JP2021114934A - Pancreatic cancer biomarker - Google Patents

Abstract

To develop a technique that can diagnose pancreatic cancer with low invasiveness and conveniently.SOLUTION: The present disclosure provides a pancreatic cancer biomarker, comprising LOC100507412 RNA; a pancreatic cancer inspecting method, including analyzing the amount of LOC100507412 RNA in a human-derived extracellular vesicle; and a pancreatic cancer diagnosis reagent, including means for analysis of the amount of LOC100507412 RNA.SELECTED DRAWING: None

Description

The present invention relates to a biomarker for pancreatic cancer, a method for testing pancreatic cancer, and a diagnostic reagent for pancreatic cancer.

In recent years, various techniques have been developed for diagnosing cancer. In particular, in order to reduce the burden on the subject, it is desired to develop a technique that is minimally invasive and can easily diagnose cancer.

Pancreatic cancer is known to be extremely malignant. Intraductal Papillary Mucinous Neoplasm (IPMN) is also known as a tumor that may be associated with pancreatic cancer. IPMN is positioned as a risk factor for pancreatic carcinogenesis, and it is known that patients with IPMN develop pancreatic cancer at an annual rate of about 1 to 2%. Since it is clinically important to distinguish between healthy subjects and pancreatic cancer patients, and between patients with intraductal papillary mucus tumor and pancreatic cancer patients, it is desirable to develop a minimally invasive and simple technique that enables such diagnosis. Has been done.

Patent Document 1 reports analysis of a plurality of long non-coding RNA (lncRNA) amounts as a minimally invasive technique capable of easily distinguishing a healthy person from a patient with a papillary mucinous tumor in the pancreatic duct or a patient with pancreatic cancer. LncRNA is a functional RNA composed of 200 or more ribonucleotide residues, but the function of many lncRNAs is unknown.

By the way, as one of the lncRNAs, the LOC100507412 RNA expressed from the genomic DNA (SEQ ID NO: 1) specified by LOCUS number: NR_038958 is registered in the database. However, as far as the present inventors know, no findings other than the sequence information of LOC100507412 RNA have been particularly reported.

JP-A-2017-158545

An object of the present invention is to develop a technique that is minimally invasive and can easily diagnose pancreatic cancer.

As a result of diligent studies, the present inventors have found that LOC100507412 RNA in human extracellular vesicles is an index of pancreatic cancer, and have completed the present invention.

That is, the present invention is as follows.
[1] A biomarker for pancreatic cancer consisting of LOC100507412 RNA.
[2] The biomarker of pancreatic cancer is a biomarker for differentiating between a healthy person and a pancreatic cancer patient, or a biomarker for differentiating an intraductal papillary mucinous tumor patient and a pancreatic cancer patient. marker.
[3] A method for testing pancreatic cancer, which comprises analyzing the amount of LOC100507412 RNA in extracellular vesicles obtained from humans.
[4] The method of [3] including the following (a) to (c):
(A) Retrieving extracellular vesicles from human blood samples;
(B) Preparing RNA samples from extracellular vesicles; and (c) analyzing LOC100507412 RNA levels in RNA samples.
[5] The method of [4], wherein the extracellular vesicles are recovered by immunoprecipitation.
[6] The method of [5], wherein immunoprecipitation is performed using an affinity substance for CD9 and / or CD63.
[7] The method of [4], wherein the extracellular vesicles are collected by ultracentrifugation.
[8] The method according to any one of [4] to [7], wherein the human blood sample is a blood sample of a pancreatic cancer patient.
[9] The method for testing pancreatic cancer is a method for distinguishing a healthy person from a pancreatic cancer patient, or a method for distinguishing an intraductal papillary mucinous tumor patient from a pancreatic cancer patient, [3] to [8]. Either way.
[10] A diagnostic reagent for pancreatic cancer, which comprises means for analyzing the amount of LOC100507412 RNA.
[11] The diagnostic reagent of [10], wherein the means for analyzing the amount of LOC100507412 RNA is one or more means selected from the group consisting of reverse transcriptase, one or more primers and probes.
[12] The diagnostic reagent according to [10] or [11], further comprising a means for recovering extracellular vesicles.

