Ichiro Kagara

The functional significance of miR-1 and miR-133a in renal cell carcinoma

PURPOSE The aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. METHODS We checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N=40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. RESULTS The expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. CONCLUSIONS Our data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.

Nuclear translocation of ADAM-10 contributes to the pathogenesis and progression of human prostate cancer.

A disintegrin and metalloproteases (ADAM) are cell membrane‐anchored proteins with potential implications for the metastasis of human cancer cells via cell adhesion and protease activities. In prostate cancer (PC), the ADAM‐10 protein showed a nuclear localization whereas in benign prostate hypertrophy (BPH) it was predominantly bound to the cell membrane. We hypothesized that the pathogenesis and progression of PC are attributable to the nuclear translocation of ADAM‐10. Immunoblotting revealed that after 5α‐dihydrotestosterone treatment, a 60‐kDa active form of ADAM‐10 was increased in the nuclear fraction but decreased in the cell membrane and cytoplasmic fractions of human androgen‐dependent PC cells. Immunocytochemistry revealed that after 5α‐dihydrotestosterone treatment, the ADAM‐10 protein was translocated from the cell membrane to the nucleus. Coimmunoprecipitation of androgen receptor and ADAM‐10 was detected in the nuclear fraction but not in the cell membrane and cytoplasmic fractions. Immunohistochemical study of 64 PC and 20 BPH samples showed that the intensity of ADAM‐10 staining was significantly higher in the nuclei of PC cells than in the nuclei of BPH cells (P < 0.0001). It was also significantly lower in the cell membrane of PC cells than in the cell membrane of BPH cells (P = 0.0017). Nuclear staining intensity was significantly correlated with the clinical T‐factor (P = 0.004), the Gleason score (P < 0.0001) and preoperative prostate‐specific antigen levels (P = 0.0061). ADAM‐10 small interfering RNA transfectants showed a significant decrease in cell growth compared to the controls. Our results suggest that in human PC, the nuclear translocation of ADAM‐10 coupled with the androgen receptor is involved in tumor growth and progression. (Cancer Sci 2007; 98: 1720–1726)

Acute Kidney Injury and Rhabdomyolysis After Protobothrops flavoviridis Bite: A Retrospective Survey of 86 Patients in a Tertiary Care Center

Acute kidney injury (AKI) is the main cause of death for victims of hematoxic snakebites. A few studies have described improvement in AKI rates in snakebite cases, but the reasons for the improvement have not been investigated. Eighty-six patients with Protobothrops flavoviridis bites admitted to a single center from January 2003 through March 2014 were included in the study. Clinical variables, including age, sex, blood pressure (BP), and serum creatinine (S-Cre), on admission were compared between patients with and without AKI. One patient died of disseminated intravascular coagulation following AKI (mortality rate 1.1%). Six patients developed AKI with rhabdomyolysis. Systolic BP, S-Cre, serum creatine kinase, white blood cell count, and platelet count differed significantly between the AKI and non-AKI groups (P = 0.01). Three of the six patients were physically challenged to a degree that made it difficult for them to move or communicate, and these difficulties likely exacerbated the severity of snakebite complications. Our study demonstrated that the risk of snakebite-induced AKI for physically challenged patients was high. To further reduce mortality due to snakebite-induced AKI, we need to make it possible for physically challenged patients to receive first aid sooner.

CPG HYPERMETHYLATION OF COL1A2 CONTRIBUTES TO PROLIFERATION AND MIGRATION ACTIVITY OF BLADDER CANCER

INTRODUCTION AND OBJECTIVE: Gene silencing due to aberrant DNA methylation plays an important role in carcinogenesis. For genome-wide screening for methylated genes in bladder cancer (BC), we performed cDNA microarray analysis of the BC cell line (BOY) treated with demethylating agent (5-Aza-dC). Collagen type 1 alpha 2 (COL1A2) gene was identified as the most up-regulated one of the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC by changing extra-cellular matrix (ECM). METHODS: To test this hypothesis, we extracted DNA and RNA from 67 BC samples and 10 normal bladder epitheliums (NBEs) from cystectomy and transurethral resection. The CpG hypermethylation of COL1A2 promoter was analyzed by quantitative methylation-specific PCR (QMSP) and confirmed by bisulfite-modified DNA sequencing. Real-time RT-PCR was carried out to evaluate COL1A2 mRNA expression. We established a stable COL1A2 transfectant for gain of function studies, for which cellular proliferation and migration were examined by MTT assay and wound healing assay, respectively. The protein secretion of type 1 collagen in culture medium was detected by immunoblot. RESULTS: Bisulfite DNA sequencing demonstrated that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index was significantly higher in the 67 BCs than in the10 NBEs (42.3 ± 28.9 vs. 0.07 ± 0.01, p = 0.0011). Conversely, COL1A2 mRNA expression was significantly lower in the BCs than in the NBEs (15.8 ± 3.8 vs. 32.0 ± 22.0, p = 0.0052). In vitro, after 5-aza-dC treatment, the COL1A2 mRNA expression was markedly increased in BOY. Our cell proliferation assays consistently demonstrated a significant growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (on day 5; 0.791 ± 0.020, 1.056 ± 0.034, and 1.107 ± 0.027, respectively, p < 0.0001). Wound healing assays also showed a significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (% of wound closure; 75.5 ± 0.2, 94.3 ± 2.9, and 96.9 ± 3.1, respectively, p = 0.0016). Immunoblot demonstrated that type 1 collagen protein markedly increased in culture medium of the COL1A2 transfectant. CONCLUSIONS: Our data suggest that inactivation of the COL1A2 gene through CpG methylation may change an ECM binding on epithelial collagen and contributes to proliferation and migration activity of human bladder cancer.