JP2013055969A - METHOD FOR DETECTING BLADDER CANCER BASED ON microRNA EXPRESSION PROFILING - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting bladder cancer including elucidation of miRNA functions, especially, in relation to canceration of cells and measurements of the variations in the expression level of miRNA.SOLUTION: The method for detecting bladder cancer includes measurements of the variations in the expression level of at least one miRNA, selected from the group consisting of miR-96, miR-190, miR-183, miR-130b, miR-124b, miR-215, miR-224, miR-106a, miR-145, miR-30a-3p, miR-100, miR-150, miR-133a, miR-320, miR-133b, miR-151, miR-195, miR-125b, miR-152, miR-218, miR-199-s, miR-199a, miR-199a*, miR-223, miR-139, miR-9, miR-140 within cells.

Description

  The present invention relates to a method for detecting bladder cancer and the like, comprising measuring fluctuations in the expression level of microRNA.

A mature microRNA (miRNA) is a 19- to 21-base non-coding RNA molecule, and consists of a precursor microRNA (pre-miRNA) having a hairpin structure of about 70 to 100 bases. As of March 2007, 500 human miRNAs have been reported to exist in the genome (miRBase :: Sequences, Sanger Institute). The miRNA function acts in a sequence-dependent manner in the untranslated region of the gene and regulates gene expression after translation or at the transcriptional level, suggesting that it is involved in development, differentiation and apoptosis. The function is still unknown. In recent years, it has been reported that the wrong gene regulation mechanism of miRNA is involved in human diseases. Particularly recently, there have been reports of miRNAs whose expression is varied in human cancer cells (review Calin and Croce, 2006).

Chronic lymphocytic leukemia was first reported to be involved in human cancer, indicating that miR-15 or miR-16 expression is reduced in cancer cells (Calin et al, 2002). Subsequently, in solid cancer, it has been reported that miR-17-92 is highly expressed in lung cancer cells (Hayashida et al, 2005). Furthermore, miRNA signatures have been reported from analysis using 6 types and 540 cases of solid cancer clinical specimens (Volinia et al, 2006).

Calin GA and Croce CM (2006) MicroRNA signatures in human cancers, Nature Reviews, 6, 857-866. Volinia S et al, (2006) A microRNA expression signature of human solid tumors define cancer gene targets, PNAS, USA, 103, 2257-2261.

Thus, about 500 types of miRNAs present in the genome have been reported at present, but the functions of many of these miRNAs are still unclear. In particular, miRNA regulates the expression of many genes, suggesting that it is involved in human diseases, and has recently attracted attention as an oncogene or tumor suppressor gene. Therefore, an object of the present invention is to elucidate the function of miRNA, particularly the relationship with cell carcinogenesis.

This time, the present inventor performed expression profiling of 156 miRNAs present in the genome using a bladder cancer clinical specimen and a cell line. As a result, it was found that the expression of 27 types of miRNAs varied in bladder cancer. These miRNAs are thought to play an important role in the canceration process of bladder cancer. The present invention has been completed based on such findings.

That is, the present invention mainly includes the following aspects.
[1] miR-96, miR-190, miR-183, miR-130b, miR-124b, miR-215, miR-224 and miR-106a in cells, and miR-145, miR-30a-3p, miR-100, miR-150, miR-133a, miR-320, miR-133b, miR-151, miR-195, miR-125b, miR-152, miR-218, miR-199-s, miR-199a, A method for detecting bladder cancer, comprising measuring a change in the expression level of at least one miRNA selected from the group consisting of miR-199a *, miR-223, miR-139, miR-9 and miR-140.
[2] miR-96, miR-190, miR-183, miR-130b, miR-124b, miR-215, miR-224 and miR-106a in cells, and miR-145, miR-30a-3p, miR-100, miR-150, miR-133a, miR-320, miR-133b, miR-151, miR-195, miR-125b, miR-152, miR-218, miR-199-s, miR-199a, A substance effective for the treatment or prevention of bladder cancer based on a change in the expression level of at least one miRNA selected from the group consisting of miR-199a *, miR-223, miR-139, miR-9 and miR-140 Screening method.
[3] A screening kit used in the screening method of the present invention.

In the present invention, bladder cancer can be detected by measuring the variation in the expression level of a specific miRNA, and further, a substance effective for the treatment or prevention of bladder cancer is screened based on the variation in the expression level of these miRNAs. It became possible to do.