According to the present invention, pancreatic cancer can be easily diagnosed with minimal invasiveness. The present invention is also useful for differentiating between healthy subjects or patients with intraductal papillary mucinous tumors and patients with pancreatic cancer.

FIG. 1 is a genomic DNA sequence encoding LOC100507412 (LOCUS number: NR_038958), which is one of long non-coding RNAs (lncRNAs). The PCR primers used in the examples were designed to correspond to the nucleotide region at positions 583 to 610 (underlined) and the nucleotide region at positions 700 to 717 (underlined). The amplified fragment of PCR was detected with a probe corresponding to the nucleotide region (italics) at positions 613 to 630. FIG. 2 shows LOC100507412 RNA in extracellular vesicle-derived RNA (EV RNA) prepared from the sera of 20 patients each of healthy subjects (Control), intraductal papillary mucinous tumor patients (IPMN), and pancreatic cancer patients (PDAC). It is a figure which shows the analysis of quantity.

The present invention provides a biomarker for pancreatic cancer consisting of LOC100507412 RNA.

LOC100507412 RNA is a long non-coding RNA (lncRNA) expressed from genomic DNA (SEQ ID NO: 1) identified by LOCUS number: NR_038958. The LOC100507412 RNA used in the present invention may have mutations (eg, substitutions, insertions, deletions) of one or more naturally occurring nucleotide residues between races and / or individuals. The number of nucleotide residues in such a mutation is, for example, 1 to 100, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably 1 or 2. It may be 3, 4, or 5.

Conventionally, no knowledge other than the sequence information of LOC100507412 RNA has been reported, and the function and use of the RNA have been completely unknown. However, this time, the present inventors have found that LOC100507412 RNA is useful for the diagnosis of pancreatic cancer, and thus the usefulness of the RNA has been clarified for the first time. For example, LOC100507412 RNA is useful for differentiating healthy individuals or patients with intraductal papillary mucinous tumors from patients with pancreatic cancer. In addition, by using the present invention in combination with other techniques capable of distinguishing a healthy person from a patient with intraductal papillary mucinous tumor, the subject is healthy, has an intraductal papillary mucinous tumor, or develops pancreatic cancer. It is also possible to determine whether it is affected.

The present invention also provides a method for testing pancreatic cancer, which comprises analyzing the amount of LOC100507412 RNA in extracellular vesicles obtained from humans.

Extracellular vesicles are tiny vesicles with a membrane structure that are secreted by various types of cells. Extracellular vesicles include, for example, exosomes, ectosomes, and apoptotic vesicles. Preferably, the extracellular vesicle is an exosome. Extracellular vesicles can also be defined by their size. The size of the extracellular vesicle is, for example, 30-1000 nm, preferably 50-300 nm, more preferably 80-200 nm. The size of extracellular vesicles can be measured by NanoSigt (manufactured by Malvern Instruments).

Extracellular vesicles can also be defined by extracellular vesicle markers. Examples of such extracellular vesicle markers include extracellular vesicle internal markers and surface markers. Examples of internal markers for extracellular vesicles include cancer fetal antigen (CEA), CA125, CA15-3, actin family, TSG101, ALIX, Charged multigenic protein (CHMP) family, and Glyceraldehyde 3-phosphate HD. , Poly (ADP-ribose) polymerase (PARP) family, AFP, CA19-9, CA125, Flotillin-I, Flotillin-II, Rab protein, promoted cell death (PDCD) 6, phosphoripid chromaze Included are the (COX) family, the lamin family, the proliferating cell nuclear antigen (PCNA), the tuberin family, the TATA binding protein (TBP), the voltage-dependent anion channel protein (VDAC), the amyloid beta, and the tau protein. Examples of surface markers for extracellular vesicles include tetraspanin membrane proteins (extracellular vesicle membrane-specific four-transmembrane proteins, eg, CD9, CD63, CD81), extracellular matrix metalloprotease inducers (eg, CD147). , Heat shock protein (HSP) 70, HSP90, major tissue compatibility complex (MHC) I, lysosome-related membrane protein (LAMP) 1, intercellular adhesion molecule (ICAM) -1, integrin, ceramide, cholesterol, phosphatidylserine, Examples include Annexins, Caveolin-I, and EpCAM. The extracellular vesicle marker is preferably an extracellular vesicle surface marker, more preferably a tetraspanin membrane protein, and even more preferably CD9 and / or CD63.

