JP6898630B2 - Methods and kits that use the amount of miRNA as an indicator of urothelial cancer - Google Patents

Description

The present invention, the amount of miRNA regarding how and kits used as an indicator of the urothelial cancer.

Currently, in clinical practice, urinary cell examination is useful for diagnosing urothelial cancer (bladder cancer and renal pelvic ureteral cancer). However, although the urine cell test has high specificity (90 to 95%), it has low sensitivity (30 to 40%), and even if it is negative, cancer cannot be ruled out. Therefore, when urothelial cancer is suspected, invasive cystoscopy and ureteroscopy are commonly performed in addition to ultrasound and CT. In addition, regular endoscopy is required to confirm recurrence after treatment. Therefore, invasiveness to patients and medical costs have become problems.

MicroRNAs (miRNAs) are short RNAs of about 21 bases that regulate target genes primarily by translational inhibition and mRNA cleavage. Many genes are thought to be regulated by miRNAs. In addition, it has been reported that abnormal gene regulation by miRNA is involved in diseases such as cancer.

The present inventors have developed a method for detecting urothelial cancer such as bladder cancer using the expression level of a specific miRNA as an index (Patent Documents 1 and 2 and Non-Patent Document 1).

Japanese Patent Application Laid-Open No. 2009-100687 Japanese Unexamined Patent Publication No. 2011-36242

Yamada et al., Cancer Sci. 2011, Vol. 102, Issue 3, p. 522-529

An object of the present invention is to provide a method and a kit using the amount of miRNA as an index of urothelial cancer.

As a result of diligent studies to solve the above problems, the present inventors have found that the amount of microRNA in a sample derived from urine is affected by the contamination of urine with blood. Furthermore, we have found that only bladder-derived cells can be isolated and recovered from a mixture of blood cells and bladder-derived cells using a cell sorter, and miRNA whose expression is increased in urothelial cancer such as bladder cancer, leading to the completion of the present invention. It was.

That is, the present invention includes the following.
[1] a step of urothelial cells urine or et blood cells of the subject is removed to obtain a sample enriched with a cell sorter,
In the sample ,
miR-200c, miR-944, miR-1323, miR-205, miR-200a, miR-200b, miR-516b, miR-203, miR-210, miR-141, miR-518b, miR-135b, miR- 519a *, miR-518a-3p, miR-523 *, miR-519c-5p, miR-518e *, miR-519b-5p, miR-522 *, miR-429, miR-200b *, miR-512-5p , MiR-1283, miR-224, miR-515-5p, miR-1246, miR-183 *, miR-517b, miR-517a, miR-200a *, miR-1307, miR-523, miR-498, miR -516a-5p, miR-519a, miR-520a-3p, miR-515-3p, miR-1293, miR-512-3p, miR-520g, miR-524-5p, miR-301b, miR-518e, miR -518f, miR-21, miR-449c, miR-522, miR-1268, miR-519b-3p, miR-526b, miR-1269, miR-371-5p, miR-519d, miR-521, miR-3687 , MiR-141 *, miR-518c, miR-520f, miR-182, miR-518c *, miR-767-5p, miR-520h, miR-548x, miR-372, miR-525-5p, miR-520c -3p, miR-519c-3p, miR-934, miR-517c, miR-520d-3p, miR-183, miR-96, miR-466, miR-205 *, miR-520b, miR-200c *, miR -335 *, miR-526b *, miR-671-5p, miR-524-3p, miR-526a, miR-518d-5p, miR-520c-5p, miR-525-3p, miR-138, miR-518f *, MiR-708, miR-1292, miR-151-3p, miR-3180, miR-3180-3p, miR-1254, miR-31, miR-520a-5p, miR-371-3p, miR-1248, miR-3145, miR-149, and miR-449b
One or more miRNA amount the measuring step a including selected from the group consisting of,
A method in which the amount of miRNA is used as an index of urothelial cancer in the subject.
[2] The sample is recovered by using the cell sorter from the cell pellet of the urine, the method described in [1].
[3] The cell pellet of the urine is stored in a polyethylene glycol solution before being subjected to the cell sorter, the method described in [2].
[4] The method according to any one of [1] to [3], wherein the urothelial cancer of the subject is indicated when the amount of the miRNA is increased more than twice as much as the control amount.
[5] The method according to any one of [1] to [4], wherein the urothelial cancer is selected from the group consisting of renal cell carcinoma, ureteral cancer, bladder cancer, and urethral cancer.
[6] Described in any of [1] to [5], wherein the miRNA is selected from the group consisting of miR-200c, miR-205, miR-200a, miR-96, miR-183 and miR-519a. the method of.
[7] Below:
miR-200c, miR-944, miR-1323, miR-205, miR-200a, miR-200b, miR-516b, miR-203, miR-210, miR-141, miR-518b, miR-135b, miR- 519a *, miR-518a-3p, miR-523 *, miR-519c-5p, miR-518e *, miR-519b-5p, miR-522 *, miR-429, miR-200b *, miR-512-5p , MiR-1283, miR-224, miR-515-5p, miR-1246, miR-183 *, miR-517b, miR-517a, miR-200a *, miR-1307, miR-523, miR-498, miR -516a-5p, miR-519a, miR-520a-3p, miR-515-3p, miR-1293, miR-512-3p, miR-520g, miR-524-5p, miR-301b, miR-518e, miR -518f, miR-21, miR-449c, miR-522, miR-1268, miR-519b-3p, miR-526b, miR-1269, miR-371-5p, miR-519d, miR-521, miR-3687 , MiR-141 *, miR-518c, miR-520f, miR-182, miR-518c *, miR-767-5p, miR-520h, miR-548x, miR-372, miR-525-5p, miR-520c -3p, miR-519c-3p, miR-934, miR-517c, miR-520d-3p, miR-183, miR-96, miR-466, miR-205 *, miR-520b, miR-200c *, miR -335 *, miR-526b *, miR-671-5p, miR-524-3p, miR-526a, miR-518d-5p, miR-520c-5p, miR-525-3p, miR-138, miR-518f *, MiR-708, miR-1292, miR-151-3p, miR-3180, miR-3180-3p, miR-1254, miR-31, miR-520a-5p, miR-371-3p, miR-1248, miR-3145, miR-149, and miR-449b
A kit for use in the method according to any one of [1] to [6], comprising a polynucleotide for measuring the amount of one or more miRNAs selected from the group consisting of.

