JP6609444B2 - Vascular lesion evaluation method and vascular lesion evaluation kit - Google Patents

Description

  The present invention relates to a method for evaluating a vascular lesion using a biomarker selected from the group consisting of miR-214 and miR-126, and a vascular lesion evaluation kit comprising a detection substance for the biomarker.

  In recent years, biomarkers have been developed for use as a means for quickly and easily detecting various diseases and disorders. For example, Patent Document 1 discloses a group consisting of miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205 and miR-214 as a method for diagnosing ovarian cancer. An invention using the amount of microRNA (miRNA) selected more as an index is disclosed.

  Vascular lesions, on the other hand, refer to any localized pathological change, disorder or disease of blood vessels or lymphatic vessels, such as hemangiomas (hemangiomas; those resulting from vascular proliferation resulting in tumors similar to neoplasms) and angiosarcomas ( Benign or malignant tumors such as Hemangiosarcoma; a malignant neoplasm composed of undifferentiated cells derived from blood vessels) and arteriosclerosis such as atherosclerosis. Among these, angiosarcoma is known as a major cancer that accounts for about 5 to 7% of cancer in dogs, although rare in humans. Angiosarcoma has a poor prognosis, and there is a strong demand for the development of early detection means. Patent Document 2 discloses an increase in the expression level of one or more miRNAs selected from the group consisting of miR-503, miR-193b, let-7e, miR-451, miR-423a, and miR-551b. A method for detecting angiosarcoma in mammals is described as an index.

JP 2010-534480 A (corresponding to International Publication No. 2009/015357) Japanese Patent Application Laid-Open No. 2014-082953

  Although the use of biomarkers is expected to detect pathological changes and the like quickly and easily, conventional biomarkers may not be sufficient in terms of specificity and sensitivity in evaluating vascular lesions. Therefore, an object of the present invention is to provide means for detecting vascular lesions such as angiosarcoma specifically and with high sensitivity.

  The present inventors have intensively studied to solve the above problems. As a result, it was found that the expression level of at least one of miR-214 and miR-126 is increased in patients suffering from vascular lesions. Thereby, using a biomarker selected from the group consisting of miR-214 and miR-126, in particular, by using a combination of miR-214 and miR-126, it is possible to detect vascular lesions specifically and with high sensitivity. As a result, the present invention has been completed.

  According to the present invention, a vascular lesion can be detected specifically and with high sensitivity.

FIG. 1 shows the expression of biomarkers (miR-214, miR-126) secreted into the medium using canine normal vascular endothelial cells (CnAOEC cells) and canine hemangiosarcoma-derived cultured cells (Re12 cells and Ud6 cells). It is the result of measuring the level. FIG. 2 shows the results of measuring the expression levels of biomarkers (miR-214, miR-126) in plasma collected from dogs in a healthy group, a non-neoplastic splenic disease group, and an angiosarcoma-affected group. FIG. 3 is a statistical analysis result when miR-214 and miR-126 are independent biomarkers as test data in Example 2. FIG. 4 shows the expression levels of miR-214 and miR-126 in each individual of a healthy group, a non-neoplastic splenic disease group, and an angiosarcoma affected group. FIG. 5 is a statistical analysis result when the test data in Example 2 is combined with miR-214 and miR-126 to be a biomarker. FIG. 6 shows the results of measuring the expression levels of biomarkers (miR-214, miR-126) in plasma before and after angiosarcoma removal. FIG. 7 shows biomarkers (miR-214, miR-126) secreted into a medium using human vascular endothelial cells (HMEC1 cells) and human hemangiosarcoma-derived cultured cells (ISO-HAS cells and HAMON cells). It is the result of measuring the expression level.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment.

  In this specification, “X to Y” indicating a range means “X or more and Y or less”. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50%.

  A first aspect of the present invention is a method for evaluating a vascular lesion in a subject, measuring a biomarker expression level in a biological sample separated from the subject, and comparing the biomarker expression level with a reference value The biomarker is selected from the group consisting of miR-214 and miR-126. According to the present invention, a vascular lesion can be detected specifically and with high sensitivity. It should be noted that “measurement” in this specification includes any concept of quantitative, semi-quantitative, and detection. Furthermore, “measuring the expression level” includes not only directly measuring miRNA, but also miRNA is converted to cDNA by RT-PCR or the like and then measuring the cDNA. Indirect measurement is also included. The “detection substance” includes both substances that hybridize and detect directly to miRNA, and substances that hybridize and detect miRNA once converted to cDNA by RT-PCR or the like. including.