The extracellular vesicles used in the present invention are not particularly limited as long as they are obtained from humans. As such extracellular vesicles, those recovered from human samples can be used. As the human sample, any sample containing extracellular vesicles containing LOC100507412 RNA can be used. Preferably, the human sample is a human blood sample. Examples of the human blood sample include a human whole blood sample, a human plasma sample, and a human serum sample, and a human serum sample is preferable.

The method of the present invention may be carried out by, for example, a method including the following (a) to (c):
(A) Retrieving extracellular vesicles from human blood samples;
(B) Preparing RNA samples from extracellular vesicles; and (c) analyzing LOC100507412 RNA levels in RNA samples.

The recovery in (a) can be performed by any method capable of recovering extracellular vesicles from a human blood sample. Examples of such a method include a guanidine cesium chloride supercentrifugation method and an AGPC (AcidGuanidinium-Phenol-Chloroform) method.

Further, for the recovery in (a), a method capable of recovering extracellular vesicles from a human blood sample with high efficiency may be used. For example, as such a highly efficient recovery method, a method including treating the extracellular-containing sample with a chelating agent and then separating the extracellular vesicles from the extracellular-containing sample treated with the chelating agent is used. (International Publication No. 2018/070479).

A chelating agent is a compound or a salt thereof having a coordination portion capable of coordinating with a metal ion. The number of coordination portions is preferably 2 or more, more preferably 3 or more (eg, 3 or 6). Examples of the coordinating atom as the coordinating portion include an oxygen atom, a phosphorus atom, a nitrogen atom, a sulfur atom, and a chlorine atom. The coordination atom is preferably an oxygen atom or a phosphorus atom, and more preferably an oxygen atom. Examples of the coordinating group as the coordinating portion include the group having the above-mentioned coordinating atom. The coordinating group is preferably a carboxylic acid group or a phosphoric acid group, and more preferably a carboxylic acid group.

Examples of the chelating agent include hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra (methylenephosphonic acid) (EDTMP), and the like. Glycol etherdiaminetetraacetic acid (EGTA), as well as salts thereof. Examples of the salt include metal salts (eg, monovalent metal salts such as sodium salt and potassium salt, and divalent metal salts such as calcium salt and magnesium salt) and inorganic salts (eg, fluoride, chloride, etc.). Halogen salts such as bromide and iodide, and ammonium salts), organic salts (eg, ammonium salts substituted with alkyl groups), and acid addition salts (eg, sulfuric acid, hydrochloric acid, hydrobromic acid, nitrate, phosphoric acid). Salts with inorganic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and organic acids such as trifluoroacetic acid).

The chelating agent may also be a chelating agent that is widely used as a component contained in a blood collection tube for clinical examination. Examples of such chelating agents include EDTA, EGTA, NTA, HEDTA, EDTMP, HIDA, citric acid and salts thereof. In the present invention, the use of such chelating agents is also desirable from the viewpoint of clinical application.

In the present invention, one kind of chelating agent may be used alone, or a plurality of kinds (eg, two kinds, three kinds, four kinds) of chelating agents may be used in combination.

The concentration of the chelating agent when the chelating agent is allowed to act on the human blood sample is not particularly limited as long as the concentration can improve the recovery amount of extracellular vesicles from the human blood sample. In the present invention, in light of the fact that mixing a human blood sample with a chelating agent produces a mixed solution containing extracellular vesicles and the chelating agent, the chelating agent when the chelating agent acts on the human blood sample The concentration is the same as the concentration of the chelating agent in such a mixed solution. Therefore, in the present invention, the concentration of the chelating agent when the chelating agent is allowed to act on the human blood sample can also be defined as the concentration of the chelating agent in such a mixed solution. Such a concentration varies depending on factors such as the type and concentration of other components used in combination with the chelating agent, but is, for example, 10 mM to 1000 mM. Preferably, the concentration of the chelating agent may be 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 80 mM or more, or 100 mM or more. Such concentrations may also be 800 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, or 300 mM or less. More specifically, the concentration of the chelating agent may be 20 to 800 mM, 30 to 600 mM, 40 to 500 mM, 50 to 450 mM, 60 to 400 mM, 80 to 350 mM, or 100 to 300 mM.