The present invention provides a method for detecting urothelial cancer.

FIG. 1 is a graph showing the results of quantitative PCR for evaluating the effect of blood contamination in urine on the amount of miRNA. FIG. 2 shows the results of cell sorter analysis of blood and bladder cancer cells expressing GFP. Figures 2a and 2b show the results of GFP-expressing bladder cancer cells. Figures 2c and 2d show blood results. Figures 2a and 2c are dot plots showing the results of analysis by the cell sorter. In FIGS. 2a and 2c, the vertical axis shows the lateral scattered light-area (SSC-A), and the horizontal axis shows the forward scattered light-area (FSC-A). 2b and 2d are graphs showing the wavelength range of light emitted by cells and the number of cells. "P6" indicates the wavelength range of green light. FIG. 3 shows the recovery of bladder cancer cells from a mixture of blood and bladder cancer cells expressing GFP. Figures 3a-3c show the layered gating used. In FIG. 3a, the vertical axis shows SSC-A and the horizontal axis shows FSC-A. In FIG. 3b, the vertical axis shows the forward scattered light-width (FSC-W), and the horizontal axis shows the forward scattered light-height (FSC-H). In FIG. 3c, the vertical axis shows the laterally scattered light-width (SSC-W), and the horizontal axis shows the laterally scattered light-height (SSC-H). FIG. 3d is a graph showing that the collected cells are cells that emit green light. FIG. 4 is a photograph showing the results of agarose gel electrophoresis to evaluate the degradation of RNA stored in 70% ethanol. FIG. 5 is a dot plot showing the results of cell sorter analysis of cells stored in different storage solutions. Figures 5a-5c show the results of the controls (without storage), Figures 5d-5f show the results of cells stored in RNAlater®, and Figures 5g-5i show the results stored in 30% polyethylene glycol. The result of the cell was shown. In FIGS. 5a, 5d and 5g, the vertical axis shows SSC-A and the horizontal axis shows FSC-A. In FIGS. 5b, 5e and 5h, the vertical axis shows FSC-W and the horizontal axis shows FSC-H. In FIGS. 5c, 5f and 5i, the vertical axis shows SSC-W and the horizontal axis shows SSC-H. FIG. 6 is a graph showing quantitative PCR results (Ct values) of miRNAs in urothelial cell fractions collected from urine and normal urine of patients with urothelial cancer using a cell sorter. Figures 6a-6c show the results of miR-200a, miR-205, and miR-200c, respectively.

(Detection method)
The present invention provides a method (or diagnostic method or method of assisting diagnosis) for detecting urothelial cancer in a subject.

As used herein, the term "urothelial cancer" refers to cancer that develops in the urothelium of the renal pelvis, ureter, bladder or urethra. Examples of urothelial cancers include renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

As used herein, the "subject" is preferably mammals such as primates (eg cynomolgus monkeys, chimpanzees and humans) and non-primates (eg cows, pigs, sheep, horses, cats, dogs, guinea pigs, rats). And mice), more preferably humans. In one embodiment, the subject may be a subject suspected of having urothelial cancer. Subjects suspected of having urothelial carcinoma can be identified by conventional testing methods (eg, urinalysis with urine test strips and urine cell testing). In one preferred embodiment, the subject may be a subject with hematuria. Hematuria can be easily determined by a person skilled in the art using, for example, a commercially available urine test paper.