27 types of miRNAs measured in the method of the present invention: miR-96, miR-190, miR-183, miR-130b, miR-124b, miR-215, miR-224 and miR-106a, and miR- 145, miR-30a-3p, miR-100, miR-150, miR-133a, miR-320, miR-133b, miR-151, miR-195, miR-125b, miR-152, miR-218, miR- Information on 199-s, miR-199a, miR-199a *, miR-223, miR-139, miR-9 and miR-140 (for example, nucleotide sequences, etc.) can be found in the already described database Readily available from the Institute).

8 miRNAs among the above 27 miRNAs (first group): miR-96, miR-190, miR-183, miR-130b, miR-124b, miR-215, miR-224 and miR-106a As for, the expression level was found to be significantly enhanced in bladder cancer cells compared to normal cells. Therefore, more specifically, bladder cancer can be detected by measuring the increase (increase) in the expression level of at least one of these miRNAs. In this case, the expression level of miRNA is significantly increased, for example, the expression level of miRNA is about 2 times or more, preferably about 5 times or more, more preferably about 10 times compared to cells derived from normal tissue. It can be determined whether or not it is bladder cancer on the basis of this.

  On the other hand, 19 other miRNAs (second group): miR-145, miR-30a-3p, miR-100, miR-150, miR-133a, miR-320, miR-133b, miR-151, miR- 195, miR-125b, miR-152, miR-218, miR-199-s, miR-199a, miR-199a *, miR-223, miR-139, miR-9 and miR-140, their expression levels Was found to be significantly reduced (decreased) in bladder cancer cells compared to normal cells. Therefore, more specifically, bladder cancer can be detected by measuring a decrease in the expression level of at least one of these miRNAs. In this case, the expression level of miRNA is significantly reduced. For example, the expression level of miRNA is about 1/2 or less, preferably about 1/5 or less, compared to cells derived from normal tissue. Preferably, it is about 1/10 or less, and based on this, it can be determined whether or not it is bladder cancer.

Therefore, a substance effective for the treatment or prevention of bladder cancer can be screened based on the increased expression level of at least one miRNA included in the first group. More specifically, the following steps:
(a) culturing cells in the presence of a test substance,
(b) measuring the expression level of each miRNA in the cell, and
(c) screening by a method comprising a step of selecting a substance that suppresses the increase in the expression level or decreases the expression level.

Furthermore, a substance effective for treating or preventing bladder cancer can be screened based on a decrease in the expression level of at least one miRNA contained in the second group. More specifically, the following steps:
(a) culturing cells in the presence of a test substance,
(b) measuring the expression level of each miRNA in the cell, and
(c) screening by a method comprising a step of selecting a substance that suppresses the decrease in the expression level or increases the expression level.

  In such a screening method, for example, when a cell derived from bladder cancer is used, it is possible to screen a substance effective for the treatment of bladder cancer. On the other hand, when cells derived from normal tissue placed under conditions that promote canceration (for example, coexistence of known carcinogens, irradiation of radiation, etc.), the test substance is bladder cancer. It is possible to screen for substances that are effective in preventing the disease.

  In the detection method and screening method of the present invention, the expression level of miRNA can be measured by a method known to those skilled in the art. For example, the expression level of miRNA can be measured using a primer or probe that specifically hybridizes to each miRNA. Such a primer or probe can be appropriately designed by those skilled in the art based on the base sequences of these miRNAs with reference to the information in the above database.

As a measuring method using such a primer or probe, the Northern blot method is a classic method. Recently, miRNA-loaded microarray (Liu et al, 2004; Lim et al, 2005), improved invader method (Allawi et al, 2004), and bead-based flow cytometer method (Lu et al, 2005) Etc. have been reported. The most quantitative method is real-time PCR (Chen et al, 2005).

The base sequence of the above-mentioned primer or probe preferably has an appropriate number of bases capable of specific binding to the template, for example, several tens of bp, about 10 to 30 bp. It is also important to have a hairpin structure or a base sequence that does not anneal the sense strand and the antisense strand to each other. For example, commercially available primer design software such as Oligo ™ (National Bioscience Inc.) can be used.

