MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of target genes. Some tumor-suppressive miRNAs are known to be epigenetically silenced by promoter DNA methylation in cancer. In the present study, we aimed to identify miRNA genes that are silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). We screened for miRNA genes with promoter DNA hypermethylation using a genome-wide methylation microarray analysis in HCC cells. It was found that miR-335, which is harbored within an intron of its protein-coding host gene, MEST, was downregulated by aberrant promoter hypermethylation via further methylation assays, including methylation-specific PCR, combined bisulfite and restriction analysis, bisulfite sequencing analysis and 5-aza-2′-deoxycytidine treatment. The expression levels of miR-335 significantly correlated with those of MEST, supporting the notion that the intronic miR-335 is co-expressed with its host gene. The levels of miR-335/MEST methylation were significantly higher in 18 (90%) out of 20 primary HCC tumors, compared to their non-tumor tissue counterparts (P<0.001). The expression levels of miR-335 were significantly lower in 25 (78%) out of 32 primary HCC tumors, compared to their non-tumor tissue counterparts (P=0.001). Furthermore, the expression levels of miR-335 were significantly lower in HCC tumors with distant metastasis compared to those without distant metastasis (P=0.02). In conclusion, our results indicate that expression of miR-335 is reduced by aberrant DNA methylation in HCC.

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sigmoid-shaped ventricular septum is one of the causes of left ventricular outflow tract (LVOT) obstruction in older patients and because it is rarely related to clinical symptoms, 1,2 medical therapy for the reduction of the left ventricular pressure gradient (LVPG) associated with sigmoid septum has not been established. It was recently reported that the class Ia antiarrhythmic drug, cibenzoline, attenuates the LVPG in patients with hypertrophic obstructive cardiomyopathy (HOCM), 3 and we describe a patient with LVPG caused by a sigmoid-shaped septum who improved after administration of cibenzoline. Case ReportA 83-year-old-female was admitted to hospital with episodes of dyspnea on effort after having breakfast. She had had hypertension for 20 years prior to admission. Her blood pressure was 154/74 mmHg, and her pulse rate was 100 beats/min and regular. Her physical examination was unremarkable, except for a grade 4/6 systolic murmur at the left sternal border in the third to fourth intercostal space. Routine blood tests were normal except for brain natriuretic peptide (BNP) of 455 pg/ml. An electrocardiography recorded 18 years prior to admission had been normal, whereas that recorded in the current admission showed normal sinus rhythm at a rate of 75 beats/min and left ventricular hypertrophy with ST-segment depression and inverted T waves characteristic of the strain pattern (Fig 1). Chest X-ray showed cardiomegaly with a cardiothoracic ratio of 56%. Two-dimensional transthoracic echocardiography revealed a sigmoid-shaped base of the interventricular septum markedly protruding into the left ventricle and calcification of both posterior mitral leaflet and aortic valve (Fig 2A,B), whereas that performed 18 years prior to admission had been normal (Fig 3). The remainder of the septum and the left ventricular free wall were concentrically hypertrophied, but no enlargement of the left ventricular cavity was observed. The angle between the mid line axis of the ascending aorta and that of the interventricular septum was 97.5°(normal range, 145±7°). M-mode echocardiography demonstrated a systolic anterior motion of the mitral valve (SAM) with fractional shortening (FS) of 39% ( Fig 4A). Color Doppler echocardiography showed turbulent systolic flow through the LVOT with a pressure gradient of 121.8 mmHg, mild aortic stenosis, and moderate mitral regurgitation (Figs 2C,4B). Cardiac catheterization was performed and angiography indicated an intermediate stenosis in the mid portion of the right coronary artery. The left ventricular end-diastolic pressure was 18 mmHg and the peak-to peak LVPG at the LVOT was 146 mmHg. These findings confirmed the diagnosis of LVOT obstruction caused by the sigmoid septum, concentric left ventricular hypertrophy and SAM.To reduce the LVOT obstruction, 60 mg/day of metoprolol was administered, but despite that treatment the LVPG remained at approximately 100 mmHg. Hence, 200 mg/day of cibenzoline, a class Ia antiarrhythmic agent, was administered in addition to 40 mg/day of metoprolol. A...

EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high-density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH-1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1–EVI1 or MDS1, was overexpressed in JHH-1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des-γ-carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15INK4B by transforming growth factor (TGF)-β and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-β-treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-β-mediated growth inhibition of HCC cells.

A primary clear cell adenocarcinoma of the colon is a rare oncologic entity. We herein report a case of such a tumor of the sigmoid colon in a 71-year-old woman who was successfully treated by an endoscopic polypectomy in our hospital. We also reviewed the published reports regarding cases of primary clear cell tumors in the colon.