In this method, a sample obtained from the urine of a subject is used. Urine has the advantage that it can be collected easily and painlessly without the need for special medical equipment. Urine contains small amounts of cells, such as urothelial cells. Urothelial cells (also called transitional epithelial cells) are cells that line the inside of the urethra from the renal pelvis through the ureter and bladder to the urethra. In one embodiment, the urothelial cells may be bladder-derived epithelial cells. In the method according to the present invention, the expression level of miRNA in a sample enriched with urothelial cells obtained from urine is used as an index.

Urine can include blood cells (eg, red blood cells and white blood cells) in addition to urothelial cells. The present inventors have found that the amount of miRNA in a sample derived from urine is affected by the contamination of blood cells with urine in the examples described later. In the method according to the present invention, in order to eliminate the influence of blood cell contamination on the amount of miRNA, a sample obtained from the urine of a subject, in which blood cells are removed and urothelial cells are enriched, is used. In the present specification, "removal" of blood cells is not only complete removal, but also 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more as compared with before treatment. Alternatively, it may include removal of 99.9% or more of blood cells. As used herein, "enrichment" (or purification or isolation) of urothelial cells means an increase in the relative amount of urothelial cells in a cell population. In one embodiment, a sample enriched with urothelial cells is urine in 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more or 99.9% or more of the total cell population. It may be a sample of urothelial cells.

Samples from which blood cells have been removed and enriched with urothelial cells may be obtained from the subject's urine by any method known in the art. The sample may be obtained, for example, using a commercially available blood cell lysing reagent. In one embodiment, the sample may have been recovered using a cell sorter. As used herein, the term "cell sorter" refers to a device that combines a flow cytometer with a function of sorting cells (sorting system). Generally, in a cell sorter, a thin fluid containing cells is irradiated with laser light, and the heights (H) of signals such as forward scatter light (Forward Scatter, FSC) and side scatter light (SSC) are generated. , Width (W) and area (A) (parameters) can be measured for individual cells and the cells of interest can be recovered based on the information obtained. The FSC provides information about cell size, and the SSC provides information about intracellular complexity and intracellular granules. As the cell sorter, a commercially available device such as BD FACSAria ™ II (BD Biosciences) can be used. The cell sorter can be operated by those skilled in the art according to the manufacturer's instructions. A person skilled in the art separates blood cells and urothelial cells by a cell sorter, and isolates (purifies) the urothelial cells based on information on cell size, internal structural complexity, and intracellular granules. Therefore, a sample in which blood cells are removed and urothelial cells are enriched can be obtained. For example, for bladder cells (eg, bladder cancer cells such as BOY-GFP cells) and blood cells, the height (H), width (W), and area (H), width (W), and area of the forward and side-scattered light signals, respectively. A) 6 parameters were measured to determine a parameter value setting that could sort only bladder cells, and the setting was used to obtain a sample from urine with blood cells removed and urothelial cells enriched. You may. The parameter value settings stored in the device in advance may be used.

Urine may be fed directly to a cell sorter to obtain a sample from which blood cells have been removed and enriched with urothelial cells. Alternatively, urine cell pellets (including blood cells and urothelial cells) may be subjected to a cell sorter to obtain a sample from which blood cells have been removed and enriched with urothelial cells. Urine cell pellets may be obtained using conventional methods, such as those used in urine cell testing. Urine cell pellets are produced, for example, by centrifuging urine (eg, 500 xg-2000 xg for 4-6 minutes, eg 5 minutes), separating into cell pellets and supernatant, and removing the supernatant. Obtainable. Removal of the supernatant may be performed, for example, by an ejector, pipette or decantation. Urine cell pellets may be delivered directly to the cell sorter. Alternatively, the urine cell pellet may be stored in a polyethylene glycol solution and then subjected to a cell sorter. The concentration of the polyethylene glycol solution is not particularly limited, but is, for example, 5 to 70 (w / v)%, 10 to 50 (w / v)%, 20 to 40 (w / v)%, or 25 to 35 (w). / v)% may be used. The polyethylene glycol solution may be a solution in an aqueous sodium chloride solution (eg, 1M-5M NaCl or 2M-4M NaCl). Storage in polyethylene glycol solution may be freezing (eg, at -20 ° C to -80 ° C). The storage period is not particularly limited, but may be, for example, in the range of 1 day to 1 year or 1 week to 1 month. Storage in a polyethylene glycol solution is preferable from the viewpoint of maintaining the quality of RNA and maintaining the separability by a cell sorter, as shown in Examples described later.

