Chihaya Maesawa

Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines

Alterations of several microRNA (miRNA) have been linked to cancer development and its biology. To search for unique miRNA that might play a role in the development of anaplastic thyroid carcinoma (ATC), we examined the expression of multiple miRNA and their functional effects on target genes in human thyroid carcinoma cell lines. We quantitatively evaluated the expression of multiple miRNA in 10 ATC and five papillary thyroid carcinoma (PTC) cell lines, as well as primary tumors from 11 thyroid carcinoma patients (three ATC and eight PTC), using the stem‐loop‐mediated reverse transcription real‐time polymerase chain reaction method. We also examined the target gene specificity of unique miRNA that showed differences in expression between ATC and PTC cell lines. One miRNA, miR‐138, was significantly downregulated in ATC cell lines in comparison with PTC (P < 0.01). Eleven miRNA (including miR‐138) potentially targeting the human telomerase reverse transcriptase (hTERT) gene were totally downregulated in both ATC and PTC cell lines in comparison with normal thyroid tissues. A tendency for an inverse correlation between miR‐138 and hTERT protein expression was observed in the thyroid cancer cell lines, although this failed to reach significance (r = –0.392, P = 0.148). We demonstrated that overexpression of miR‐138 induced a reduction in hTERT protein expression, and confirmed target specificity between miR‐138 and the hTERT 3′‐untranslated region by luciferase reporter assay. These results suggest that loss of miR‐138 expression may partially contribute to the gain of hTERT protein expression in ATC, and that further multiple miRNA targeting hTERT mRNA might be involved in the development of thyroid carcinoma. (Cancer Sci 2008; 99: 280–286)

Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease.

Background  Endothelial progenitor cells (EPCs) play an important role in the maintenance of vascular integrity. Lipid lowering therapy (LLT) with statins may contribute to biologically relevant activities including the proliferation of endothelial cells. The physiological role of microRNA (miR)‐221/222, a newly discovered class of small RNA, is closely linked to the proliferation of endothelial cells. We therefore investigated whether LLT with statins might affect miR‐221/222 expression in EPCs obtained from patients with coronary artery disease (CAD).

Tumor Necrosis Factor-α–Converting Enzyme and Tumor Necrosis Factor-α in Human Dilated Cardiomyopathy

Background—Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of dilated cardiomyopathy (DCM). TNF-α–converting enzyme (TACE) has recently been purified and its complementary DNA cloned. The expression of TACE results in the production of a functional enzyme that has precursor TNF-α in the mature form. The aim of this study was to determine whether TACE is expressed with TNF-α in myocardium and whether levels of TACE and TNF-α are related to clinical severity of DCM. Methods and Results—Endomyocardial tissues were obtained from 30 patients with DCM and 5 control subjects. TNF-α and TACE mRNA levels were measured by a novel real-time quantitative reverse transcriptase–polymerase chain reaction method. Expression of TNF-α and TACE proteins was determined by immunohistochemical analysis. TNF-α mRNA was expressed in DCM patients (TNF-α/GAPDH ratio 0.85±0.24) but not in control subjects. TACE mRNA expression was significantly greater in DCM patients than in control subjects (TACE/GAPDH rat...

Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.

The non-coding control region of mitochondrial DNA (mtDNA), containing the hypervariable regions HV1 and HV2 and the D-loop region, was screened for mutations in 45 gastric tumours (15 tumours each of adenoma, differentiated adenocarcinoma and undifferentiated carcinoma). We found mutations in two of the 45 tumours (4%); a 1 bp A deletion at nucleotide position 248 in a differentiated adenocarcinoma and a G to A transition at nucleotide position 16,129 in an adenoma. We also observed 10 polymorphisms, four of which were not previously recorded. Both mtDNA mutations were present in replication error negative (RER-) tumours. Short mono- or dinucleotide repeats in the control region, such as (C)7, (A)5 or (CA)5, were not altered regardless of nuclear genetic instability. In summary, mtDNA is mutated in a subset of benign and malignant gastric tumours, but, disruption of the mtDNA repair system appears not to be significantly involved in gastric tumours of Japanese patients.

