Expression profiling of human thyroid cell line stably expressing BRAF V600E mutation

Abstract

Introduction: BRAF V600E mutation is the most common somatic mutation in papillary thyroid carcinoma (PTC) and act as an initiator of cancer development. Gene expression change caused by BRAFV600E mutation might play an important role for thyroid cancer development. Methods: To study the genomic alteration caused by BRAF V600E mutation, we made human thyroid cell lines which harbor wild type BRAF gene (Nthy/WT) and V600E mutant type BRAF gene (Nthy/V600E). Successful transduction of BRAF gene was evaluated by flow cytometry and Sanger sequencing. Phosphorylation of ERK was anlayzed by flow cytometry and western blot. Functional changes of the cells according to BRAFV600E mutation were observed by soft agar and invasion assay. Microarray analysis was performed to find differentially expressed genes between BRAF wild and BRAFV600E cells. Results: BRAF gene was successfully transfected into Nthy-ori cell, confirmed by Sanger sequencing in both Nthy/WT and Nthy/V600E. Flow cytometry and Western blot showed stable transfection amount of BRAF gene. In functional experiments, Nthy/V600E cells have increased phophorylated ERK levels, higher anchorage-independent grow rate, and enhanced Matrigel invasion in vitro. Microarray analysis reveals that 2441 genes were up-regulated in Nthy/V600E cells

top 3 up-regulated genes were IL1B, ANO1, and SERPINE2. Up-regulated genes are associated with cell adhesion, migration, motility, ERK and MAPK cascade in gene ontology. Cancer related pathways also enriched in pathway analysis. Conclusions: Our Nthy/WT and Nthy/V600E cell lines, with its correlation with human BRAFV600E PTCs, may be used as a source for basic experiments to further research for molecular characteristics of thyroid cancer.