In the method according to the present invention, the following:
miR-200c, miR-944, miR-1323, miR-205, miR-200a, miR-200b, miR-516b, miR-203, miR-210, miR-141, miR-518b, miR-135b, miR- 519a *, miR-518a-3p, miR-523 *, miR-519c-5p, miR-518e *, miR-519b-5p, miR-522 *, miR-429, miR-200b *, miR-512-5p , MiR-1283, miR-224, miR-515-5p, miR-1246, miR-183 *, miR-517b, miR-517a, miR-200a *, miR-1307, miR-523, miR-498, miR -516a-5p, miR-519a, miR-520a-3p, miR-515-3p, miR-1293, miR-512-3p, miR-520g, miR-524-5p, miR-301b, miR-518e, miR -518f, miR-21, miR-449c, miR-522, miR-1268, miR-519b-3p, miR-526b, miR-1269, miR-371-5p, miR-519d, miR-521, miR-3687 , MiR-141 *, miR-518c, miR-520f, miR-182, miR-518c *, miR-767-5p, miR-520h, miR-548x, miR-372, miR-525-5p, miR-520c -3p, miR-519c-3p, miR-934, miR-517c, miR-520d-3p, miR-183, miR-96, miR-466, miR-205 *, miR-520b, miR-200c *, miR -335 *, miR-526b *, miR-671-5p, miR-524-3p, miR-526a, miR-518d-5p, miR-520c-5p, miR-525-3p, miR-138, miR-518f *, MiR-708, miR-1292, miR-151-3p, miR-3180, miR-3180-3p, miR-1254, miR-31, miR-520a-5p, miR-371-3p, miR-1248, miR-3145, miR-149, and miR-449b
The amount of one or more miRNAs selected from the group consisting of is used as an indicator of urothelial carcinoma. As used herein, microRNA (miRNA) also includes miRNA star (miRNA *) strands produced during its biosynthesis.

In the present invention, the amount of miRNA can also be used as an index for determining (predicting) the prognosis of urothelial cancer.

Sequence information for the above miRNAs from humans and other species can be found in the miRBase database (Kozomara A, Griffiths-Jones S. NAR 2014 42: D68-D73; Kozomara A, Griffiths-Jones S. NAR 2011 39: D152-D157. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ. NAR 2008 36: D154-D158; Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. NAR 2006 34: D140-D144; It is available from Griffiths-Jones S. NAR 2004 32: D109-D111). As an example, the base sequence of the above human miRNA is shown in Table 1. The present inventors have found that in the examples described later, 99 types of miRNAs shown in Table 1 are increased in bladder cancer as compared with normal bladder.

In the method according to the present invention, one or more of the above miRNAs (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or all 99) may be measured. In one embodiment, the miRNA to be measured is selected from the group consisting of miR-200c, miR-205, miR-200a, miR-96, miR-183 and miR-519a. In another embodiment, the miRNA to be measured is selected from the group consisting of miR-200c, miR-944, miR-1323, miR-205 and miR-200a. In a preferred embodiment, the miRNA to be measured is selected from the group consisting of miR-200c, miR-205 and miR-200a.

In one embodiment, the measured amount of miRNA is increased compared to the control amount (eg, 2x or greater, 4x or greater, 6x or greater, 8x or greater, 10x or greater, 15x or greater, 20x or greater, 25x. If more than double or more than 30 times), the subject's urothelial cancer is indicated. The control amount may be a normal and healthy individual or population, or an amount measured in an individual or population known not to have urothelial cancer. The control amount may be the average value of the amounts obtained in a plurality of individuals. The control amount may be a previously measured value stored in the database.

Further, the cutoff value (threshold value) may be set in advance. If the cutoff value is exceeded, a subject's urothelial carcinoma may be indicated. The cutoff value can be determined by, for example, ROC (receiver operating characteristic curve) analysis. In addition, the diagnostic accuracy (sensitivity and specificity) according to the method of the present invention can be determined by ROC analysis. In ROC analysis, for example, the amount of miRNA is measured in a patient sample and a healthy person sample, the sensitivity and specificity at each cutoff value are calculated, and the sensitivity and false positive rate (1-specificity) are shown on the vertical axis. And plot on the horizontal axis respectively to create a ROC curve. The cutoff value can be determined using the area under the curve (AUC) as an index.

In the method according to the present invention, the amount of RNA is typically measured by extracting total RNA from a sample obtained from urine from which blood cells have been removed and enriched with urinary tract epithelial cells, and the extracted RNA is used. It can be carried out by using a nucleic acid quantification method known in the art, for example, a quantitative PCR method using a PCR primer (for example, a real-time PCR method), a Northern hybridization method using a nucleic acid probe, a microarray method, or the like. RNA extraction may be performed using a commercially available RNA extraction reagent such as ISOGEN (Nippon Gene). Real-time PCR methods use, for example, kits such as the TaqMan microRNA Assay (Thermo Fisher Scientific) and equipment such as the ABI 7300 Sequence Detection System (Thermo Fisher Scientific). You may go.

The method according to the present invention includes any one or more of urinary cell examination, cystoscopy, ureteroscopy, ultrasonography, CT examination, and MRI examination, which are conventional examination methods for urothelial cancer. It may be done in combination. Such a combination can improve the sensitivity and specificity of the test.

If a subject's urothelial cancer is indicated by the method according to the invention, the cancer may be treated in the subject. Treatment includes, for example, excision of the affected area, chemotherapy with anticancer agents (eg, gemcitabine, cisplatin, methotrexate, vinblastine, adriamycin, and / or cisplatin), and radiation therapy.