Recovery of extracellular vesicles from human blood samples can be performed, for example, by immunoprecipitation or ultracentrifugation. Analyzing the amount of LOC100507412 RNA after recovery of human extracellular vesicles can prevent potential contamination with substances that can inhibit the detection of the RNA and also cause differences between human samples (eg, in human blood samples). The influence of existing impurities) can be reduced. Preferably, recovery can be performed by immunoprecipitation. When the recovery is performed by immunoprecipitation, the potential contamination can be further prevented, and the influence of the difference can be further prevented.

If the extracellular vesicles are recovered from a human blood sample by immunoprecipitation, the immunoprecipitation can be performed using an affinity substance for the surface markers of the extracellular vesicles.

Examples of the substance having an affinity for the surface marker of the extracellular vesicle include an antibody against the above-mentioned extracellular vesicle surface marker, an aptamer, a phosphatidylserine binding protein, and a ceramide binding protein. In the present invention, a single substance or a plurality of types (eg, 2 types, 3 types, 4 types) of substances can be used as the affinity substance for the surface marker of extracellular vesicles.

Preferably, the substance having an affinity for the surface marker of the extracellular vesicle is an antibody against the surface marker of the extracellular vesicle from the viewpoint of ensuring specificity for the surface marker of the extracellular vesicle and easiness of preparation. You may. Antibodies include, for example, full-length antibodies (eg, monoclonal antibodies, polyclonal antibodies) and antigen-binding fragments thereof. The antigen-binding fragment may be any antibody fragment that maintains binding to the extracellular vesicle surface marker of interest, and examples thereof include Fab, Fab', F (ab') ₂ , scFv and the like. In the present invention, a single antibody or a plurality of types (eg, 2 types, 3 types, 4 types) of antibodies can be used.

The affinity substance (preferably an antibody) for the surface marker of the extracellular vesicle may be immobilized on a solid phase to facilitate the separation of the extracellular vesicle. Examples of the solid phase include beads and particles (eg, cepharose beads, agarose beads, magnetic beads (magnetic particles)) and supports (eg, plates such as plastic plates, membranes). The substance can be fixed to the solid phase by any method. The substance that is immobilized on the solid phase and has an affinity for the surface marker of the extracellular vesicle is mixed with the mixed solution containing the extracellular vesicle, and the substance that binds to the surface marker of the extracellular vesicle and the extracellular vesicle The extracellular vesicles can be separated from the mixed solution by forming the complex of the above and separating the complex from the mixed solution. For example, when beads or particles are used as the solid phase, extracellular vesicles can be separated from the mixed solution by centrifuging after forming the complex and removing the supernatant. When magnetic beads are used as the solid phase, extracellular vesicles may be separated from the mixed solution by collecting magnetism after forming the complex and removing the supernatant. When a support is used as the solid phase, extracellular vesicles can be separated from the mixed solution by removing the mixed solution after the formation of the complex.

The affinity substance for the surface marker of extracellular vesicles is preferably an affinity substance for a tetraspanin membrane protein, and even more preferably an affinity substance for CD9 and / or CD63.

The temperature at which the human blood sample is mixed with the extracellular vesicle surface marker affinity substance is at a temperature at which the extracellular vesicle can bind to the affinity substance and the extracellular vesicle can be recovered using the affinity substance. As long as there is no particular limitation. Such a temperature may be, for example, 4 to 80 ° C, preferably 35 to 60 ° C. The time for treating a human blood sample with an affinity substance for the surface marker of the extracellular vesicle is not particularly limited as long as the extracellular vesicle and the affinity substance can bind to each other for a sufficient time.

When the extracellular vesicles are collected from a human blood sample by ultracentrifugation, the ultracentrifuge can be performed using an ultracentrifuge. The gravity applied by ultracentrifugation is, for example, 10,000 × g to 200,000 × g, preferably 70,000 × g to 150,000 × g. The time for ultracentrifugation is, for example, 0.5 to 24 hours, preferably 1 to 5 hours. The temperature in ultracentrifugation is, for example, 4 to 30 ° C. Ultracentrifugation may be performed once or multiple times (eg, two or three times).