  In the present invention, miRNA (microRNA) selected from the group consisting of miR-214 and miR-126 is used as a biomarker. miRNA is a non-coding RNA of about 20 to 25 bases that is endogenous to cells. miRNA is first transcribed from a miRNA gene on genomic DNA as a primary transcript (pri-miRNA) having a length of about several hundred to several thousand bases. Then, it is processed to become a pre-miRNA having a hairpin structure of about 60 to 70 bases. Thereafter, the miRNA moves from the nucleus into the cytoplasm and is further processed to become a mature miRNA composed of a dimer (guide strand and passenger strand) of about 20 to 25 bases. In mature miRNA, the guide strand (antisense strand) of the mature miRNA forms a complex with a protein called RISC (RNA-Induced Silencing Complex) and acts on the mRNA of the target gene, thereby inhibiting the translation of the target gene. It is known to do.

  Certain cells are known to secrete vesicles encapsulating molecules such as proteins and nucleic acids in lipid bilayer structures, and these vesicles are known as microvesicles, nanovesicles, etc. Yes. Secreted vesicles are considered to have a function of fusing with other cells and transferring molecules such as encapsulated proteins and nucleic acids to other cells. Molecules contained in vesicles are thought to differ depending on the cell type. Therefore, the use of a molecule that is specifically encapsulated in a vesicle secreted by a tumor cell as an index is also considered as a biomarker for tumor detection. It is also known that certain miRNAs are contained in a cell type-specific manner as molecules contained in vesicles.

  On the other hand, the present inventors have found that damaged vascular endothelial cells (particularly vascular endothelial tumor cells) secrete a large amount of miR-214 and miR-126 to the outside of the cells. Without limiting the technical scope of the present invention, this is because damaged vascular endothelial cells (especially vascular endothelial tumor cells) utilize miR-214 and miR-126 as signals to other cells. It is thought that these molecules are contained in vesicles that are secreted as a means of transmission. In the present invention, the miRNA that is an expression level measurement target or a detection substance detection target is pri-miRNA, pre-miRNA (pri-miRNA and pre-miRNA are also referred to as “precursor miRNA”) and. Although it may be any mature miRNA, it is preferably a mature miRNA.

  The “subject” in the present specification is not particularly limited as long as it is an animal including human and non-human animals that express miR-214 and / or miR-126. For example, human, dog, cat, mouse, rat, hamster It is preferably a mammal such as guinea pig, monkey, horse, cow, pig, rabbit, sheep, goat. The presence of miR-214 and miR-126, which are measurement targets, has been confirmed in many mammals as described above. Among these, the method for evaluating a vascular lesion according to the present invention is suitably used for humans or dogs. By way of example, human and canine mature miR-214 (SEQ ID NO: 1) and mature miR-126 (SEQ ID NO: 2) sequences are shown. The nucleotide sequences of mature miR-214 and mature miR-126 are common in mammals including humans and dogs.

  The “biological sample” used in the present invention means a cell, tissue or body fluid taken out from a subject ex vivo, such as a biopsy, a puncture aspirate, a exfoliated material, a cell exudate, and the like. Although not particularly limited, more specifically, for example, urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymph fluid, airway fluid, intestinal fluid, genitourinary tract fluid, Examples include breast milk, semen, cerebrospinal fluid, endotracheal fluid, ascites, cystic tumor fluid, and amniotic fluid, but may be selected from the group consisting of blood, plasma, and serum from the viewpoint of availability. preferable. A biological sample separation method may be performed by a conventionally known means in this technical field.

  As used herein, “vascular lesion” refers to a localized pathological change, disorder or disease of a blood vessel or lymphatic vessel, and is not particularly limited. For example, hemangioma (for example, hemangioblastoma, Hemangioendothelioma, angioderma, capillary hemangioma, infantile hemangioma, spongiform hemangioma, venous hemangioma, sclerosing hemangioma, senile hemangioma, spider hemangioma, strawberry hemangioma , Wart angioma, lymphangioma, tortuous hemangioma), angiosarcoma (eg malignant hemangioendothelioma, malignant hemangioderma, Kaposi sarcoma), atherosclerosis, hypertrophic arteriosclerosis, hypertensive arteriosclerosis Examples thereof include endometriosis, coronary artery disease, stroke, myocardial infarction, restenosis, and diabetic vascular disorder. The present invention is particularly preferably used for detection of hemangiomas or angiosarcomas, and more preferably for detection of hemangiosarcomas.

