B-RafV600E mutant is found in 40-70% of papillary thyroid carcinoma (PTC) and has an important role in the pathogenesis of PTC. The sodium iodide symporter (NIS) is an integral plasma membrane glycoprotein that mediates active iodide transport into the thyroid follicular cells, and B-RafV600E has been known to be associated with the loss of NIS expression. In this study, we found that B-RafV600E inhibited NIS expression by the upregulation of its promoter methylation, and that specific regions of CpG islands of NIS promoter in B-RafV600E harboring PTC were highly methylated compared with surrounding normal tissue. Although DNA methyltransferase 3a and 3b (DNMT3a,3b) were not increased by B-RafV600E, DNMT1 expression was markedly upregulated in PTC and B-RafV600E expressing thyrocytes. Furthermore, DNMT1 expression was upregulated by B-RafV600E induced NF-kappaB activation. These results led us to conclude that NIS promoter methylation, which was induced by B-RafV600E, is one of the possible mechanisms involved in NIS downregulation in PTC.
Base Sequence ; Carcinoma/*genetics/metabolism/pathology ; Cells, Cultured ; DNA (Cytosine-5-)-Methyltransferase/analysis/*genetics/metabolism ; DNA Methylation ; Down-Regulation ; *Gene Expression Regulation, Neoplastic ; Humans ; Molecular Sequence Data ; *Point Mutation ; Promoter Regions, Genetic ; Proto-Oncogene Proteins B-raf/*genetics/metabolism ; Symporters/analysis/*genetics/metabolism ; Thyroid Gland/cytology/metabolism/pathology ; Thyroid Neoplasms/*genetics/metabolism/pathology ; Up-Regulation

OBJECTIVETo observe the sensitivity change of SMMC-7721 cells transfected with antisense DNMT1 gene fragment to tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its mechanism.

METHODSCell survival rate was measured by trypan blue, apoptosis rate by TUNEL method and the expression of bcl-2, bax and bad by flow cytometry.

RESULTSCell survival rate of SMMC-7721 cells transfected with antisense DNMT1 gene fragment was markedly lower than that transfected with sense DNMT1 gene fragment or empty vector (P < 0.05 and 0.01), but the apoptosis rate was on the contrary (P < 0.05 or 0.01). The expression of bax and bad (especially the former), but not bcl-2 of SMMC-7721 cells transfected with antisense DNMT1 gene fragment was markedly higher than those of SMMC-7721 cells transfected with sense DNMT1 gene fragment or empty vector.

CONCLUSIONThe sensitivity of SMMC-7721 cells to TRAIL can be enhanced by the transfection of antisense DNMT1 gene fragment, which may be related to the increase of bax and bad expression.

Antisense Elements (Genetics) ; genetics ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Carrier Proteins ; analysis ; Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; antagonists & inhibitors ; genetics ; Flow Cytometry ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Membrane Glycoproteins ; pharmacology ; Proto-Oncogene Proteins ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha ; pharmacology ; bcl-2-Associated X Protein ; bcl-Associated Death Protein