The preparation in (b) can be performed by any method capable of preparing an RNA sample from extracellular vesicles. As such a method, any RNA extraction method widely used in the art can be preferably used.

The analysis in (c) can be performed by any method capable of analyzing the amount of RNA. Examples of the method for analyzing the amount of RNA include a gene amplification method, a hybridization method, and a sequencing method. Examples of the gene amplification method include a temperature-changing gene amplification method (eg, PCR) and an isothermal gene amplification method (eg, LAMP, ICAN). In the gene amplification method, reverse transcriptase can be used in combination. Examples of the hybridization method include microarray and Northern blotting. As the sequencing method, for example, the technique described in JP-A-2017-158545 may be used.

Based on the analysis of the amount of LOC100507412 RNA, the possibility of developing pancreatic cancer is determined. The result of the analysis of the amount of RNA is used as an index for comparison with the reference value. The group of subjects suffering from pancreatic cancer has a significantly higher amount of LOC100507412 RNA than the group of subjects not suffering from pancreatic cancer. Therefore, according to the method of the present invention, when the amount of RNA is equal to or higher than the reference value, it can be determined that the subject is highly likely to have pancreatic cancer. In addition, when the amount of RNA is less than the reference value, it can be determined that the subject is unlikely to have pancreatic cancer.

As the above reference value, for example, a cutoff value appropriately set so as to be able to distinguish between a group not suffering from pancreatic cancer (healthy subjects and / or a papillary mucus tumor in the pancreatic duct) and a group suffering from pancreatic cancer is used. It can be used. The cutoff value is a value that satisfies both high diagnostic sensitivity (prevalence correct diagnosis rate) and high diagnostic specificity (disease-free correct diagnosis rate) when the disease is determined based on the value. Diagnostic sensitivity (or simply sensitivity) is the percentage of subjects with a particular condition that are correctly diagnosed. If a "positive" result is obtained for all subjects with a particular condition, the sensitivity is 100%. Diagnostic specificity (or simply specificity) is the percentage of subjects who do not have a specific condition and are correctly diagnosed. If a "negative" result is obtained for all subjects without a particular condition, the specificity is 100%. Methods for calculating cutoff values are well known in the art.

The method of the present invention may also include administering a therapeutic agent for pancreatic cancer (eg, an anticancer agent for pancreatic cancer) to a patient whose LOC100507412 RNA amount is equal to or higher than a reference value. The method of the present invention further comprising such administration is useful as a therapeutic method for pancreatic cancer.

The present invention also provides diagnostic reagents for pancreatic cancer, including means for analyzing LOC100507412 RNA levels.

Examples of means for analyzing the amount of RNA include reverse transcriptase, one or more primers, and a probe. These analysis means may be used in combination. For example, when RNA levels are measured, RT-PCR may be performed in combination with reverse transcriptase and two or more primers (and probes as needed). In addition, a hybridization method may be performed using a probe. Further, a sequencing method may be performed using one primer. The primer and probe may be labeled with a labeling substance such as a fluorescent substance.

The diagnostic reagent of the present invention may further include means for recovering extracellular vesicles. As a means for recovering extracellular vesicles, an affinity substance for a surface marker of extracellular vesicles can be mentioned. Affinities for extracellular vesicle surface markers are as described above. The affinity substance for the surface marker of extracellular vesicles may be immobilized on a solid phase to facilitate the separation of extracellular vesicles. Alternatively, the diagnostic reagents of the present invention may further comprise a solid phase if the substance that has an affinity for extracellular vesicle surface markers is not immobilized on the solid phase. The solid phase is as described above. The diagnostic reagents of the present invention are useful, for example, for the rapid and convenient implementation of the methods of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1: Analysis of RNA amount of LOC100507412, which is a biomarker for pancreatic cancer RNA is present in multiple modes in blood. For example, RNA includes (a) cell-free RNA (cfRNA) which is free RNA in blood, (b) RNA existing in free cells in blood, and (c) factors in blood (eg, protein, etc.). RNA that binds to (stabilizing factor), (d) RNA derived from extracellular vesicles (EV RNA), and the like. Further, as the EV RNA, RNA existing inside and / or on the surface of the EV, RNA existing in a specific EV (eg, RNA derived from an EV positive for tetraspanin such as CD9 and / or CD60) and the like are used. be. Therefore, in order to evaluate the influence of the difference in the detection sensitivity of the biomarker depending on the type of RNA used as the detection target, a method for recovering RNA from the blood sample was examined. The methods examined were (1) a method capable of recovering the cfRNA of (a) above as a main RNA component, and (2) a method using ExoQuick ™ capable of recovering various RNAs (a) to (d) above. 3) The method capable of recovering the EV RNA of the above (d) was examined. As a blood sample, sera of 5 patients with pancreatic cancer were used.

