Using Microarrays to Identify Biomarkers and to Investigate the Mechanism of Carcinogenesis

Abstract

The major research interests in my lab are using microarrays to explore the cancer genomics. Our main goals are to identify biomarkers and to understand the mechanism of carcinogenesis. Approaches to these goals include investigating gene expression profiling, copy number variation, single nucleotide polymorphism, DNA methylation and microRNA in several cancers. Two studies done in my lab will be presented herein. Firstly, in search of biomarkers for prognosis, we identified that several semaphorin gene family members can be used as potential therapeutic targets, and that SEMA5A may be useful as a prognostic biomarker for non-smoking women with non-small cell lung carcinoma (NSCLC). Sixty pairs of tumor and adjacent normal lung tissue specimens were analyzed by using Affymetrix U133plus2.0 expression arrays. Differentially expressed genes in tumor tissues were identified by a paired t test and validated by reverse transcriptase-PCR and immunohistochemistry. Functional analysis was conducted by using Ingenuity Pathway Analysis as well as gene set enrichment analysis and sigPathway algorithms. Kaplan-Meier survival analyses were used to evaluate the association of SEMA5A expression and clinical outcome. We identified 687 differentially expressed genes in non–small cell lung carcinoma. Many of these genes, most notably the semaphorin family, were participants in the axon guidance signaling pathway. The down-regulation of SEMA5A in tumor tissue, both at the transcriptional and translational levels, was associated with poor survival among nonsmoking women with NSCLC. Therefore, SEMA5A can be used as a prognostic biomarker for NSCLC. Regarding the exploration of carcinogenesis mechanism, we identified that down-regulation of NDRG1 can promote migration of breast cancer cells during reoxygenation. The breast cancer MCF-7 cells were cultured under 0.5% oxygen for 24 h followed by 24 h of reoxygenation in normoxia. Cells were harvested at 0, 1, 4, 8, 12, and 24 h during reoxygenation. The transcriptional profile of MCF-7 cells upon reoxygenation was examined using Illumina Human-6 v3 BeadChips. We identified 127 differentially expressed genes, of which 53.1% were up-regulated and 46.9% were down-regulated upon reoxygenation. Pathway analysis revealed that the HIF-1-alpha transcription factor network and validated targets of C-MYC transcriptional activation were significantly enriched in these differentially expressed genes. Among these genes, a subset of interest genes was further validated by quantitative reverse-transcription PCR. In particular, human N-MYC down-regulated gene 1 (NDRG1) was highly suppressed upon reoxygenation. NDRG1 is associated with a variety of stress and cell growth-regulatory conditions. To determine whether NDRG1 plays a role in reoxygenation, NDRG1 protein was overexpressed in MCF-7 cells. Upon reoxygenation, overexpression of NDRG1 significantly inhibited cell migration. Our results revealed the dynamic nature of gene expression in MCF-7 cells upon reoxygenation and demonstrated that NDRG1 is involved in tumor adaptation to reoxygenation.