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Method for detecting carcinoma and agent for suppressing carcinoma

Inactive Publication Date: 2009-08-13
FUJIFILM CORP +1
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AI Technical Summary

Benefits of technology

[0004]Successful elucidation of the mechanism of malignant transformation of oral-cavity-derived cells and mainly oral mucous membrane epithelium-derived cells at the gene level will enable detection of malignant transformation of oral mucous membrane epithelium cells at the gene level, diagnosis of the malignancy of oral squamous-cell carcinoma, and suppression of the advancement thereof. Furthermore, it will also enable establishment of methods for selecting and developing drugs and methods for therapy, based on such mechanisms. Specifically, identifying genes exhibiting characteristic behavior observed in oral squamous-cell carcinoma cases and then carrying out technical examination mainly targeting such genes can achieve this object. Hence, an object to be achieved by the present invention is to identify genes exhibiting characteristic behavior in the cases of carcinoma such as oral squamous-cell carcinoma, so as to provide a method for detecting carcinoma and an agent for suppressing carcinoma.
[0005]The real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR) is the best method for conveniently and rapidly analyzing the amount of transcripts resulting from gene expression. In order to analyze changes in expression of genes associated with carcinoma, TaqMan MicroRNA Assays was used, and the carcinoma-associated microRNA genes, which would accelerate malignant transformation of oral mucous membrane epithelium cells, shown in Tables 1 and 2 were successfully identified. The expression levels of miR-34b, miR-132, miR-137, miR-193a, and miR-203 located on or around the CpG islands are down-regulated also by methylation of cytosine that is present on CpG islands. Specifically, down-regulated expression levels of such genes result in acceleration of oral squamous-cell carcinoma growth. Further, the present inventors discovered that the up-regulated expression levels of such genes in oral squamous-cell carcinoma would significantly down-regulate carcinoma growth. This has led to the completion of the present invention.
[0018]The present invention enables accurate understanding of malignant transformation in cell specimens derived from the oral mucosa. Also, introduction of synthetic double-stranded RNA containing the microRNA sequence into the oral squamous-cell carcinoma would be able to suppress the growth of carcinoma, such as oral squamous-cell carcinoma.

Problems solved by technology

Specifically, down-regulated expression levels of such genes result in acceleration of oral squamous-cell carcinoma growth.

Method used

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  • Method for detecting carcinoma and agent for suppressing carcinoma
  • Method for detecting carcinoma and agent for suppressing carcinoma
  • Method for detecting carcinoma and agent for suppressing carcinoma

Examples

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example 1

Changes in miRNA Gene Expression in Oral Squamous-Cell Carcinoma

[0043]To detect changes in miRNA gene expression in oral squamous-cell carcinoma, 18 types of oral squamous-cell carcinoma cell lines (Ca9-22, HO-1-N-1, HSC-2, HSC-3, HSC-4, KOSC-2 c13-43, HOC-313, HOC-815, HSC-5, HSC-6, HSC-7, KON, NA, OM1, OM2, SKN3, TSU, and ZA) were used. As a control, a normal oral mucosal epithelium-derived immortalized cell line, RT7, was used. The oral squamous-cell carcinoma cell line was cultured in DMEM medium containing streptomycin (100 μg / ml), penicillin (100 units / ml), 2 mM glutamine, and 10% fetal bovine serum (FBS). The RT7 cell line was cultured using the KGM-2 Bullet Kit (Cambrex). Genomic DNA was extracted therefrom using the Genome DNA Purification kit (Gentra, Minneapolis, Minn.), and RNA was extracted using Isogen (Nippon Gene), in accordance with manufacturers' instructions.

[0044]FIG. 1A shows the strategy for isolating antioncogenic miRNA, the gene expression level of which has ...

example 2

Analysis of Candidate miRNA and Methylation in Oral Squamous-Cell Carcinoma Cell Lines

[0048]The human genome database (http: / / genome.ucsc.edu / ) was screened for the presence of CpG islands in the vicinities of 157 types of miRNA genes. As a result, 21 types of miRNA genes were found to be located on or around (within 1,000-bp) the CpG islands (Table 3).