(A) Recovery of RNA from blood sample (1) cfRNA (major RNA component)
CfRNA was recovered from serum using a miRNeasy Mini Kit (QIAGEN). First, 700 μL of QIAzol Lysis Regent was added to 300 μL of serum, and then RNA was extracted according to the protocol. Although this method allows contamination of the RNA components (a) to (d) above, cfRNA can be extracted as the main RNA component.

(2) ExoQuick ™
According to ExoQuick ™ (System Biosciences), it is explained that various RNAs (a) to (d) above can be recovered. Therefore, various RNAs were recovered from serum using ExoQuick ™. 100 μL of ExoQuick ™ was added to 300 μL of serum, incubated for 12 hours at 4 ° C. after mixing, and centrifuged at 1,500 xg for 30 minutes. After centrifugation, the supernatant was discarded and pellets were collected. Various RNAs were extracted from this pellet using SeraMir Kit (System Biosciences) according to the protocol.

(3) EV RNA
Equal amounts of 300 μL of EDTA / EGTA / PBS solution were added to serum (both EDTA and EGTA had a final concentration of 50 mM), and the mixture was centrifuged at 15,000 × g for 15 minutes. The supernatant after centrifugation was transferred, and ultracentrifugation was performed at 100,000 × g for 1 hour. The supernatant was then discarded and the precipitate was resuspended in EDTA / EGTA / PBS solution to give an EV RNA-containing sample. For this EV RNA-containing sample, EV RNA was extracted using miRNeasy in the same procedure as in (1).

(B) Analysis of LOC100507412 RNA amount in RNA sample Next, LOC100507412 RNA amount was analyzed in each RNA sample extracted in (1) to (3). Each RNA sample was subjected to a reverse transcription reaction according to the protocol using iScript (BIO-RAD) to obtain a cDNA sample. These cDNA samples were used to analyze the amount of LOC100507412 RNA. The analysis of the amount of LOC100507412 RNA was performed using digital PCR (Quant Studio 3D Digital PCR System (Thermo Fisher Scientific)) (a pair of primers used in digital PCR and a simple position of the primer used in the digital PCR. See the explanation). The results are shown in Table 1. The unit is copies / μL.

As a result, in EV RNA, the amount of LOC100507412 RNA increased (Table 1). This indicates that LOC100507412 RNA is present in extracellular vesicles. It was also confirmed that the extracellular vesicle sample is useful for improving the measurement sensitivity of LOC100507412 RNA.

Example 2: Evaluation of LOC100507412 RNA as a biomarker for pancreatic cancer Extracellular vesicles were used from 20 cases each of healthy subjects (Control), intraductal papillary mucinous tumor patients (IPMN), and pancreatic cancer patients (PDAC). The amount of LOC100507412 RNA in the vesicle-derived RNA (EV RNA) was analyzed.
First, an equal amount of EDTA / EGTA / PBS solution was added to 300 μL of serum (the final concentration of both EDTA and EGTA was 50 mM), and the mixture was centrifuged at 15,000 × g for 15 minutes. The supernatant after centrifugation was transferred, and ultracentrifugation was performed at 100,000 × g for 1 hour. The supernatant was then discarded and the precipitate was resuspended in EDTA / EGTA / PBS solution to obtain an EV sample. For this EV sample, EV RNA was extracted using miRNeasy in the same procedure as in Examples 1 (A) and (1). Next, the analysis of the amount of LOC100507412 RNA in EV RNA was performed in the same manner as in Example 1 (B). The results are shown in FIG.
As a result, in multiple cases of PDAC, the amount of LOC100507412 RNA in EV RNA was significantly higher than that in Control and IPMN (Fig. 2).
Therefore, LOC100507412 RNA is expected to be useful as a biomarker for pancreatic cancer.

