TW201502277A - Detection method for urothelial carcinoma - Google Patents

Abstract

The purpose of the present invention is to provide a novel method for detecting urothelial carcinoma. According to several aspects of the present invention, the followings are provided: a detection method for urothelial carcinoma comprising a step of measuring the changes of expression level of miR-519a in urine sample; a screening method for a substance effective in prophylaxis and/or treatment of urothelial carcinoma based on the changes of expression level of miR-519a in cells; and a kit useful in the above detection method and above screening method.

Description

Method for detecting urothelial carcinoma

The present invention relates to a method for detecting urothelial carcinoma.

The urothelial carcinoma is a general term for malignant tumors that occur in the urothelium. The site of urothelial cancer occurs throughout the urinary tract, which includes the renal pelvis, ureter, bladder, and urethra. The incidence of bladder cancer in urothelial carcinoma in Japan is about 20 (male) relative to the population of 100,000 people, and it has a tendency to increase in recent years. The prevalence rate in Europe and the United States is significantly higher, and the urothelial cancer system is globally demanding countermeasures. If it is non-invasive, for example, as a part of a health check to assess the risk of cancer caused by urothelial cancer, preventive measures to limit urothelial cancer such as smoking, which are called risk factors, may be used for the risk group. Countermeasures for early diagnosis such as urine cell diagnosis.

Urinary tract carcinoma has a characteristic temporal/spatial multipleity of the urinary tract as a whole. This is thought to be caused by a field effect that causes cancer (Non-Patent Document 1). It is known that in patients with upper urothelial carcinoma such as renal pelvic cancer and ureteral cancer, even if the primary cancer is cured by total ureteral resection, 10 to 30% of patients will develop urothelial carcinoma in the bladder from time to time. . Therefore, in the current situation, patients with postoperative renal pelvic/ureteral cancer must repeat an invasive cystoscopy. In order to alleviate the burden of these patients It is required to improve the diagnostic efficiency of metachronous urothelial carcinoma by predicting renal pelvis/ureteral cancer with a non-invasive prognosis.

In the past, urothelial carcinoma was examined by invasive endoscopy. Check the biopsy to diagnose. However, endoscopy is highly reliable in the diagnosis of bulging lesions, but it is difficult to diagnose carcinoma in situ (CIS; intraepithelial neoplasia). Further, in the case of cancer of the upper urinary tract (kidney/ureter), the endoscope method is invasive and it is not possible to use the biopsy forceps, and thus it is particularly difficult to diagnose. On the other hand, urine cell diagnosis, which is useful as a non-invasive test, is highly specific (90 to 95%) but low in sensitivity (about 30%). Therefore, there is a problem that the presence of cancer cannot be denied even if the examination is negative, and the result may vary depending on the subjective state of the diagnosis.

Urinary NMP-22 developed for the detection of urothelial carcinoma The sensitivity/specificity of the new marker such as BTA is about 50 to 60%, and even if it is a urinary tract infection, it does not exceed the level of the auxiliary diagnosis, and it is not popular. In addition, other markers of Urovysion are detected by fluorescence in situ hybridization (FISH) to detect genetic abnormalities in urinary exfoliated cells, which has a high cost of examination. For these reasons, the development of novel diagnostic markers for urothelial cancer is a priority.

In addition, miRNA (microRNA; microRNA) is intracellular Non-coding RNA of about 20 to 25 bases. The miRNA system is first transcribed from a miRNA gene on the genomic DNA into a primary transcript (pri-miRNA) of several hundred to several thousand bases in length. Next, it is processed to become a pre-miRNA having a hairpin structure of about 60 to 70 bases. Thereafter, it moves from the nucleus to the cytoplasm and is further processed into a mature miRNA comprising a dimer of about 20 to 25 bases (including a guide strand and a passenger strand). It is known that mature miRNAs form a complex with a protein called RISC (RNA-Induced Silencing Complex) through a guide strand (antisense strand) and act on the target. The mRNA of the gene inhibits the translation of the target gene (postogenic transcriptional regulation). Hundreds of human miRNAs are now reported in human genomes (miRBase::Sequences, Sanger Institute).

The inventors have established in the past the urine that will be taken from humans. The change in the amount of expression of miR-96 or miR-183 in the cells in the sample (especially hyperactivity) is used as an indicator for detecting urothelial carcinoma and is patented (Patent Document 1).

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. 2011-036242

[Non-patent literature]

[Non-Patent Document 1] Harris, A.L. and Neal, D.E., N. Engl. J. Med., 1992, 326: 759-761

As mentioned above, although the proposal to date has used miRNA detection The technique of measuring urothelial carcinoma, but the current situation is decisively effective as a detection technique for urothelial carcinoma has not yet been developed. Moreover, from the point of view of the calibration of the enrichment detection technique, it is important to develop a novel detection method for urothelial cancer.

