Differential Gene Expression Profiling Of Laryngeal Squamous Cell Carcinoma By Laser Capture Microdissection And Complementary DNA Microarrays

Objective:Laryngeal squamous cell carcinoma (LSCC) is the second most common cancer worldwide of squamous cell carcinoma of the head and neck (SCCHN) and accounts for approximately 60% of glottic carcinoma of larynx in China. The management of LSCC, one type of SCCHN, depends mainly on early detection and appropriate surgical resection. Many aspects of the current management such as the benefit and optimal timing of combining chemotherapy to the radiation are still unknown. Thus, to improve its detection and treatment, understanding of the pathogenesis and biologic features of LSCC is crucial. Most of the previous studies on genetic alterations of LSCC have focused on selected genes or chromosomal regions known in other cancers, and in LSCC, stromal and inflammatory cells usually intermingled with cancer cells. These obstacles may be overcome by using techniques of laser capture microdissection (LCM) and oligonucleotide microarray. Therefore, in this study, we aimed to examine the gene expression profiles of LSCC by combining these two techniques. These efforts will provide a better understanding of molecular mechanisms underlying the carcinogenesis of larynx, as well as facilitate the identification of novel biomarkers and therapeutic targets.Methods:(1) To establish tissue specimen bank and clinical information database of LSCC, Eight matched surgical tissue specimens from these patients, including pairs of eight normal epithelium tissues adjacent to the carcinoma and eight glottic carcinoma of larynx were identified to collect from four different stage and each stage had two sambles.(2) The tissue samples from LSCC preserved in the RNAlater reagent was microdissected to derive pure populations of malignant cells and the pure normal laryngeal epithelial pillar cells by LCM. line amplification mRNA in vetro gain enough aRNA for the study. Then we constructed the gene expression profiling by using HG-U133.Plus.2.0 using this aRNA(3) qRT-PCR was performed to validate the gene expression profiling(4) We made high-quality tissue microarray (TMA) with fifty matched pairs of the tumor samples including fifty all stages of LSCC and fifty normal epithelium tissues adjacent to the tumors for immunohistochemistry.Results:(1) Hierarchical clustering of gene expression demonstrated that a total of 2351 differentially expressed genes in patients groups with LSCC and their normal adjacent epithelium after the SAM analysis. But we identified 761 genes that were differentially expressed between early stage (stage 1,2) and later stage (stage 3,4) by SAM.(2) We applied these genes for gene ontologies to divide into three groups: cellular component, molecular function and biological process. further focused on biological process branch to investigate the deregulated genes, we found that genes encoding for proteins involved in regulation of transcription, signal transduction; cytoskeletal and extracellular matrix proteins and proteins involved in cell cycle, cell adhesion and proteolysis were most frequently identified.(3) We found that the several major pathways were involved in cell cycle, matrix metalloproteinases, and integrin mediated cell adhesion in cancerous versus normal groups.(4) qRT-PCR demonstrated that the expression levels of MMP12, KRT16, HMGA2 and KRT19 were higher in tumor cells, especially, KRT19 was more highly expressed in later stage tissue than early stage tissue, whereas the expression of KRT23, RARB, PRBlwere higher in normal tissues and thus under expressed in tumor cells.(5) TMA for immunohistochemistry showed MMP12,. HMGA2 were higher in tumor cells. RARB were higher in normal tissues and thus under expressed in tumor cells.Conclusion: (1) We have successfully combined RNAlater reagent, LCM, RNA amplification, and microarray technologies to generate globle gene expression profiles in LSCC(2) Three importment pathways identified in this study and prospective translational studies to determine their potential clinical value may lead to a deep insight into the pathogenesis of LSCC and facilitate the development of novel biomarkers for diagnostic and therapeutic applications.(3) The results of qRT-PCR and Immunohistochemistry supported the validity of combined use of LCM and microarray as an experimental approach for the profiling of LSCC.