Example 3: Improvement of PDAC determination accuracy by improving RNA detection sensitivity It was examined whether the type of extracellular vesicle recovery method affects the PDAC determination accuracy. Immunoprecipitation and ultracentrifugation were compared as methods for collecting extracellular vesicles. As blood samples, sera of 20 cases each of a healthy person (Control), an intraductal papillary mucinous tumor patient (IPMN), and a pancreatic cancer patient (PDAC) were used.

First, EV samples were prepared by immunoprecipitation. Equal amounts of carboxycellulose (CMC) / EDTA / EGTA / PBS solution were added to 300 μL of serum (CMC, final concentration 0.5%, final concentration 50 mM for both EDTA and EGTA), and anti-CD9 antibody (manufactured in-house) or anti-CD63. The antibody (manufactured in-house) fixed Dynabeads M-280 tosylacticated (Life Technologies) was added in equal amounts to 0.26 mg / mL. After a rotation reaction at 4 ° C. overnight, the cells were washed 3 times with PBS to obtain an EV RNA-containing sample (IP-EV).

Next, the EV sample was prepared by the ultracentrifugation method in the same manner as in Example 2 (UC-EV).

For each EV sample prepared by the immunoprecipitation method and the ultracentrifugation method, EV RNA was extracted using miRNeasy in the same procedure as in Examples 1 (A) and (1). Next, the analysis of the amount of LOC100507412 RNA in EV RNA was performed in the same manner as in Example 1 (B). The results are shown in Table 2. The unit is copies / μL.

As a result, in both immunoprecipitation (IP-EV) and ultracentrifugation (UC-EV), PDAC was significantly higher in LOC100507412 RNA in EV RNA than in Control and IPMN, but in particular, In the immunoprecipitation method, the determination accuracy of PDAC improved as the detection sensitivity of LOC100507412 RNA improved (Table 2). It is not clear why the immunoprecipitation method is superior to the ultracentrifugation method in determining PDAC, but considering that the immunoprecipitation method can reduce impurities compared to the ultracentrifugation method, the impurities by the immunoprecipitation method. The reduction may have prevented contamination with substances that could interfere with the detection of LOC100507412 RNA.
Therefore, detection of LOC100507412 RNA in RNA samples using extracellular vesicles recovered by immunoprecipitation is expected to be useful as a biomarker for pancreatic cancer with excellent determination accuracy.

The present invention is useful, for example, in clinical examination of pancreatic cancer.

Claims (12)

LOC100507412 A biomarker for pancreatic cancer consisting of RNA. The biomarker according to claim 1, wherein the pancreatic cancer biomarker is a biomarker for differentiating a healthy person from a pancreatic cancer patient, or a biomarker for differentiating an intraductal papillary mucinous tumor patient from a pancreatic cancer patient. A method for testing pancreatic cancer, which comprises analyzing the amount of LOC100507412 RNA in extracellular vesicles obtained from humans. The method according to claim 3, which includes the following (a) to (c):
(A) Retrieving extracellular vesicles from human blood samples;
(B) Preparing RNA samples from extracellular vesicles; and (c) analyzing LOC100507412 RNA levels in RNA samples. The method according to claim 4, wherein the extracellular vesicles are recovered by immunoprecipitation. The method of claim 5, wherein immunoprecipitation is performed with an affinity for CD9 and / or CD63. The method according to claim 4, wherein the extracellular vesicles are collected by ultracentrifugation. The method according to any one of claims 4 to 7, wherein the human blood sample is a blood sample of a pancreatic cancer patient. Any one of claims 3 to 8, wherein the pancreatic cancer test method is a method for distinguishing a healthy person from a pancreatic cancer patient, or a method for distinguishing an intraductal papillary mucinous tumor patient from a pancreatic cancer patient. The method described. A diagnostic reagent for pancreatic cancer, which comprises means for analyzing LOC100507412 RNA levels. The diagnostic reagent according to claim 10, wherein the means for analyzing the amount of LOC100507412 RNA is one or more means selected from the group consisting of reverse transcriptase, one or more primers and probes. The diagnostic reagent according to claim 10 or 11, further comprising a means for recovering extracellular vesicles.

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