Accordingly, it is an object of the present invention to provide a novel method for detecting urothelial carcinoma.

The present inventors conducted intensive studies in view of the above problems. As a result, it was found that urothelial carcinoma can be detected with high specificity/sensitivity by measuring the change in the amount of expression of miR-519a in the urine sample, and the present invention has been completed.

That is, according to an aspect of the present invention, a method for detecting urothelial carcinoma comprising the step of measuring a change in the amount of expression of miR-519a in a urine sample is provided.

Further, according to another aspect of the present invention, there is provided a method for screening a substance effective for the prevention and/or treatment of urothelial cancer, which is based on a change in the amount of expression of miR-519a in a cell.

According to another aspect of the present invention, a kit for use in the above detection method or screening method is further provided.

In accordance with the present invention, novel methods for detecting urothelial carcinoma are provided. Further, screening methods for substances effective for prevention and/or treatment of urothelial cancer, and kits for such methods are also provided.

Fig. 1 is a diagram showing the statistical comparison (man-Whitney U test) of urothelial carcinoma patients (30 persons) and non-cancer patients (59 persons) of hsa-miR-519a. A graph of the results of the performance.

Figure 2 is a graph showing the results of the performance of hsa-miR-519a in patients with urothelial carcinoma (30 patients) and non-cancer patients (59 patients) by Receiver Operating Characteristic (ROC) curve analysis in the examples. .

Figure 3 is a graph showing the statistical comparison (U-test of the Mann-Whitney) in patients with urothelial carcinoma (30 patients), among patients with high grade (10) and the degree of progression. A graph of the results of the performance of hsa-miR-519a in patients with low grade (14 patients).

Hereinafter, the form for carrying out the invention will be described. Furthermore, in the following description, the miRNA, pri-miRNA, and pre-miRNA lines will be paired with the mRNA of the final target gene, and the mRNA is an antisense strand (or an RNA region containing the antisense strand) share". On the other hand, a meaningful share (or an RNA field containing the meaningful share) for the aforementioned antisense stock is expressed as a "passenger share".

Detection method of urinary tract cancer

According to an aspect of the present invention, a method for detecting urothelial carcinoma comprising the step of measuring a change in the amount of expression of miR-519a in a urine sample is provided.

Here, information about the miR-519a measured in the method of the present invention (for example, a base sequence, etc.) can be obtained from the above database. (miRBase::Sequences, Sanger Institute) is easy to obtain.

As shown in the examples below, it is determined that the urothelial carcinoma is fine. Compared with normal cells, the expression of miR-519a in urine samples was significantly increased. Therefore, more specifically, urothelial carcinoma can be detected by a method of measuring the increase in the amount of expression in the urine sample of miR-519a. Here, "the amount of expression of miR-519a is significantly increased (increased)" means, for example, the miR-519a of the cells in the urine sample when determined based on the cutoff value in the ROC curve analysis described in the examples below. The ratio of the amount of expression to the amount of expression of cells derived from normal tissues is a predetermined value or more. The "predetermined value" is, for example, about 10 (in this case, the expression amount of miR-519a is increased by about 10 times or more compared with the cells derived from normal tissues), preferably about 20, and more preferably about 30. The urothelial cancer can be detected by using the predetermined value or more as a (determination) criterion.

Here, it is used as an inspection object in the embodiment described later. When the urine sample of a patient with hematuria is visually observed, the previously known detection method for urothelial carcinoma such as miR-96 or miR-183 cannot be sensitive or specific when measuring the performance of the miRNA. Sexuality can be satisfied, and the identification of cancer and non-cancer is not sufficient. The present inventors presume that the reason is that red blood cells miR-96 and miR-183 originally exhibit a considerable amount, and non-cancer diseases (urinary tract infections, stones) often have hematuria, which makes it difficult to correctly diagnose the concurrent Non-cancer patients with hematuria. In contrast, according to the detection method of the present invention, urothelial carcinoma can be detected as described above with extremely high sensitivity and specificity.

From the above knowledge, can be based on the performance of miR-519a The amount of sputum is introduced to screen for substances effective for the prevention and/or treatment of urothelial cancer. More specifically, it may be screened by the following steps: (a) a step of culturing the cells in the presence of the test substance; (b) a step of measuring the amount of expression of the miR-519a of the test cells; and (c) The step of selecting a substance that inhibits the increase or decrease in the amount of performance.

In such a screening method, for example, when cells derived from urothelial carcinoma are used, a substance effective for the treatment of urothelial carcinoma can be screened. On the other hand, when a cell derived from a normal tissue in a state in which canceration is promoted (for example, in the case where a known carcinogen is present, irradiation of radiation, or the like) is used, a substance effective for prevention of urothelial cancer can be screened.

