Decreased expression of haemoglobin beta (HBB) gene in anaplastic thyroid cancer and recovory of its expression inhibits cell growth

Abstract

Anaplastic thyroid cancer (ATC) is one of the most fulminant and foetal diseases in human malignancies. However, the genetic alterations and carcinogenic mechanisms of ATC are still unclear. Recently, we investigated the gene expression profile of 11 anaplastic thyroid cancer cell lines (ACL) and significant decreased expression of haemoglobin beta (HBB) gene in ACL. Haemoglobin beta is located at 11p15.5, where loss of heterozygosity (LOH) was reported in various kinds of cancers, including ATC, and it has been suggested that novel tumour suppressor genes might exist in this region. In order to clarify the meaning of decreased expression of HBB in ATC, the expression status of HBB was investigated with ACL, ATC, papillary thyroid cancer (PTC) and normal human tissues. Haemoglobin beta showed significant decreased expression in ACLs and ATCs; however, in PTC, HBB expressed equal to the normal thyroid gland. In addition, HBB expressed in normal human tissues ubiquitously. To validate the tumour-suppressor function of HBB, cell growth assay was performed. Forced expression of HBB in KTA2 cell, which is a kind of ACL, significantly suppressed KTA2 growth. The mechanism of downregulation of HBB in ATC is still unclear; however, our results suggested the possibility of HBB as a novel tumour-suppressor gene.

Discovery of microarray-identified genes associated with ovarian cancer progression

“…HBB was found to be significantly downregulated in 11 anaplastic thyroid cancer cell lines, and a cell proliferation assay suggested that HBB might be a novel TSG (41). Analysis of primary and post-treatment samples (12 tumor and eight normal) by the Affymetrix platform (HG U133 plus 2) identified 119 genes as differentially regulated in recurrent tumors, of which HBB alone, and collagen, type V, α1 (COL5A1) and HBB in combination, have the best predictive power for treatment response in patients with oral tongue cancer (42).…”

Down-regulation of an inhibitor of cell growth, transmembrane protein 34 (TMEM34), in anaplastic thyroid cancer

“…To adjust the amount of transcribed cDNA, GAPDH was selected as an internal control and SQ-PCR was performed as previously described (Onda et al 2005); the primer sequences for GAPDH were 5¢-ggaaggtgaaggtcggagt-3¢ (forward) and 5¢-tgggtggaatcatattggaa-3¢ (reverse). After adjustment of concentrations, SQ-PCR experiments for TMEM34 were performed using primers 5¢-ggcttggtttattgctggaa-3¢ (forward) and 5¢-gtgtgggcccaatattcatc-3¢ (reverse); all primers were designed with primer 3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) after sequence information was obtained from NCBI GenBank (http://www.ncbi.nlm.nih.gov/).…”

“…Q-PCR experiments were performed with an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA) according to the comparativethreshold (Ct) cycle method, as previously described (Onda et al 2005). All quantitative RT-PCR products were visualized on a 2% agarose gel to ensure a single product.…”

“…A TMEM34 expression vector, pcDNA TMEM34, was constructed using a pcDNA3.1 directional TOPO expression kit (Invitrogen, Carlsbad, CA, USA) as described previously (Onda et al 2005). To generate a full-length coding sequence of TMEM34, PCR was performed using the cloning forward primer: 5¢-caccatgccttgcacttgtacctgga-3¢ and the reverse primer: 5¢-ggaatccacggatttatct-3¢.…”

“…PCR analysis of each exon of TMEM34 was performed as previously described (Onda et al 2005), using the primers shown in Table 1. PCR products were denatured for 10 min at 95°C and placed immediately on ice, to prevent annealing of single-stranded products.…”