  In the present invention, at least one of miR-214 and miR-126 is used as a biomarker. Preferably, miR-214 and miR-126 are used as biomarkers from the viewpoint that the specificity and sensitivity can be significantly improved. The expression level is measured. For the measurement of the expression level of a biomarker, a conventionally known quantitative / semi-quantitative technique can be employed. For example, the expression level may be measured by a nucleic acid amplification method, a microarray method, or a Northern blot method. More preferably, from the viewpoint of high sequence specificity, the expression level is measured by a nucleic acid amplification method. Hereinafter, the nucleic acid amplification method, the Northern blot method, and the microarray method will be further described as typical expression level measurement methods, but the expression level measurement method is not limited to the following.

  When using a nucleic acid amplification method, a nucleic acid amplification method using a primer set to include a region (for example, 7 to 25 consecutive residues) corresponding to a mature miRNA of a biomarker (miR-214, miR-126). , Specifically amplifying a partial region of RNA or cDNA thereof containing a mature miRNA base sequence of a biomarker (miR-214, miR-126), and measuring the amplified product. Here, “set to a region” means that it hybridizes with the region under stringent conditions, and a reverse primer for the guide strand of a biomarker (miR-214, miR-126) is used. And the case where the forward primer is used are included. Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, the conditions described in the textbook (edited by Sambrook et al., “Molecular Cloning, Laboratory Manual”). 2nd edition ", 1989, Cold Spring Harbor Laboratory, in particular Section 11.45" Conditions for Hybridization of Oligonucleotide Probes "etc.), and is not particularly limited. Stringent conditions depend on salt concentration, temperature, and other conditions. For example, the lower the salt concentration, the higher the temperature, the higher the stringency, and the primer and RNA (or its cDNA) hybridize. It becomes difficult to do. The salt concentration is generally adjusted by adjusting the concentration of the SSC solution (NaCl + trisodium citrate), and the stringent salt concentration is, for example, about 250 mM or less NaCl and about 25 mM or less trisodium citrate. The stringent temperature is generally 15 to 25 ° C. lower than the melting temperature (Tm) of the complete hybrid, for example, about 30 ° C. or more. The temperature can be lowered by adding an organic solvent (eg, formamide) to the solution. Other conditions include hybridization time, detergent (eg, SDS) concentration, presence or absence of carrier DNA, and various stringencies can be set by combining these conditions. As a preferred example, hybridization is performed at a temperature of 42 ° C. under conditions of 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / mL denatured salmon sperm DNA. In addition, washing conditions after hybridization also affect stringency. This wash condition is also defined by salt concentration and temperature, and wash stringency increases with decreasing salt concentration and increasing temperature. As a preferred example, washing is performed at a temperature of 68 ° C. under conditions of 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. In addition, “specifically amplify” means that an RNA containing the base sequence of either miR-214 or miR-126 or a partial region of its cDNA is amplified, but the miR that is the subject of measurement. This means that an RNA containing a base sequence of mature miRNA other than -214 or miR-126 or a partial region of cDNA thereof is not amplified. In addition, although the size of a primer is not specifically limited, Usually, it is about 13-30 bases, Preferably it is about 15-25 bases.

  A preferred example of obtaining DNA (cDNA) having the same base sequence as the template partial RNA region (where u is t) is reverse transcription PCR (RT-PCR). In RT-PCR, RNA is extracted from a biological sample by a conventional method, a first strand cDNA is generated using RNA as a template by the action of reverse transcriptase, and a complementary strand is generated using this first strand cDNA as a template. Double stranded cDNA is obtained. Next, PCR is performed using the obtained double-stranded cDNA as a template to amplify the cDNA or a partial region thereof. RT-PCR itself is a well-known technique, and kits and devices therefor are also commercially available, and can be easily implemented.

  When RT-PCR is performed, amplification using a pseudogene contained in genomic DNA mixed at the time of initial RNA extraction as a template may occur. Accordingly, the primer set to be used is preferably a set in which primers that do not cause amplification due to such pseudogenes are selected. Whether or not amplification due to the pseudogene occurs, for example, when PCR is performed using the primer set and genomic DNA as a template, only amplification products of the same size as when PCR is performed using cDNA as a template. It can be determined by whether or not it is generated.