The present invention is also a method for detecting urothelial cancer in a subject.
A step of obtaining a sample from the subject's urine from which blood cells have been removed and enriched with urothelial cells, and in the sample:
miR-200c, miR-944, miR-1323, miR-205, miR-200a, miR-200b, miR-516b, miR-203, miR-210, miR-141, miR-518b, miR-135b, miR- 519a *, miR-518a-3p, miR-523 *, miR-519c-5p, miR-518e *, miR-519b-5p, miR-522 *, miR-429, miR-200b *, miR-512-5p , MiR-1283, miR-224, miR-515-5p, miR-1246, miR-183 *, miR-517b, miR-517a, miR-200a *, miR-1307, miR-523, miR-498, miR -516a-5p, miR-519a, miR-520a-3p, miR-515-3p, miR-1293, miR-512-3p, miR-520g, miR-524-5p, miR-301b, miR-518e, miR -518f, miR-21, miR-449c, miR-522, miR-1268, miR-519b-3p, miR-526b, miR-1269, miR-371-5p, miR-519d, miR-521, miR-3687 , MiR-141 *, miR-518c, miR-520f, miR-182, miR-518c *, miR-767-5p, miR-520h, miR-548x, miR-372, miR-525-5p, miR-520c -3p, miR-519c-3p, miR-934, miR-517c, miR-520d-3p, miR-183, miR-96, miR-466, miR-205 *, miR-520b, miR-200c *, miR -335 *, miR-526b *, miR-671-5p, miR-524-3p, miR-526a, miR-518d-5p, miR-520c-5p, miR-525-3p, miR-138, miR-518f *, MiR-708, miR-1292, miR-151-3p, miR-3180, miR-3180-3p, miR-1254, miR-31, miR-520a-5p, miR-371-3p, miR-1248, miR-3145, miR-149, and miR-449b
A method may be provided that comprises the step of measuring the amount of one or more miRNAs selected from the group consisting of.

Since a sample derived from urine is used, the detection method according to the present invention has an advantage that it is non-invasive. In addition, the method according to the present invention can bring about high specificity because the influence of blood contamination in urine on the amount of markers can be eliminated. In particular, the method according to the present invention may be useful for distinguishing a patient other than cancer having hematuria, for example, a patient with urolithiasis and a patient with a urinary tract infection, and a patient with urothelial cancer.

(kit)
The present invention also provides a kit for use in a method for detecting urothelial cancer in the above-mentioned subjects. The kit is below:
miR-200c, miR-944, miR-1323, miR-205, miR-200a, miR-200b, miR-516b, miR-203, miR-210, miR-141, miR-518b, miR-135b, miR- 519a *, miR-518a-3p, miR-523 *, miR-519c-5p, miR-518e *, miR-519b-5p, miR-522 *, miR-429, miR-200b *, miR-512-5p , MiR-1283, miR-224, miR-515-5p, miR-1246, miR-183 *, miR-517b, miR-517a, miR-200a *, miR-1307, miR-523, miR-498, miR -516a-5p, miR-519a, miR-520a-3p, miR-515-3p, miR-1293, miR-512-3p, miR-520g, miR-524-5p, miR-301b, miR-518e, miR -518f, miR-21, miR-449c, miR-522, miR-1268, miR-519b-3p, miR-526b, miR-1269, miR-371-5p, miR-519d, miR-521, miR-3687 , MiR-141 *, miR-518c, miR-520f, miR-182, miR-518c *, miR-767-5p, miR-520h, miR-548x, miR-372, miR-525-5p, miR-520c -3p, miR-519c-3p, miR-934, miR-517c, miR-520d-3p, miR-183, miR-96, miR-466, miR-205 *, miR-520b, miR-200c *, miR -335 *, miR-526b *, miR-671-5p, miR-524-3p, miR-526a, miR-518d-5p, miR-520c-5p, miR-525-3p, miR-138, miR-518f *, MiR-708, miR-1292, miR-151-3p, miR-3180, miR-3180-3p, miR-1254, miR-31, miR-520a-5p, miR-371-3p, miR-1248, miR-3145, miR-149, and miR-449b
Contains a polynucleotide for measuring the amount of one or more miRNAs selected from the group consisting of. The polynucleotide may include a base sequence complementary to the miRNA, a fragment thereof containing 15 or more (eg, 19 or more) consecutive bases, or a base sequence that hybridizes to the miRNA under stringent conditions. ..

Stringent hybridization conditions can be appropriately selected by those skilled in the art. Hybridization conditions include, for example, low stringent conditions and high stringent conditions. The low stringent conditions may be, for example, 30 ° C., 2 × SSC, 0.1% SDS. High stringent conditions may be, for example, 65 ° C., 0.1 × SSC, 0.1% SDS. The stringency of hybridization can be adjusted by changing conditions such as temperature and salt concentration. Here, 1 × SSC comprises 150 mM sodium chloride and 15 mM sodium citrate.