The kit used in the screening method of the present invention can have an appropriate configuration depending on the measurement object or measurement principle. The kit can contain, for example, the above-described primers for amplification of mRNA (cDNA) and a probe for hybridization used in a DNA chip or the like. Further, the kit includes other elements or components known to those skilled in the art, such as various reagents, enzymes, buffers, reaction plates (containers), and the like, depending on the configuration and purpose of use. In order to facilitate detection after the PCR reaction, it is preferable that a labeling substance such as an arbitrary fluorescent substance known to those skilled in the art is bound to at least one end of these primers. For example, suitable fluorescent substances include 6-carboxyfluorescein (FAM), 4,7,2 ′, 4 ′, 5 ′, 7′-hexachloro-6-carboxyfluorescein (HEX), NED (Applied Systems Japan) And 6-carboxy-X-rhodamine (Rox).

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is limited and is not interpreted at all by description of a following example. A person skilled in the art can make many variations and modifications based on the description of the present specification without departing from the technical scope of the present invention. Also, unless otherwise stated, the following examples are described in, for example, Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols. It can be carried out according to standard genetic engineering and molecular biology techniques known to those skilled in the art, as described in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. In addition, the description content of the literature referred in this specification comprises the indication of this specification, and a part of content.

(Method)
miRNA-specific reverse transcription and quantitative PCR
Trizol (Gibco BRL, Life Technologies, Gaitherburg, MD) total RNA from 19 clinical specimens of bladder cancer patients (14 specimens from cancer and 5 specimens from non-cancerous part) and 3 kinds of cell lines derived from bladder cancer (BOY, KK47, T24) USA) and extracted. cDNA was synthesized from the obtained total RNA using miRNA-specific primers (TaqMan microRNA Assay Protocol, PE, Applied Biosystems, Foster City, CA, UAS). The reverse transcription reaction was performed as follows. 10 nM RNA samples, 50 nM stem-loop RT primer, 1 x RT buffer, 0.25 mM each of dNTPs, 3.33 U / μl MultiScribe reverse transcriptase and 0.25 U / μl RNase Inhubitor. Further, these 7.5 μl reaction solutions were incubated according to the following protocol. It was stored at 4 ° C after the reaction at 16 ° C for 30 minutes, 42 ° C for 30 minutes, 85 ° C for 5 minutes. Quantitative PCR was performed in the following reaction system using ABI 7300 Sequence Detection System. After incubation at 95 ° C for 10 minutes, the reaction was carried out 40 times at 95 ° C for 15 seconds and at 60 ° C for 10 minutes.

Quantification of 1156 miRNAs and selection of miRNAs that change in expression in bladder cancer Database of 19 clinical specimens of bladder cancer patients (14 specimens of cancer and 5 specimens of non-cancerous part) and 3 types of cell lines derived from bladder cancer (miRBase :: Sequences, Sanger Institute) 156 miRNAs and control genes were analyzed. The analysis results were normalized using the global median normalization method used in microarrays. From this analysis data, genes having a difference in expression predominantly (P <0.01) in cancerous and non-cancerous parts were selected. As a result, it was found that the expression of 27 types of miRNAs was significantly changed in the cancerous part (eight kinds of miRNAs increased in expression in the cancerous part and 19 kinds of miRNAs decreased in expression in the cancerous part). The list is shown in Table 1 and Table 2 below.

Furthermore, as a result of cluster analysis using 27 miRNAs whose expression was significantly changed in bladder cancer (P <0.01), 5 normal transitional epithelia and 3 cancer cell lines belonged to the same cluster. The 14 cancer samples were divided into two clusters, 11 samples and 3 samples, but they were found to be different from the normal cluster.

(Discussion)
In this analysis, 27 miRNAs were listed as miRNAs related to bladder cancer. Among them, miR-30a-3p, miR-133b, and miR-145 among miRNAs whose expression is decreased in the cancerous part compared to normal transitional epithelium are also colorectal cancer profiles using the ABI TaqMan method. Was also reported to be decreased in the cancer site (Bandres et al, 2006). miR-145 has been reported to decrease in lung cancer (Yanaihara et al, 2006) and breast cancer (Iorio et al, 2005) as well as colorectal cancer (Cummins et al, 2006). It is suggested that it is miRNA that plays a role.