  A common technique for nucleic acid amplification is PCR. PCR itself can be performed according to a conventional method. Then, after amplification by PCR, the expression level of the amplification product is measured. Such expression level can also be measured by a conventional method. Furthermore, simple determination or semi-quantification such as measurement of the fluorescence intensity of the electrophoresis band of the amplification product and visual determination of the thickness is also included in the concept of “measurement”. The expression level can be measured, for example, by subjecting a reverse transcription product to electrophoresis and detecting an amplification band. The expression level can also be measured by fluorescently labeling the reverse transcription product by performing PCR in the presence of fluorescently labeled nucleotide triphosphate and measuring the fluorescence intensity of the amplified band. Furthermore, the electrophoresis pattern is transferred to a membrane made of nylon or nitrocellulose, and a labeled probe that hybridizes to the reverse transcription product is hybridized to detect or quantify the label (PCR-Southern method). be able to. Furthermore, a probe that hybridizes to the reverse transcription product is immobilized, the amplification step is performed in the presence of labeled nucleotide triphosphate, the reverse transcription product is bound to the immobilized probe, and the reverse phase bound to the solid phase. It can also be done by measuring the transcript. These are all conventional methods, and known knowledge can be referred to appropriately.

  The expression level of the biomarker may be measured by real-time PCR (quantitative PCR) using the RT-PCR reverse transcription product described above. Alternatively, the expression level of the biomarker may be measured by so-called one-step RT-PCR in which the reverse transcription reaction and quantification by real-time PCR (quantitative PCR) are performed in the same tube. Real-time PCR (quantitative PCR) may be performed by a conventionally known method using a fluorescent dye, and may be either TaqMan (registered trademark) probe method or intercalator method (SYBR (registered trademark) Green I). In the present invention, the expression level of the biomarker is preferably measured by the TaqMan (registered trademark) probe method because of its high sequence specificity.

  As described above, the case where the measurement method of the present invention is performed by the PCR method has been described in detail. However, the measurement method of the present invention uses the above-described primers to mature the biomarkers (miR-214, miR-126). Any other nucleic acid amplification method may be used as long as it is a method for amplifying an RNA containing a miRNA base sequence or a partial region of its cDNA.

  The measurement of the expression level of the biomarker may be performed by the Northern blot method. The probe used for Northern blotting is a probe set to include a region (for example, continuous 7 to 25 residues) corresponding to a mature miRNA of a biomarker (miR-214, miR-126). The probe is capable of hybridizing under stringent conditions with the biomarker (miR-214, miR-126) to be detected, as with the above-described primer. For example, the probe is 70% or more (for example, 80% or more, 90% or more, 92% or more, 95% or more, 99% or more, 100%). The probe is not particularly limited as long as it can hybridize with a biomarker to be detected (miR-214, miR-126) under stringent conditions, and may be either a DNA probe or an RNA probe, Alternatively, it may be chemically modified such as a cross-linked nucleic acid such as LNA (registered trademark), or may contain a non-natural base (artificial base). The probe length is, for example, 5 to 30 residues, preferably 10 to 25 residues. The probe is detectably labeled with a labeling substance such as a radiolabel, a fluorescent dye, digoxigenin (DIG), biotin, or an enzyme.

  Northern blots can be performed by techniques well known in the art. Specifically, RNA extracted from a biological sample is fractionated by gel electrophoresis and transferred to a membrane such as a nitrocellulose membrane or a nylon membrane. The membrane is then incubated in buffer in the presence of the probe. Thereafter, miRNA hybridized with the probe can be detected by detecting the label of the probe. The expression level may be measured by digitizing the band intensity. For example, when a probe labeled with a radioactive substance is used, it may be performed by a technique such as autoradiography.

  The expression level of the biomarker may be measured by a microarray method. The probe used for the microarray is as described above, which can hybridize with the biomarker (miR-214, miR-126) to be detected under stringent conditions. A plurality of probes that hybridize to the same detection target may be arranged as separate spots in the same array.

  Array techniques are known and have been used to analyze gene expression (Chee, M. et al. (1996) Science, .274, 610-613). For example, the probe described above is disposed on a base material such as glass, plastic, polymer, latex, or metal that has been surface-treated with poly-L-lysine or the like so that the position can be specified. As for the arrangement of the probes, the probes may be attached by a spotter or an arrayer, or the probes may be synthesized on the substrate by means such as photolithography or inkjet printing.

  In the evaluation method according to the present invention, the expression level of the biomarker (miR-214, miR-126) in the biological sample measured as described above is compared with a reference value.