The polynucleotide contained in this kit may be DNA or RNA, but is preferably DNA from the viewpoint of stability. This polynucleotide can be used as a probe and primer for detecting the above-mentioned miRNA which is a target nucleic acid. The kit may further include, for example, enzymes for PCR, substrates and buffers, reagents for Northern hybridization, and the like. The kit may be a microarray chip in which a probe for detecting the above-mentioned miRNA, which is a target nucleic acid, is arranged on a substrate. The polynucleotide included in this kit can be prepared by using a general technique such as a DNA recombination technique, a PCR method, or a synthesis method using a commercially available nucleic acid automatic synthesizer.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the technical scope of the present invention is not limited to these examples.
[Example 1]
Effect of blood contamination in urine on the amount of microRNA The present inventors have previously reported a method for detecting urothelial cancer using the amount of miR-96 and miR-183 in urine as an index (). Yamada et al., Cancer Sci. 2011, Vol. 102, Issue 3, p. 522-529; Japanese Patent Application Laid-Open No. 2011-36242; Japanese Patent Application Laid-Open No. 2009-100687). However, prospective studies in patients with gross hematuria suggested that the specificity of the test may be reduced. In this example, it was investigated whether the amount of miRNA was affected by the contamination of urine with blood.

The following four samples were examined: urine sample (30 ml), hematuria sample (mixture of 30 ml urine and 100 μl blood), serum sample (400 μl), and blood sample (400 μl). The sample was centrifuged at 3000 rpm (1710 × g) for 5 minutes, and the obtained precipitate was used in the following steps.

Total RNA extraction, cDNA synthesis and quantitative PCR were performed as follows. Total RNA was extracted by the phenol-chloroform method using ISOGEN (Nippon Gene, Tokyo, Japan). Complementary DNA from the resulting total RNA using miRNA-specific primers (TaqMan microRNA Assay, PE Applied Biosystems; now Thermo Fisher Scientific) Synthesized by photoreaction. For the reverse transcription reaction, 100 nM total RNA, 50 nM stem-loop RT primer, 1 × RT buffer, 0.25 mM dNTPs each, 3.33 U / μl MultiScribe reverse transcriptase and 0.25 U / μl RNase inhibitor reaction solution were used. The reaction was prepared and incubated at 16 ° C. for 30 minutes, 42 ° C. for 30 minutes, and 85 ° C. for 5 minutes (the reaction solution after the reverse transcription reaction was stored at 4 ° C.). Quantitative PCR was performed using the ABI 7300 Sequence Detection System by 45 reactions at 95 ° C for 15 seconds and 60 ° C for 10 minutes after incubation at 95 ° C for 10 minutes. Ct values were obtained from the results of quantitative PCR for each sample. A 2 ^ (40-Ct) value was calculated from the Ct values obtained for each sample, and this value was used to evaluate the expression level of each miRNA. The results are shown in Table 2 and FIG.

Hematuria samples showed increased amounts of both miR-96 and miR-183 compared to urine samples. In addition, the amounts of miR-96 and miR-183 were much higher in the blood sample than in the serum sample (the sample from which blood cell components in the blood had been removed), and thus the blood cells (erythrocytes) in the blood. And blood cell components such as leukocytes) have been shown to affect the amount of miR-96 and miR-183.

From the above results, it was shown that the amount of microRNA is affected by the contamination of urine with blood, especially blood cells.

[Example 2]
Separation of blood cells and bladder cancer cells by cell sorter and recovery of bladder cancer cells In this example, blood cells and bladder cancer cells are separated using a cell sorter, and whether or not only bladder cancer cells can be recovered from a mixture of both is determined. Examined.

Bladder cancer cells expressing GFP (green fluorescent protein) (BOY-GFP strain, AntiCancer Japan Co., Ltd.) and blood were analyzed using cell sorters (BD FACSAria ™ II, BD Biosciences), respectively. The result is shown in figure 2. It was shown that GFP-expressing bladder cancer cells and blood differ in the location of population, the size of the cells contained, the complexity of the internal structure, and the intracellular granules (Fig. 2a (Bladder cancer)). (Cells) and Figure 2c (blood)). In addition, it was confirmed that a large number of cells emitting green light were detected in bladder cancer cells expressing GFP, whereas cells emitting green light were not detected in blood (Fig. 2b (bladder cancer cells) and Fig. 2d). (blood)).

Subsequently, a sample in which GFP-expressing bladder cancer cells and blood were mixed was subjected to a cell sorter gated so that only bladder cancer cells could be sorted, and whether or not only bladder cancer cells could be sorted was examined. Figures 3a to 3c show the layered gating that was actually used. In layered gating, the six parameters obtained by scattered light (side scattered light (SSC) and forward scattered light (FSC) signals are of height (H), area (A), and width (W), respectively. The parameter) is combined to analyze the cell size, internal structural complexity and intracellular granules, and the cells within the region set for the parameter combination are sorted hierarchically. The P1 to P3 regions having the following vertices were set.
P1 (Fig. 3a): (FSC-A / SSC-A, value x 1000) Vertex 1; 31/135, Vertex 2; 65/222, Vertex 3; 254/250, Vertex 4; 216/18, Vertex 5; 89/28, apex 6; 44/59
P2 (Fig. 3b): (FSC-H / FSC-W, value x 1000) Vertex 1; 34/118, Vertex 2; 110/118, Vertex 3; 110/86, Vertex 4; 39/74
P3 (Fig. 3c): (SSC-H / SSC-W, value x 1000) Vertex 1; 21/138, Vertex 2; 117/138, Vertex 3; 120/80, Vertex 4; 22/76

It was shown that the cells after sorting by stratified gating were only the cells emitting green light, and only the bladder cancer cells could be sorted with an accuracy (purity) of 99% or more (Fig. 3d).