In the above detection method and screening method of the present invention, the amount of expression of miR-519a can be determined by any method known to those skilled in the art. For example, a primer or probe that specifically hybridizes to miR-519a can be used to determine the amount of expression of miR-519a. Such a primer or a probe can be appropriately designed according to the base sequence of miR-519a by referring to the information of the above-mentioned database as long as it is a person in the field. Further, the extraction of the miRNA from the urine sample and the preparation of the sample can also be carried out by any method known to those skilled in the art.

For the measurement method using such a primer or a probe, the northern ink dot method is a classical method. Recently, microarrays carrying miRNAs have been reported (Liu CG et al, (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues, PNAS, USA, 101, 9740-9744; Lim LP et al, (2005) Microarray analysis shows that some microRNAs downregulate large Numbers of target mRNAs, Nature, 433, 769-773), Modified Invaders Detection (Allawi HT et al, (2004) Quantitation of microRNAs using a modified Invader assay, Rna., 10, 153-1161), according to the flow of beads Cytometry (Lu J et al, (2005) MicroRNA expression profiles classify human cancers, Nature, 435, 834-838) and the like. Moreover, in terms of the most quantitative method, there is a real-time PCR method of quantitative PCR (Chen C et al, (2005) Real-time quantification of microRNAs by stem-loop RT-PCR, Nucleic Acids Res, 33, e179). When miR-519a is quantified by the real-time PCR method, a stem-loop RT primer that specifically hybridizes to miR-519a can be used.

The base sequence of the above primer or probe has a modulo The number of appropriate bases to which the template is specifically bound is, for example, preferably several tens of bp and about 10 to 30 bp, and a commercially available primer design software such as OligoTM (manufactured by National Bioscience Inc.) can be used.

Set used in the detection method or screening method of the present invention The group can take an appropriate configuration depending on the object to be measured, the measurement principle, and the like. The kit may include, for example, a primer for amplification of the above-described mRNA (cDNA) and a probe for hybridization such as a DNA wafer (microarray). Further, the above-mentioned kits include other elements or components known to those skilled in the art in view of the constitution and purpose of use, such as various reagents, enzymes, buffers, reaction trays (containers) and the like. Further, in order to facilitate the detection after the PCR reaction, it is preferred to bind at least one of the primers to a labeling substance such as any fluorescent substance known to those skilled in the art. For example, just Examples of the fluorescent substance include 6-carboxyfluorescein (FAM), 4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein (HEX), NED. (Applied Systems Japan) and 6-carboxy-X-rosin (Rox).

Furthermore, by combining (using) the above-mentioned urothelial cancer test The method of measurement and urine cell examination can determine urothelial carcinoma with high detection rate and non-invasiveness. The urinary cell test can be carried out by a method/means known to those skilled in the art as described in, for example, "Cell Diagnostic Specimen Production Manual (Urbilis)" (published by the Cell Inspector Association, 2004). For example, about 50 mL or more of urine is collected, and the urine sediment is obtained by centrifugation, and Papanicolaou stain is observed by a microscope, and the cancer is determined by the abnormality of the nucleus. Usually judged by the 5-stage method, class I, II is judged to be negative, class III is judged to be false positive, class IV, and V is judged to be positive. Therefore, the method for detecting urothelial carcinoma of the present invention preferably further comprises the step of performing a urine cell test.

(Example)

Hereinafter, the present invention will be described in detail by way of examples, but the technical scope of the present invention is not limited by the description below. Many variations and modifications can be made without departing from the spirit and scope of the invention. Further, the following examples are based on, 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 in Molecular Biology, John. Standard gene known to those in the field as described by Wiley & Sons, New York, NY, 1995, etc. Implemented in engineering and molecular biology techniques. Furthermore, the contents of the documents cited in the present specification form part of the disclosure and contents of the present specification.

(miRNA-specific reverse transcription reaction and quantitative PCR)

From the time of external examination, 89 patients who presented with hematuria by naked eyes took about 10 mL of each urine sample. Next, the urine sample taken was centrifuged at 3000 rpm x 15 minutes (4 ° C), leaving about 1 mL of sediment and the supernatant was removed by decantation. Then, it was stored at -80 ° C until the RNA extraction treatment described later.

Total RNA extracted from the urine sample (Total RNA) lines were used mirVana ^TM PARIS ^TM Kit (Ambion (registered trademark)) from a urine specimen 50μL.

Then, from the extracted total RNA, Stem-loop RT-PCR using a stem-loop RT primer specific for hsa-miR-519a by using real-time PCR (7300HT Real-Time PCR System; Applied Biosystems (registered trademark)) The method (TaqMan (registered trademark) MicroRNA Assays), and hsa-miR-519a was specifically amplified and quantified. The PCR conditions were set to (95 ° C for 15 seconds → 60 ° C for 1 minute) × 40 cycles.