  In certain embodiments, the reference value is a biomarker expression level in a biological sample isolated from a healthy individual. The reference value may be an average value or a median value of biomarker expression levels in a biological sample separated from a plurality (for example, 5 to 1000) of healthy individuals. In this case, when the biomarker expression level is lower than the reference value, it can be considered as an index having no vascular lesion, and when it exceeds the reference value, it can be considered as an index having vascular lesion. The reference value is a healthy individual. It is a biomarker expression level in the biological sample isolate | separated from. In a further embodiment, the reference value is a biomarker expression level in a biological sample separated from healthy individuals (or a biomarker expression level in a biological sample separated from multiple (eg, 5-1000 individuals) healthy individuals. (Average value or median value) is 1.2 times (or 1.5 times).

  In certain embodiments, the reference value may be a biomarker expression level in a biological sample separated from the same subject. For example, in a subject suffering from a vascular lesion, the therapeutic treatment (eg, medication, surgical treatment) is performed using the biomarker expression level in the biological sample before the therapeutic treatment (eg, medication, surgical treatment) as a reference value. With respect to the same subject after application, detection of a vascular lesion may be performed according to the present invention. In this case, when the biomarker expression level in the biological sample separated from the subject after the therapeutic treatment is less than the reference value, it is an index that does not have vascular lesions, and when the biomarker expression level is greater than or equal to the reference value, it is an index that has vascular lesions. As such, the reference value is the biomarker expression level in a biological sample separated from the same subject prior to therapeutic treatment. In another embodiment, the reference value is a value obtained by multiplying the biomarker expression level in a biological sample separated from the same subject by 0.8 (or 0.6). Medically appropriate therapeutic treatment is appropriately selected and known by those skilled in the art. Since the biomarker expression level serves as an index for evaluating vascular lesions in a subject, in one embodiment of the present invention, the biomarker expression level in a biological sample separated from the subject is measured, and the biomarker expression level is a reference value. And a method for acquiring data of vascular lesions in a subject, wherein the biomarker is selected from the group consisting of miR-214 and miR-126. In another embodiment, the method comprises measuring a biomarker expression level in a biological sample isolated from a vascular lesion patient, and comparing the biomarker expression level to a reference value, wherein the biomarkers are miR-214 and miR-126. A method of managing treatment of a vascular lesion patient selected from the group consisting of:

  In the present invention, vascular lesions may be evaluated by comparing the expression level measured for at least one of the biomarkers selected from the group consisting of miR-214 and miR-126 with a reference value. It is preferable to evaluate the vascular lesion by comparing the expression levels measured for both 214 and miR-126 with reference values, respectively, from the viewpoint of remarkable specificity and sensitivity. At this time, if the expression level of at least one of miR-214 and miR-126 is lower than or equal to the reference value (or less than the reference value), it is assumed that the index has no vascular lesion, and the expression level of miR-214 and miR-126 Can exceed the reference value (or more than the reference value), it can be an indicator having a vascular lesion. Thereby, the specificity as a biomarker becomes high. Alternatively, when the expression levels of miR-214 and miR-126 are both lower than or equal to the reference value (or less than the reference value), the expression level is not an vascular lesion, and the expression level of at least one of miR-214 and miR-126 Can be an indicator having a vascular lesion. Thereby, the detection sensitivity as a biomarker becomes high. Alternatively, when the expression levels of miR-214 and miR-126 are both lower than the reference value (or higher than the reference value), pathological condition level 1 (healthy), and the expression level of at least one of miR-214 and miR-126 is the reference If the value exceeds (or exceeds the reference value) level 2, and if the expression levels of miR-214 and miR-126 both exceed the reference value (or above the reference value), level 3 It can also be used to assess the disease state in stages. The above judgment methods can be selected and used according to the purpose of evaluation.

  In one embodiment of the present invention, in order to correct the measurement value of the biomarker, the endogenous control expression level in the same biological sample used for measurement of the biomarker expression level may be further measured. That is, one embodiment of the present invention includes measuring the endogenous control expression level in the biological sample, and comparing the biomarker expression level corrected by the endogenous control expression level with a reference value. By comparing the biomarker expression level corrected by the endogenous control expression level with a reference value, more reliable evaluation can be performed. The expression level of the endogenous control can be measured by the same method as the above-described biomarker expression level. In the present embodiment, the expression level used as the reference value is also a value corrected in the same manner by the endogenous control expression level.