Based on the above results, blood cells and bladder cancer cells are separated using a cell sorter, and bladder cancer is derived from a mixture of blood cells and bladder cancer cells based on information on cell size, internal structural complexity, and intracellular granules. It was shown that only cells can be recovered.

[Example 3]
Examination of sample storage method In this example, a sample storage method that can maintain the separability by the cell sorter and the quality of RNA was examined. Three types of preservatives (30% polyethylene glycol (PEG), RNAlater® and 70% ethanol) were examined.

It is already known that samples stored in 70% ethanol can be sorted. We investigated whether RNA quality was preserved in samples stored in 70% ethanol. ISOGEN (Nippon Gene) from 786-o cells, T24 cells and A498 cells purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and BOY cells, a bladder cancer cell line established at the Graduate School of Medical and Dental Sciences, Kagoshima University. RNA samples were prepared according to the manufacturer's protocol. RNA samples from 786-o cells were mixed with 70% ethanol and incubated overnight at -80 ° C. RNA samples after incubation were analyzed by agarose gel electrophoresis. The results are shown in Fig. 4. In the ethanol-treated sample (786-o RNA (70% ethanol storage)), the 28s rRNA and 18s rRNA bands found in 786-o RNA (no storage) disappeared. It was shown to decompose.

It has already been found that RNA quality is preserved in samples stored in RNAlater® or polyethylene glycol. It was examined whether the sample stored in RNAlater® or polyethylene glycol could retain the separability by the cell sorter. RNAlater® was purchased from Thermo Fisher Scientific. A 30% polyethylene glycol solution was prepared by dissolving polyethylene glycol 8000 (Sigma-aldrich) in 3M NaCl at a concentration of 30 (w / v)%. Bladder cancer cells (BOY-GFP strain) were mixed with RNAlater® or a 30% polyethylene glycol solution and incubated overnight at -20 ° C. After incubation, samples were analyzed using a cell sorter (BD FACSAria ™ II, BD Biosciences). The results are shown in Fig. 5. Samples stored in RNAlater® (FIGS. 5d-5f) showed different populations compared to controls (without preservation) (FIGS. 5a-5c). On the other hand, the samples stored in polyethylene glycol (Figs. 5g-5i) showed similar populations to the controls (no storage) (Figs. 5a-5c).

From the above results, it was shown that the preservation method using polyethylene glycol can maintain the separation ability of the sample by the cell sorter and the quality of RNA.

[Example 4]
Selection of miRNAs whose expression is increased in bladder cancer In this example, whole genome analysis of microRNAs was performed with a next-generation sequencer using 10 human clinical specimens (5 bladder cancer specimens and 5 normal bladder mucosa specimens). The analysis was commissioned by Riken Genesis Co., Ltd. The results were analyzed by the Z-score method. MicroRNAs whose expression was increased in bladder cancer samples compared to normal bladder samples were sorted based on the p-value, which is an index of significance, and 99 types of microRNAs were selected (Table 3).

[Example 5]
Expression of miRNAs in urothelial cell fractions recovered from urine using a cell sorter In this example, 3 of the 99 microRNAs selected in Example 4 (miR-200c, miR- For 205 and miR-200a), the expression level in the urothelial cell fraction collected from urine using a cell sorter was measured using quantitative PCR.

We used urine from 10 cases of human urothelial cancer (bladder cancer and renal pelvis ureteral cancer) and 11 cases of normal cases. Urine was transferred to a 50 ml centrifuge tube and centrifuged (1500 rpm, 5 minutes). After centrifugation, the supernatant was discarded, 30% polyethylene glycol (PEG) was added to the pellet fraction (cell fraction), and the mixture was stored at -20 ° C.

The sample stored at -20 ° C was dissolved, centrifuged, and the pellet was washed with phosphate buffer (PBS). Then, using a cell sorting device (BD FACSAria ™ II, BD Biosciences), urothelial cells (cells derived from the bladder) and blood cells are examined by laser irradiation, and the cell size and internal structure are complicated. The cells were separated based on the information of the intracellular granules, and the fraction containing the urothelial cells was collected. The setting of cell separation was the same as the setting in which only bladder cancer cells could be recovered from the mixture of the bladder cancer cell line expressing GFP and blood in Example 2. The urothelial cell fraction recovered by cell sorting retained a sufficient amount and quality of RNA.