The characteristic of miRNA is that gene expression is controlled at the post-translational or transcriptional level, and it is suggested that one miRNA controls several tens to several hundreds of genes. However, the prediction of the target gene is insufficient only by sequence homology, and the target gene for each miRNA is still unknown. It is expected that miRNA target genes whose expression is reduced in the cancer site are involved in the development and progression of cancer. In other words, it can be easily imagined that the genes that miRNAs originally control become involved in canceration because they become uncontrollable due to miRNA expression decrease. A typical example is that the target gene of let-7 whose expression is decreased in lung cancer is RAS of oncogene. The target gene of miR-145 is expected to be MYCN, FOS, YES of the oncogene, cyclin D2 of the cell cycle gene, or MAP3K3 or MAPK4K4 which are MAPK signals (Iorio et al, 2005).

Similarly, this analysis suggests that miR-133b, whose expression was reduced in cancerous sites, is targeted for RAS (Bandres et al, 2006). More interestingly, YES and MAP3K3 may be targets for both miR-133b and miR-145 (Bandres et al, 2006). In bladder cancer, expression of miR-133a, which is very similar in sequence to miR-133b, is also reduced in the cancerous part. This is very interesting because both miRNAs may jointly control the same target gene.

On the other hand, miRNA whose expression is enhanced in the cancerous part is considered to have a function as an oncogene. That is, it is highly possible that miRNA is excessively expressed, thereby causing a loss of function by further controlling the target gene. In this analysis, eight miRNAs were found to be upregulated in the cancerous part. Among them, miR-96 and miR-183 have been reported to be highly expressed in colorectal cancer (Bandres et al, 2006). These two miRNAs are known to form a cluster on chromosome 7q32.2, and it is necessary to investigate the relationship with chromosome amplification in bladder cancer cells.

In addition, the well-known literature quoted with the number in a parenthesis in this specification is as follows.
1. Calin GA and Croce CM (2006) MicroRNA signatures in human cancers, Nature Reviews, 6, 857-866.
2. Calin GA et al, (2002) Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia, PNAS, USA, 99, 15524-15529.
3.Hayashida Y et al, (2005) A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation.Cancer Res, 65, 9628-9632.
4.Volinia S et al, (2006) A microRNA expression signature of human solid tumors define cancer gene targets, PNAS, USA, 103, 2257-2261.
5. Liu CG et al, (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues, PNAS, USA, 101, 9740-9744.
7. Lim LP et al, (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs, Nature, 433, 769-773.
8. Allawi HT et al, (2004) Quantitation of microRNAs using a modified Invader assay, Rna., 10, 153-1161.
9. Lu J et al, (2005) MicroRNA expression profiles classify human cancers, Nature, 435, 834-838.
10. Chen C et al, (2005) Real-time quantification of microRNAs by stem-loop RT-PCR, Nucleic Acids Res, 33, e179.
11. Bandres E et al, (2006) Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues, Molecular Cancer, 5, 29.
12. Cummins JM et al, (2006) The colorectal microRNAome, PNAS, USA, 103, 3687-3692.
13. Hayashita Y et al, (2006) A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation, Cancer Res, 65, 9628-9632.
14.Iorio MV et al, (2006) MicroRNA gene expression deregulation in human breast cancer, Cancer Res, 65, 7065-7070.

In this study, we searched for miRNAs involved in bladder cancer from analysis using clinical specimens of bladder cancer. As a result, the identified miRNAs include miRNAs that have been reported in other cancers so far, indicating the validity of our analysis. These miRNAs are very useful as targets for treatment as well as diagnosis of bladder cancer.

Claims (5)

measuring the increase in the expression level of miR-183 in the cell, using as a criterion for determination of bladder cancer cells that the expression level of miR-183 is more than 10 times that of cells derived from normal tissues A method for detecting bladder cancer cells. The detection method according to claim 1, wherein the expression level of miR-183 is measured using a primer that specifically hybridizes to miR-183. The detection method according to claim 2, wherein the expression level of miR-183 is measured by real-time PCR. A screening method for a substance effective for treating or preventing bladder cancer, based on the increased expression level of miR-183 in a cell. A screening method according to claim 4, wherein
(a) culturing cells in the presence of a test substance,
(b) measuring the expression level of miR-183 in the cells, and
(c) selecting the substance which suppresses the increase in the expression level of miR-183 or decreases the expression level.