  As an endogenous control, housekeeping genes such as β-actin, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyltransferase 1), and 5S rRNA may be used. From the viewpoint of ease, it is preferably a small RNA such as let-7a, miR-16, RNU48, U6, U47, RNU6B, snoRNA202, snoRNA234, RNU24, RNU38B, or Z30, or a cDNA thereof. These endogenous controls can be appropriately selected depending on the biological sample to be measured.

  In order to correct the biomarker expression level by the endogenous control expression level, the measured value of the detected biomarker is divided by the measured value of the detected endogenous control, and the calculated value is used as the biomarker expression level. Use it.

  The second aspect of the present invention relates to a vascular lesion evaluation kit comprising a biomarker detection substance selected from the group consisting of miR-214 and miR-126. According to the present invention, a vascular lesion can be detected specifically and with high sensitivity.

  The “biomarker detection substance” used in the present invention is a detection substance used for measuring the expression level of miRNA, such as nucleic acid amplification method, Northern blotting method, and microarray method. Primers and probes can be exemplified. The kit includes a detection substance for at least one biomarker selected from the group consisting of miR-214 and miR-126. Preferably, since the specificity and sensitivity as a biomarker can be significantly improved by measuring the expression levels of both miR-214 and miR-126 as described above, the kit can be used as a biomarker detection substance. -214 detection material and miR-126 detection material. More preferably, the kit according to the present invention further includes the above-described endogenous control detection substance.

  The kit may be in a dry state or a detection substance dissolved in a solution. Alternatively, the detection substance may be immobilized on the carrier using a microarray substrate, a microtiter plate, or resin or metal beads as a carrier. In addition to the above detection substances, the kit includes reverse transcriptase, DNA polymerase (eg, Taq polymerase), dNTP, oligo dT primer, random primer, RNase inhibitor, RNase H, labeling substance, buffer, and the like. To make a measurement kit. Furthermore, various devices that can be used for executing the evaluation method of the present invention and instructions for use may be included.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.

Example 1
Biomarkers (miR-214, miR-126) and endogenous secreted into the culture medium during culture using canine normal vascular endothelial cells (CnAOEC cells) and canine hemangiosarcoma-derived cultured cells (Re12 cells and Ud6 cells) The expression level of the control (RNU6B) was measured.

(Cell culture)
Canine normal vascular endothelial cells (CnAOEC cells) were purchased from CELL APPLICATIONS.

Canine hemangiosarcoma-derived cultured cells (Re12 cells and Ud6 cells) were prepared as follows. That is, tumor cells collected from two dogs affected with angiosarcoma were transplanted subcutaneously into nude mice at 1 × 10 ⁶ cells / mouse. Two to four weeks after the transplantation treatment, the formed tumor was removed, and in a DME medium supplemented with 10% (v / v) heat-inactivated FBS (Sigma-Aldrich) in a flask for cell culture. Tumor cells were cultured. Thereafter, cell lines were cloned from the tumor cell population, and Re12 cells and Ud6 cells were isolated from the two individuals as strains having high proliferation activity.

The cells were cultured in an incubator at 37 ° C. (95% air / 5% CO ₂ ).

(Measurement of marker)
Cells seeded in a 6-well plate at a concentration of 1 × 10 ⁶ cells / ml were cultured for 48 hours under the above conditions. Subsequently, all the culture media after culture | cultivation were collect | recovered, and low molecular RNA was collect | recovered using NucleoSpin miRNA (Takara Bio Inc.). Using PrimeScript (registered trademark) RT reagent kit (Takara Bio Inc.), reverse transcription reaction of the recovered RNA was performed, and the obtained cDNA was used as a biomarker (miR-214, miR-126) and endogenous control (RNU6B). RNA, U6B small nucleotide) expression level measurement sample.

  The expression levels of biomarkers (miR-214, miR-126) and endogenous control (RNU6B) were measured by quantitative PCR using a TaqMan (registered trademark) probe. Specifically, Premix EX Taq (Probe qPCR) (Takara Bio Inc.) was used as a reaction reagent, and measurement was performed with a 7500 real-time PCR system (Applied Biosystems). Moreover, TaqMan (registered trademark) MicroRNA Assays (Applied Biosystems) was used as a primer set and a probe.

  FIG. 1 shows the biomarker (miR-214, miR-126) expression level corrected with the endogenous control (RNU6B) expression level. As shown in FIG. 1, compared with normal vascular endothelial cells (CnAOEC cells), the amount of miR-214 secreted into the medium was higher in cultured cells derived from angiosarcoma. Of the angiosarcoma-derived cultured cells, Re12 cells also had a large amount of miR-126 secretion.