Using the obtained urothelial cell fraction, total RNA extraction, cDNA synthesis and quantitative PCR were performed from the sample as described in Example 1. The Ct values obtained by quantitative PCR are shown in Fig. 6 and Table 4.

The lower the Ct value, the higher the expression level of microRNA. All of miR-200c, miR-205 and miR-200a showed significantly lower Ct values in the urothelial cell fraction obtained from the urine of patients with urothelial cancer compared to normal urine. It was confirmed that the expression of these microRNAs was significantly increased in the urothelial cell fraction obtained from the urine of patients with urothelial cancer as compared with normal urine (Fig. 6).

Claims (7)

A step of urothelial cells urine or et blood cells of the subject is removed to obtain a sample enriched with a cell sorter,
In the sample ,
miR-200c, miR-944, miR-1323, miR-205, miR-200a, miR-200b, miR-516b, miR-203, miR-210, miR-141, miR-518b, miR-135b, miR- 519a *, miR-518a-3p, miR-523 *, miR-519c-5p, miR-518e *, miR-519b-5p, miR-522 *, miR-429, miR-200b *, miR-512-5p , MiR-1283, miR-224, miR-515-5p, miR-1246, miR-183 *, miR-517b, miR-517a, miR-200a *, miR-1307, miR-523, miR-498, miR -516a-5p, miR-519a, miR-520a-3p, miR-515-3p, miR-1293, miR-512-3p, miR-520g, miR-524-5p, miR-301b, miR-518e, miR -518f, miR-21, miR-449c, miR-522, miR-1268, miR-519b-3p, miR-526b, miR-1269, miR-371-5p, miR-519d, miR-521, miR-3687 , MiR-141 *, miR-518c, miR-520f, miR-182, miR-518c *, miR-767-5p, miR-520h, miR-548x, miR-372, miR-525-5p, miR-520c -3p, miR-519c-3p, miR-934, miR-517c, miR-520d-3p, miR-183, miR-96, miR-466, miR-205 *, miR-520b, miR-200c *, miR -335 *, miR-526b *, miR-671-5p, miR-524-3p, miR-526a, miR-518d-5p, miR-520c-5p, miR-525-3p, miR-138, miR-518f *, MiR-708, miR-1292, miR-151-3p, miR-3180, miR-3180-3p, miR-1254, miR-31, miR-520a-5p, miR-371-3p, miR-1248, miR-3145, miR-149, and miR-449b
One or more miRNA amount the measuring step a including selected from the group consisting of,
A method in which the amount of miRNA is used as an index of urothelial cancer in the subject. The sample is recovered by using the cell sorter from the cell pellet of the urine, the method according to claim 1. Cell pellets of the urine is stored in a polyethylene glycol solution before being subjected to the cell sorter method of claim 2. The method according to any one of claims 1 to 3, wherein the urothelial cancer of the subject is indicated when the amount of the miRNA is increased more than twice as much as the control amount. The method according to any one of claims 1 to 4, wherein the urothelial cancer is selected from the group consisting of renal cell carcinoma, ureteral cancer, bladder cancer, and urethral cancer. The method according to any one of claims 1 to 5, wherein the miRNA is selected from the group consisting of miR-200c, miR-205, miR-200a, miR-96, miR-183 and miR-519a. Less than:
miR-200c, miR-944, miR-1323, miR-205, miR-200a, miR-200b, miR-516b, miR-203, miR-210, miR-141, miR-518b, miR-135b, miR- 519a *, miR-518a-3p, miR-523 *, miR-519c-5p, miR-518e *, miR-519b-5p, miR-522 *, miR-429, miR-200b *, miR-512-5p , MiR-1283, miR-224, miR-515-5p, miR-1246, miR-183 *, miR-517b, miR-517a, miR-200a *, miR-1307, miR-523, miR-498, miR -516a-5p, miR-519a, miR-520a-3p, miR-515-3p, miR-1293, miR-512-3p, miR-520g, miR-524-5p, miR-301b, miR-518e, miR -518f, miR-21, miR-449c, miR-522, miR-1268, miR-519b-3p, miR-526b, miR-1269, miR-371-5p, miR-519d, miR-521, miR-3687 , MiR-141 *, miR-518c, miR-520f, miR-182, miR-518c *, miR-767-5p, miR-520h, miR-548x, miR-372, miR-525-5p, miR-520c -3p, miR-519c-3p, miR-934, miR-517c, miR-520d-3p, miR-183, miR-96, miR-466, miR-205 *, miR-520b, miR-200c *, miR -335 *, miR-526b *, miR-671-5p, miR-524-3p, miR-526a, miR-518d-5p, miR-520c-5p, miR-525-3p, miR-138, miR-518f *, MiR-708, miR-1292, miR-151-3p, miR-3180, miR-3180-3p, miR-1254, miR-31, miR-520a-5p, miR-371-3p, miR-1248, miR-3145, miR-149, and miR-449b
A kit for use in the method of any one of claims 1-6, comprising a polynucleotide for measuring the amount of one or more miRNAs selected from the group consisting of.

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