In the subsequent confirmed diagnosis, 30 of these patients were diagnosed with urothelial carcinoma, 24 were diagnosed with urinary tract infection, 17 were diagnosed with urinary calculi, and 18 were diagnosed with other benign disease. The results of the statistical comparison (U-test of Mann-Whitney) for hsa-miR-519a in urothelial carcinoma patients (30 patients) and non-cancer patients (59 patients) are shown in Fig. 1. From the results shown in Fig. 1, it was found that the performance of hsa-miR-519a was significantly increased in urothelial patients compared with non-cancer patients.

Further, the performance of hsa-miR-519a in the above urothelial carcinoma patients (30 patients) and non-cancer patients (59 patients) was evaluated by Receiver Operating Characteristic (ROC) curve analysis. The results are shown in Figure 2. As shown in Fig. 2, the sensitivity is 70.0% and the specificity is 74.8%, which can distinguish between cancer and non-cancer.

Furthermore, in the same manner as above, in the statistical comparison (U-Hitney's U-test) urothelial cancer patients (30 patients), patients with high grade (10) and progress were compared. The results of the performance of hsa-miR-519a in patients with low grade (14 persons) are shown in Fig. 3. From the results shown in Fig. 3, it was found that patients with High grade showed a significant increase in the performance of hsa-miR-519a compared with patients with Low grade.

Furthermore, although the diagnosis of urothelial carcinoma was negative by the urine cell diagnosis used in the past, 11 patients who were diagnosed as urothelial carcinoma by pathology were finally detected, and hsa-miR- was detected in the same manner as described above. 519a, 9 people were correctly diagnosed as positive. Thus, the sensitivity of the detection method of the present invention is high.

As described above, the detection of miRNA can be carried out stably from the urine sediment sample. Further, since it is a detection method based on the PCR method, it can be measured as long as it is a general inspection institution. Furthermore, the cost of detection is about 1500 yen per sample, which is very inexpensive, and the time required for the measurement is as fast as about 4 hours. Moreover, since the diagnosis uses a clear cutoff value, there is an advantage that the subject is not subjectively subject to the diagnosis.

In the present embodiment, when a urine sample of a patient who presents hematuria as a subject to be examined is used, it is conventionally known to detect The detection method of urothelial carcinoma such as miR-96 or miR-183 cannot obtain the sensitivity and specificity when the expression of these miRNAs is measured, and the cancer and non-cancer identification systems are insufficient. The present inventors presume that the reason is that red blood cells miR-96 and miR-183 originally exhibit a considerable amount, and non-cancer diseases (urinary tract infections, stones) often have hematuria, which makes it difficult to correctly diagnose the concurrent Non-cancer patients with hematuria. In contrast, according to the detection method of the present invention, urothelial carcinoma can be detected as described above with extremely high sensitivity and specificity.

Claims (12)

A method for detecting urothelial carcinoma, comprising the step of measuring a change in the amount of expression of miR-519a in a urine sample. The method for detecting urothelial carcinoma according to claim 1, which comprises the step of measuring the amount of expression of miR-519a. A method for detecting urothelial carcinoma, which is characterized in that the amount of expression of miR-519a in a urine sample is more than 10 times higher than that of cells derived from normal tissues as a criterion for urothelial carcinoma. The method for detecting urothelial carcinoma according to any one of claims 1 to 3, wherein the expression amount of miR-519a is measured using a primer that specifically hybridizes with miR-519a. The method for detecting urothelial carcinoma according to the fourth aspect of the invention, wherein the amount of expression of miR-519a is measured by an instant PCR method. The method for detecting urothelial carcinoma according to any one of claims 1 to 3, further comprising the step of performing a urine cell test. A method for screening a substance effective for the prevention and/or treatment of urothelial carcinoma, which is based on a change in the amount of expression of miR-519a in a cell. A method for screening a substance effective for prevention and/or treatment of urothelial cancer according to claim 7 of the patent application, comprising the steps of: (a) culturing a cell in the presence of a test substance; (b) a step of measuring the amount of expression of the miR-519a of the test cell; and (c) a step of selecting a substance that inhibits the amount of expression or decreases the amount of expression. Prevention of urothelial carcinoma as described in item 8 of the patent application scope and/or Or a method for screening a therapeutically effective substance, wherein in the step (b), the expression amount of miR-519a is measured using a primer which specifically hybridizes with miR-519a. A method for screening a substance effective for the prevention and/or treatment of urothelial carcinoma according to claim 9 of the invention, wherein the amount of expression of miR-519a is measured by an instant PCR method. A kit for use in the method of any one of claims 1 to 3, 7 to 10. A kit according to claim 11 which contains a primer which specifically hybridizes to miR-519a.