially expressed in human primary lung squamous cell carcinoma

https://doi.org/10.1016/j.lungcan.2007.01.016Get rights and content

Summary

To identify differentially expressed genes in lung squamous cell carcinomas (SCCs), the suppression subtractive hybridization method (SSH) was performed comparing six lung tumour tissues and 10 morphologically normal bronchial epithelial tissues. A cDNA library consisting of 220 upregulated genes in tumour tissue was established and named as LSCC (lung squamous cell carcinoma). Of them, six were tested using semi-quantitative reverse transcription-PCR on 27 pairs of tumour tissue and normal lung tissue. Differential expression was confirmed in five of these six genes, including IGFBP5, SQLE, RAP2B, CLDN1, and TBL1XR1. The elevated mRNA expression of RAP2B, CLDN1 and TBL1XR1, three genes located on chromosome 3q, were further validated in 64.3% (18/28), 82.1% (23/28), and 75.0% (21/28) of lung SCC tumour tissues, respectively, by quantitative real-time reverse transcription-PCR analysis. Moreover, western blot analysis showed that the protein expression of TBL1XR1 was also upregulated in 53.3% (8/15) of lung SCC tumour samples, as well as in five lung cancer cell lines and in one human immortalized bronchial epithelial cell line. All the initial characteristics of these genes were first reported in the lung SCCs. The differentially expressed genes reported in this study will provide a valuable resource for understanding the pathogenesis of lung SCCs and for discovery of novel diagnostic or therapeutic targets.

Introduction

Lung cancer is the leading cause of cancer death in the world, estimated to be responsible for 1.1 million deaths each year [1]. Lung carcinomas are classified as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC constitutes 75% of lung cancer cases, and squamous cell carcinoma (SCC) is a large subset of this group. Lung SCC originates from the bronchial epithelium. Increasing evidence suggests that lung cancer is the result of an accumulation of molecular changes, including the activation of oncogenes (i.e., kRas, EGFR and Bcl2), inactivation of tumour suppressor genes (i.e., p53, p16, and Rb), and a high number of recurrent chromosomal aberrations (i.e., amplification of chromosome 3q, deletion of chromosome 3p) [2]. However, the molecular mechanism underlying this malignant progression is still poorly understood.

The identification of differentially expressed genes can better elucidate the disease process. Techniques for identifying differentially expressed genes include representational difference analysis, differential display, serial analysis of gene expression, suppression subtractive hybridization (SSH) and microarray hybridization. SSH is a PCR-based cDNA subtraction technique that allows selective amplification of target cDNA while simultaneously suppressing non-target cDNA amplification [3]. SSH has been successfully applied to a wide variety of malignant diseases [4], [5]. In our previous studies using SSH, genes that were differentially expressed in SV40T immortalized human bronchial epithelial cells or primarily cultured lung SCC tumour cells in comparison with their normal counterparts were described [6], [7]. However, either the SV40T immortalized cells or primarily cultured cells differ from primary tissue cells that have been removed from their in vivo environment and selected for growth characteristics at in vitro culture conditions, thus bringing into question the clinical relevance of these findings.

In this study, SSH was performed to identify the differentially expressed genes in human primary lung SCC tumour tissues, compared to normal bronchial epithelial tissues. Here we reported 177 known genes that are overexpressed in lung SCC. Further, a subset of these genes was validated by independent assays using human lung tumour tissues and cancer cell lines. Our data showed that at least three genes, located on chromosome 3q, were overexpressed in tumour tissues compared to their matched normal lung tissues. Further characterization of these genes and other genes identified in this study will be helpful to defining a panel of diagnostic and therapeutic targets for lung SCC and better our understanding into the biology of this malignancy.

Section snippets

Patients and tissue samples

For construction of the SSH library, six lung SCC tumour tissues and 10 biopsies of morphologically normal human bronchial epithelia were collected. The carcinoma samples were obtained from the tumours immediately after resection, quick-frozen in liquid nitrogen and stored at −80 °C for molecular analyses. The characteristics of the tumour samples were detailed in Table 1. The 10 bronchial epithelial specimens were obtained after surgeries for three lung adenocarcinomas, five lung SCCs, one

Generation of SSH cDNA libraries

To enrich for differentially expressed genes in lung SCCs, SSH was performed using two pools of total RNA from six lung SCC tumour tissues and 10 morphologically normal bronchial epithelial tissues. The subtraction efficiency test, carried out as described in the manufacturer's protocol, showed that the subtraction was successful. Two cDNA libraries were generated, consisting of preferentially up- and down-regulated genes in the tumour tissues, and named as LSCC (lung squamous cell carcinoma)

Discussion

In this study, to understand the molecular mechanism associated with lung SCC carcinogenesis, the expression profiles between fresh tumour tissues and morphologically normal bronchial epithelial tissues were compared by using the suppression subtractive hybridization method. Two cDNA libraries, LSCC and BRE, were established, comprising the upregulated genes and downregulated genes respectively. Among the LSCC library, genes such as AKR1B10, AKR1C1, AKR1C3, KRT6C, BPAG1, TPX2, GSTM3, TFRC, NME1

Conflict of interest

None declared.

Acknowledgements

This work was supported by grants from the National Basic Research Program of China (no. 2004CB518707), the National High-Tech R&D Program of China (nos. 2002BA711A06, 2002BA711A11 and 2002AA2Z2011), the Specialized Research Fund for the Doctoral Program of Higher Education (no. 20030023005), the National Natural Science Foundation of China (no. 30270582). We thank Dr. Sam Kuo and Dr. Steven Chang for the editorial assistance.

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