Inactivation of the E-Cadherin Gene in Primary Gastric Carcinomas and Gastric Carcinoma Cell Lines

We investigated the E (epithelial)‐cadherin gene for mutations and loss of heterozygosity (LOH) in 24 primary gastric carcinomas (12 differentiated and 12 undifferentiated types, including 3 signet‐ring cell carcinomas), as well as 4 gastric carcinoma cell lines of the undifferentiated type (MKN‐45, GCIY, HGC‐27 and GT3TKB). We utilized PCR‐SSCP and RT‐PCR followed hy direct sequencing to detect gene mutations and skipped exons, and RT‐PCR‐SSCP to examine LOH. In primary carcinomas, gene mutations or skipped exons, were detected in 4 of 9 (44%) undifferentiated carcinomas of the scattered type, including 2 signet‐ring cell carcinomas, and in none of the 3 undifferentiated carcinomas of the adherent type and 12 differentiated carcinomas. Demonstrated mutations of the E‐cadherin gene included an 18 bp deletion (codon 418‐423) and a 3 bp deletion (codon 400, calcium‐binding domain), both located in exon 9. Skipping of exon 9 with a 1 bp insertion at codon 337, and skipping of exon 8 with a 1 bp deletion at codon 336, also were detected. LOH was confirmed in all of the carcinomas in which gene mutations or skipped exons (3/3 informative cases) were demonstrated. The MKN‐45 cell line exhibited an 18 bp deletion at the exon 6‐intron 6 boundary with loss of the wild‐type allele, and 2 of the remaining 3 cell lines (HGC‐27 and GT3TKB) had lost expression without detectable structural alteration of the E‐cadherin gene. These data provide support for classic two‐hit inactivation of the E‐cadherin gene in a high percentage of undifferentiated carcinomas of the scattered type.

ALLELOTYPE OF ADENOMA AND DIFFERENTIATED ADENOCARCINOMA OF THE STOMACH

The molecular mechanism of gastric tumourigenesis has not yet been clarified, although investigators have postulated that differentiated adenocarcinoma may arise from pre‐existing adenoma, similarly to the colorectal adenoma–carcinoma sequence. An allelotype analysis has been performed to identify chromosomal regions which are frequently deleted in gastric tumours and to examine the significance of the adenoma–carcinoma sequence in gastric tumourigenesis. Forty‐five gastric tumours, 20 adenomas, and 25 differentiated adenocarcinomas were examined for loss of heterozygosity (LOH) using 39 microsatellite markers covering each non‐acrocentric chromosome arm. Frequent LOH in the adenocarcinomas was observed on chromosomes 2q (33 per cent), 4p (33 per cent), 5q (50 per cent), 6p (33 per cent), 7q (43 per cent), 11q (36 per cent), 14q (38 per cent), 17p (45 per cent), 18q (36 per cent), and 21q (40 per cent). In contrast, the incidence of LOH in adenomas did not exceed 10 per cent at any of the loci examined. In addition to the p53 gene on 17p and the DCC gene on 18q, which are known to be frequently deleted in differentiated adenocarcinomas of the stomach, other unknown tumour suppressor genes on the above‐mentioned chromosomes may also be inactivated. These observations suggest that the adenoma–carcinoma sequence is not a major pathway in gastric tumourigenesis.

Functionally inactivating point mutation in the tumor‐suppressor IRF‐1 gene identified in human gastric cancer

Loss of heterozygosity (LOH) observed in human tumors strongly suggests the existence of (a) tumor‐suppressor gene(s) at the concerned locus. A series of studies has revealed that LOH on the long arm of chromosome 5 (5q) frequently occurs in differentiated gastric adenocarcinomas. Furthermore, it has been shown that the interferon regulatory factor‐1 (IRF‐1) locus on chromosome 5q31.1 is one of the common minimal regions of LOH in these cancers. IRF‐1 is a transcriptional activator that shows tumor‐suppressor activity in the mouse. In the present study, we examined the sequence of the IRF‐1 gene in 9 cases of histologically differentiated gastric adenocarcinomas, all of which exhibited LOH at the IRF‐1 locus. We identified a mis‐sense mutation in the residual allele in one case. This mutated form of IRF‐1 showed markedly reduced transcriptional activity. In addition, over‐expression of wild‐type IRF‐1 induced cell‐cycle arrest, whereas such activity was attenuated in the mutant IRF‐1. These results suggest that the loss of functional IRF‐1 is critical for the development of human gastric cancers. Int. J. Cancer 77:522–527, 1998.