TABLE 321 miRNAs located on / around CpG islandsmiRNALocusmiR-9miR-9-1, 1q22; miR-9-3, 15q26.1miR-9*miR-9-1, 1q22; miR-9-3, 15q26.1miR-34b11q23.1miR-92miR-92-1, 13q31.3; miR-92-2, Xq26.2; miR-92b, 1q22miR-124amiR-124a-1, 8p23.1; miR-124a-2, 8q12.3; miR-124a-3,20q13.33miR-1269q34.3miR-12714q32.31miR-129miR-129-1, 7q32.1; miR-129-2, 11p11.2miR-13217p13.3miR-1371p21.3miR-1492q37.3miR-15217q21.32miR-1899q22.32 (Replaced by miR-24-1)miR-1913p21.31miR-193a17q11.2miR-20314q32.33miR-21011p15.5miR-219miR-219-1, 6p21.32; miR-219-2, 9q34.11miR-3208p21.3miR-3397p22.3let-7i12q14.1

[0049]Among the 21 types of miRNA genes, the miR-34b, miR-132, miR-137...

example 3

Analysis of miRNA Expression and Methylation in Specimen from Patient with Oral Squamous-Cell Carcinoma

[0056]Whether or not methylation of four types of miRNA genes has occurred in cancerous tissue of a patient with oral squamous-cell carcinoma and the correlation between the state of DNA methylation of the four types of miRNA genes and the expression patterns in cancerous tissue of a patient with oral squamous-cell carcinoma were analyzed by using the COBRA method and TaqMan real-time RT-PCR analysis.

[0057]Regarding cancerous and noncancerous tissue samples of patients with oral squamous-cell carcinoma, 11 cases of frozen samples (T1: 0 cases; T2: 10 cases; T3: 0 cases; T4: 1 case) were obtained with the approval of the Ethics Committee of Tokyo Dental and Medical University, followed by acquisition of written agreements from patients with oral squamous-cell carcinoma who had undergone surgery at the Tokyo Dental and Medical University Hospital, Faculty of Dentistry. The relevant s...

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Abstract

An object of the present invention is to identify genes exhibiting characteristic behavior in the cases of carcinoma such as oral squamous-cell carcinoma using a change of expression level of micro RNA in oral squamous-cell carcinoma as an indicator, so as to provide a method for detecting carcinoma and an agent for suppressing carcinoma. The present invention A method for detecting oral squamous-cell carcinoma, which comprises detecting malignant transformation of specimens by employing a change of expression level of micro RNA as an indicator, and an agent for suppressing oral squamous-cell carcinoma using micro RNA.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of detecting carcinomas, such as oral squamous-cell carcinoma by utilizing changes in the expression levels of microRNA genes that are present in the human chromosome, and more particularly to an agent for suppressing growth of carcinoma, such as oral squamous-cell carcinoma.BACKGROUND ART[0002]Oral squamous-cell carcinoma (OSCC) is classified as a head and neck carcinoma and is a tumor that is mainly generated from oral mucous membrane epithelia and the like. Among head and neck carcinomas, OSCC incidence is as high as about 35% and it is assumed to develop in about 270,000 people worldwide every year (Parkin, D. M., et al., CA Cancer J. Clin. 55, 74-108, 2002). In Japan, 5,500 or more people had died thereof in 2003. The most common site of the origin of oral squamous-cell carcinoma is tongue and the second most common site thereof is gingiva (gum). Oral squamous-cell carcinoma is known to be developed at other mucous ...

Claims

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Application Information

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IPC IPC(8): A61K9/127C40B30/00C12Q1/68G01N33/00A61K31/7088
CPCC12Q1/6886C12Q2600/154Y10T436/143333C12Q2600/178C12Q2600/16A61P1/02A61P35/00
Inventor INAZAWA, JOHJIKOZAKI, KEN-ICHIIMOTO, ISSEI
Owner FUJIFILM CORP
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