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Senescence marker, method for evaluating senescence inhibitor, and cancer inhibitor

a senescence inhibitor and cancer technology, applied in the field of senescence markers, can solve the problems that the use of hsa-mir-22 for diseases such as cancer has not been elucidated in detail, and achieve the effect of inhibiting the growth, invasion and/or metastasis of cancer, and being easy to evalua

Inactive Publication Date: 2012-12-13
HIROSHIMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0031]With the senescence marker of the first embodiment of the present invention, the degree of senescence of a sample can be judged. By the method of the second embodiment of the present invention for evaluating a senescence inhibitor, a substance that inhibits senescence can be easily evaluated. With the cancer inhibitor of the third embodiment of the present invention, cellular senescence can be promoted to inhibit the growth, invasion and / or metastasis of cancer.

Problems solved by technology

The mechanism and meaning of the above-mentioned induction of cellular senescence due to replication, including the relationship with aging of individuals, have been controversial for a long time.
However, whether (hsa-)miR-22 can be used for diseases such as cancer has not been elucidated in detail based on Examples or in view of the mechanism.

Method used

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  • Senescence marker, method for evaluating senescence inhibitor, and cancer inhibitor
  • Senescence marker, method for evaluating senescence inhibitor, and cancer inhibitor
  • Senescence marker, method for evaluating senescence inhibitor, and cancer inhibitor

Examples

Experimental program
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example 1

[0100]Example 1 describes an example in relation to the expression levels of hsa-miR-22* in young and senescent human fibroblasts. The present inventors investigated the expression levels of hsa-miR-22* in young human fibroblasts and senescent human fibroblasts (cells that experienced a larger number of times of division than young cells) by quantitative real-time RT-PCR. Details of the investigation are described below.

[0101]The expression level of hsa-miR-22* was investigated for human fibroblasts TIG-3 cells, TIG-1 cells, TIG-112 cells, TIG-114 cells and MRC-5 cells. The TIG-3 cells, TIG-1 cells, TIG-1 12 cells and TIG-114 cells were cultured using DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS (Fetal Bovine Serum) and an antibiotic. The MRC-5 cells were cultured in DMEM / F-12 medium (1:1, v / v) supplemented with SAP (0.2 mM serine, 0.1 mM aspartic acid, 1.0 mM pyruvate), 10% FBS and an antibiotic. All these cells were cultured using a humidified incubator at 3...

example 2

[0104]The present Example 2 describes an example in relation to cases where double-stranded hsa-miR-22 was introduced into human fibroblasts. The present inventors investigated whether or not phenomena found in senescent cells are observed when double-stranded hsa-miR-22 was introduced into human fibroblasts (MRC-5 cells). Details of the investigation are described below.

[0105]First, the present inventors investigated whether or not formation of senescence-associated heterochromatic foci (hereinafter referred to as SAHF), which is a marker for senescent cells, is observed in cases where double-stranded hsa-miR-22 was introduced into human fibroblasts (MRC-5 cells). According to the protocol for Lipofectamine RNAi Max (Invitrogen), 10 nM double-stranded hsa-miR-22 (B-bridge, this also applies to later-mentioned Examples) or AllStars Negative Control siRNA (Qiagen) was introduced into MRC-5 cells. The introduction efficiency was assumed to be not less than 90% based on measurement usi...

example 3

[0117]The present Example 3 describes an example in relation to cases where Pre-hsa-miR-22 (see FIG. 1) was introduced into human fibroblasts (MRC-5 cells) by lentivirus infection. The present inventors confirmed whether or not phenomena that specifically accompany cellular senescence (the SA-β-gal activity and inhibition of the cell growth) are observed when Pre-hsa-miR-22, which is a precursor of mature hsa-miR-22, was introduced into human fibroblasts.

[0118]First, Lipofectamine LTX Plus reagent (Invitrogen) was used to cotransfect 293T cells with 0.9 μg of Pre-hsa-miR-22 or a lentivirus vector as a control empty vector (these were manufactured by System Biosciences), and 0.9 μg of a packaging plasmid mix (pPACK-H1-GAG, pPACK-H1-Rev and pVSV-G), thereby producing a lentivirus. Forty-eight hours after the transfection, the supernatant obtained by filtration through a 0.45 μm filter was collected, and the collected supernatant was directly used for infection of MRC-5 cells. Although...

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Abstract

The present invention aims to elucidate a miRNA involved in cellular senescence and to provide a method of use thereof. The senescence marker of the present invention comprises a gene transcript of miR-22. Further, the method for evaluating a senescence inhibitor of the present invention comprises the step of measuring the expression level of a gene transcript of miR-22 in a sample in the presence of a test compound and in the absence of the test compound; and the step of comparing the expression level of the gene transcript of miR-22 in the sample in the presence of the test compound with the expression level of the gene transcript of miR-22 in the sample in the absence of the test compound. Further, the cancer inhibitor of the present invention comprises as an effective component a gene transcript of miR-22, which cancer inhibitor promotes cellular senescence and inhibits invasion and / or metastasis of cancer.

Description

CROSS REFERENCES TO RELATED APPLICATIONS[0001]This application is a U.S. National Phase application of International Patent Application No. PCT / JP2010 / 072592, filed Dec. 15, 2010, and claims priority to Japanese Patent Application No. 2009-288707, filed Dec. 21, 2009, and Japanese Patent Application No. 2010-049928, filed Mar. 5, 2010, each of which is incorporated herein by reference in its entirety.TECHNICAL FIELD [0002]The present invention relates to a senescence marker and a method for evaluating a senescence inhibitor using as an index the expression level of hsa-miR-22, and a cancer inhibitor using a cellular senescence promoting action of hsa-miR-22.BACKGROUND ART [0003]MicroRNAs (hereinafter referred to as miRNAs) are short non-coding RNAs identified in the genomes of a wide variety of species. miRNAs were first discovered in 1993 in Caenorhabditis elegans, and this was followed by discovery of miRNAs in many other multicellular organisms. A miRNA is a negative regulator of...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/02C12Q1/68
CPCC12Q1/6886C12Q2600/136C12Q1/6876C12N15/113C12N2310/141C12Q2600/158
Inventor TAHARA, HIDETOSHI
Owner HIROSHIMA UNIVERSITY
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