Commonly deleted regions on the long arm of chromosome 21 in differentiated adenocarcinoma of the stomach

During an allelotype analysis of differentiated adenocarcinoma of the stomach, we observed frequent loss of heterozygosity (LOH) on several chromosomes including the long arm of chromosome 21 (21q). Therefore, we analyzed DNA isolated from 45 tumors for LOH at 10 loci on 21q by using polymorphic microsatellite markers. In 20 (44%) of 45 tumors, we detected LOH at single or multiple loci on 21q. Deletion mapping of these 20 tumors revealed two separate commonly deleted regions. Our findings suggest that 21q contains at least two potential tumor suppressor genes which play crucial roles in the development of differentiated adenocarcinoma of the stomach. Genes Chromosom. Cancer 18:318–321, 1997.

Disruption of cell-type-specific methylation at the Maspin gene promoter is frequently involved in undifferentiated thyroid cancers

Cancer-associated DNA hypomethylation is as prevalent as cancer-linked hypermethylation, but the biological significance of DNA hypomethylation in carcinogenesis is less understood. The expression of Maspin (mammary serpin) in differentiated normal cells is regulated by epigenetic modifications in a cell-type-specific manner. Paradoxical Maspin expression due to epigenetic modification has been addressed in several cancer cell types. To elucidate the role of the Maspin gene in thyroid cancer, we studied methylation status in the promoter region and its expression in six human undifferentiated thyroid cancer cell lines and in specimens from 92 primary thyroid tumors, consisting of six follicular adenomas, 56 well-differentiated thyroid cancers (WDTCs), 17 poorly differentiated thyroid cancers (PDTCs) and 13 undifferentiated thyroid cancers (UDTCs). Three of the six cell lines overexpressed Maspin mRNA and its protein product, but the remaining three did not. The methylation status at the promoter region was inversely correlated with Maspin expression. In Maspin-negative cell lines, Maspin expression was induced by treatment with 5-aza-2′-deoxycytidine, a DNA demethylating agent. Immunoreactivity for Maspin protein was frequently detected in UDTCs (8/13, 62%) and PDTCs (7/17, 41%). Immunoreactivity for Maspin was diffusely positive in UDTCs, and was restricted to dedifferentiated components of the tumor in PDTCs. Positive immunoreactivity was infrequent in WDTCs (1/56, 2%), and all follicular adenomas and normal thyroid glands were completely negative. Their methylation status evaluated by the methylation-specific PCR method showed a good inverse correlation with their immunoreactivity in surgically resected specimens. Our data suggest that overexpression of Maspin by DNA hypomethylation is closely associated with morphological dedifferentiation in thyroid cancers.

Loss of HOXD10 expression induced by upregulation of miR-10b accelerates the migration and invasion activities of ovarian cancer cells

Small and large non-coding RNAs (ncRNAs) contribute to the acquisition of aggressive tumor behavior in diverse human malignancies. Two types of ncRNAs, miRNA‑10b (miR-10b) and homemobox (HOX) transcript antisense RNA (HOTAIR), can suppress the translation of the HOXD10 gene, an mRNA encoding a transcriptional repressor that inhibits the expression of cell migration/invasion-associated genes. Using epithelial ovarian cancer cell lines and primary tumors, we investigated whether miR‑10b and/or HOTAIR can regulate the expression of HOXD10, and whether it permits gain of pro‑metastatic gene products, matrix metallopeptidase 14 (MMP14) and ras homolog family member C (RHOC). Overexpression of miR-10b induced a decrease in HOXD10 protein expression, and upregulated the migration and invasion abilities in ovarian cancer cell lines (P<0.05). In these cells, a significant increase of MMP14 and RHOC protein was observed. No significant upregulation of the HOXD10 protein was observed in cells with the treatment of HOTAIR-siRNA. Positive signals for HOXD10 and MMP14 proteins were observed in 47 (69%) and 25 (37%) of 68 patients with epithelial ovarian cancers. An inverse correlation between HOXD10 and MMP14 immunoreactivities was observed (P<0.05), and miR-10b expression was also inversely correlated with HOXD10 protein expression (P<0.05). These results suggested that downregulation of HOXD10 expression by miR-10b overexpression may induce an increase of pro-metastatic gene products, such as MMP14 and RHOC, and contribute to the acquisition of metastatic phenotypes in epithelial ovarian cancer cells.