Example 2
3 to 4 ml of blood was collected from the dog healthy group (n = 10), the non-neoplastic splenic disease group (n = 9), and the angiosarcoma affected group (n = 10), respectively. Recovered). Plasma was quickly collected from the blood and stored at −80 ° C. until use. Low molecular RNA was recovered from the plasma sample using NucleoSpin miRNA (Takara Bio Inc.). The expression levels of the biomarkers (miR-214, miR-126) and the endogenous control (miR-16) were measured by the same method as in Example 1 except that miR-16 was used as the endogenous control.

  The results for the biomarker (miR-214, miR-126) expression level corrected by the endogenous control (miR-16) expression level are shown in FIG. 2 (Control: canine healthy population, NNS: canine non-neoplastic spleen) Disease group, HSA: dog angiosarcoma affected group). As shown in FIG. 2A, in the angiosarcoma affected group, there were many individuals showing high expression levels in plasma of miR-214 and / or miR-126. In an angiosarcoma-affected group, individuals with high miR-214 and miR-126 expression levels such as HSA3; individuals with high miR-214 expression levels and slightly higher miR-126 expression levels, such as HSA10; Individuals with low miR-214 expression level and high miR-126 expression level, such as HSA9, were confirmed.

  FIG. 2B shows the result of statistical analysis by the Steel method for the result of FIG. 2A (in FIG. 2B, “**” indicates that there is a statistically significant difference). As shown in FIG. 2B, in the angiosarcoma-affected group, the expression level of biomarkers (miR-214, miR-126) in plasma was increased compared to the healthy group and the non-neoplastic splenic disease group.

  3A (miR-214) and B (miR-126) show the results of analyzing the above results using medical statistical analysis software GraphPad Prism 6 (GraphPad Software). In FIG. 3, the vertical axis (Sensitivity%) indicates the sensitivity as a biomarker (the higher the value, the lower the probability of showing false negatives), and the horizontal axis (Specificity%) indicates the specificity (value) as a biomarker. The higher the value, the lower the probability of showing false positives). In FIG. 3, the larger the AUC (area under the curve), the higher the reliability. AUC (area under the curve) may be 0.9 or more, but 0.95 or more is preferable from the viewpoint of specificity and sensitivity (maximum value: 1.0). As shown in FIG. 3, it can be seen that miR-214 (AUC = 0.9) and miR-126 (AUC = 0.94) can be used for detection of vascular lesions.

  The result of plotting the miR-214 expression level corrected by the endogenous control (miR-16) expression level on the horizontal axis and the miR-126 expression level corrected by the endogenous control (miR-16) expression level on the vertical axis is shown in FIG. 4 shows. As shown in FIG. 4, in the angiosarcoma affected group individuals, the expression level of at least one of miR-214 and miR-126 tended to increase. Furthermore, FIG. 5 shows the results of analysis using medical statistical analysis software GraphPad Prism 6 (GraphPad Software Co., Ltd.) for the case where both miR-214 and miR-126 are combined into a biomarker. As shown in FIG. 5, by combining both miR-214 and miR-126, blood vessels with superior specificity and sensitivity compared to the case where miR-214 and miR-126 are each used alone as a biomarker. It turns out that it becomes a lesion biomarker (AUC = 0.97).

Example 3
Tumor excision surgery was performed on 3 individuals of the angiosarcoma affected group used in Example 2. The expression level of the biomarker in plasma before and after the tumor removal operation was measured. Specifically, blood was collected from HSA3, HSA6, and HSA8, which are affected by hemangiosarcoma, from the first visit to the day before surgery on the day of surgery as day 0. Tumor removal surgery was performed from each individual, and blood was collected again on day 20 (HSA3), day 21 (HSA6), or day 15 (HSA8). Plasma was prepared from the collected blood, and the expression levels of biomarkers (miR-214, miR-126) and endogenous control (miR-16) were measured in the same manner as described above.

  The biomarker (miR-214, miR-126) expression level corrected with the endogenous control (miR-16) expression level in plasma before and after tumor excision surgery is shown in FIG. 6 (FIG. 6A: miR-214, FIG. 6B: miR-126, vertical axis is logarithmic display). In FIG. 6, the numerical value under “FC” means Fold-Change (magnification change; expression level after surgery when the expression level before surgery is 1). In FIG. 6, “**” indicates that there is a statistically significant difference.

  As shown in FIG. 6, for HSA3 and HSA6, the expression level of plasma biomarkers (miR-214, miR-126) was decreased. On the other hand, with respect to HSA8, although a decreasing tendency was observed in the expression level of miR-126, miR-214 hardly changed. Therefore, it can be seen that by measuring the expression levels of both miR-214 and miR-126 as biomarkers of vascular lesions, it is possible to evaluate vascular lesions with higher sensitivity.

Example 4
Biomarkers (miR-214, miR-126) and endogenous secreted into the culture medium using human vascular endothelial cells (HMEC1 cells) and human hemangiosarcoma-derived cultured cells (ISO-HAS cells and HAMON cells) The expression level of the sex control (miR-16) was measured.

(Cell culture)
HMEC1 cells (human vascular endothelial cells) were cultured in MCDB131 medium (containing 10 ng / ml EGF, 1 μg / ml hydrocortisone, 10 mM glutamine, 10% (v / v) fetal bovine serum). ISO-HAS cells (cultured cells derived from human hemangiosarcoma, see International Journal of Cancer (1999), 81 (2) 305-308) are composed of DME medium (15% (v / v) fetal bovine serum, 50% (v / v v) ISOS-1 cell culture supernatant (including ISOS-1 cells cultured in DME medium containing 15% (v / v) fetal bovine serum for 3-4 days and collected by filtration through a 0.22 μm filter) Incubated in HAMON cells (human hemangiosarcoma-derived cultured cells, see Journal of Dermatological Science (2013), 70 (2), 116-122) were cultured in DME medium (containing 10% fetal bovine serum).

(Measurement of marker)
Cells seeded in a 6-well plate at a concentration of 1-2 × 10 ⁵ cells / ml were cultured for 48 hours in a 37 ° C. incubator (95% air / 5% CO ₂ ). The cultured medium was collected, and the expression levels of biomarkers (miR-214, miR-126) and endogenous control (miR-16) were measured according to Examples 1 and 2 using the following primers and probes. .

  The biomarker (miR-214, miR-126) expression level corrected by the endogenous control (miR-16) expression level is shown in FIG. As shown in FIG. 7, compared to human vascular endothelial cells (HMEC1 cells), human hemangiosarcoma-derived cultured cells secreted more miR-214 into the medium. Among the angiosarcoma-derived cultured cells, HAMON cells also had a large amount of miR-126 secretion.

Claims (8)

A method for evaluating a vascular lesion in a subject,
Measuring a biomarker expression level in a biological sample separated from the subject, and comparing the biomarker expression level to a reference value,
When the biomarker expression level is equal to or lower than the reference value, it is an index that does not have a vascular lesion, and when it exceeds the reference value, it is an index that has a vascular lesion, and the reference value is separated from a healthy individual The level of biomarker expression in the biological sample,
The vascular lesion is an angiosarcoma;
The method for evaluating a vascular lesion, wherein the biomarker is selected from the group consisting of miR-214 and miR-126. A method for evaluating a vascular lesion in a subject,
Measuring a biomarker expression level in a biological sample separated from the subject; and
Comparing the biomarker expression level to a reference value,
When the expression level of the biomarker in the biological sample separated from the subject after the therapeutic treatment is less than the reference value, it is an index that does not have a vascular lesion, and when it is equal to or higher than the reference value, the index that has a vascular lesion And the reference value is a biomarker expression level in a biological sample separated from the same subject prior to the therapeutic treatment,
The vascular lesion is an angiosarcoma;
The method for evaluating a vascular lesion, wherein the biomarker is selected from the group consisting of miR-214 and miR-126. The evaluation method of the vascular lesion of Claim 1 or 2 which measures the expression level of miR-214 and miR-126 as said biomarker. The method for evaluating a vascular lesion according to any one of claims 1 to 3 , wherein the expression level is measured by a nucleic acid amplification method, a microarray method, or a Northern blot method. Measuring the endogenous control expression level in the biological sample, and the biomarker expression level has been corrected by the endogenous control expression level is compared with the reference value, according to any one of claims 1-4 Method for evaluating vascular lesions. The method for evaluating a vascular lesion according to any one of claims 1 to 5 , wherein the biological sample is selected from the group consisting of blood, plasma, and serum. An angiosarcoma evaluation kit comprising a biomarker detection substance that is miR-214 and miR-126. The kit for angiosarcoma evaluation according to claim 7 , wherein the detection substance is a primer for nucleic acid amplification or a probe.