WO2022124564A1 - Method for predicting and diagnosing diabetic neuropathy using micro-rna, and kit therefor - Google Patents

Abstract

Provided are: a method for diagnosing or predicting diabetic neuropathy using microRNA, which can be an indicator of diabetic neuropathy, and a kit for the method. A composition for predicting or diagnosing diabetic neuropathy comprises at least one biomarker measurement reagent selected from the group consisting of miR-122, miR-199b, and miR-8485.

Description

Method for predicting and diagnosing diabetic neuropathy using microRNA and kit therefor

The present invention relates to a method for diagnosing diabetic neuropathy and a diagnostic kit using the same. In detail, it relates to a method for diagnosing or predicting diabetic neuropathy using microRNA, which can be an indicator of diabetic neuropathy, and a kit for the same. More specifically, it relates to a method and a kit capable of increasing the accuracy of diagnosis of diabetic neuropathy by using microRNAs showing opposite expression levels.

Diabetic neuropathy, a common microvascular complication of type 1 diabetes mellitus and type 2 diabetes mellitus, is a symptom of peripheral nerve dysfunction in diabetic patients. defined by existence. Population- and clinical-based studies suggest that the prevalence of diabetic neuropathy after 20 years of type 1 diabetes is 20% and increases by about 10% to 15% to 50% at 10 years of type 2 diabetes. Despite these studies, the pathophysiology of diabetic neuropathy has not been clearly defined because of numerous closely related causal mechanisms and difficulties in establishing a definitive diagnostic method.

Diagnostic approaches for diabetic neuropathy are complex and not yet standardized and have generally consisted of a combination of various qualitative and quantitative methods to increase the sensitivity and specificity of the results.

In particular, effective screening for early abnormalities prior to identifying patients with obvious clinical symptoms, asymptomatic disease course, or diabetic neuropathy development candidates was not possible by conventional methods. As well as the lack of a reliable diagnostic method for diabetic neuropathy, there was also a complexity to the conventional treatment regimens. In the absence of a causal treatment for the development of diabetic neuropathy, conventional therapies have often consisted of a combination of glycemic control and pain management.

In order to solve this problem, the present inventors focused on a method of using micro-RNA (micro-RNA or miRNA, hereinafter abbreviated as 'miRNA') involved in diabetic neuropathy.

miRNA is an untranslated RNA composed of several to several tens of nucleotides, and performs the functions of RNA silencing in the transcriptional stage and regulating gene expression in the post-transcriptional stage. It is known that the function of these miRNAs is performed through the formation of base pairs with complementary sequences in mRNA (Bartel, D.P., Cell, 136(2): 215-233, 2009).

After miRNA was first discovered in C. elegans, various types of miRNAs have been reported one after another as it is known that it is being used as a regulatory mechanism for gene expression in animals, plants and viruses, due to mutations in miRNA genes, etc. It is known that certain diseases, such as cancer, may occur due to the dissonance of the functions of : 834-838, 2005).

As described above, accurate and rapid diagnosis is required for effective treatment of diabetic neuropathy. To solve this problem, a diagnostic method using microRNA associated with diabetic neuropathy is presented.

That is, the technical problem to be solved by the present invention is to measure the expression level of miRNA that is over-expressed or under-expressed under diabetic neuropathy and compared with a normal control group to treat diabetic neuropathy. It is to provide a simple method for diagnosing or predicting expression.

In addition, it is to increase the accuracy of diagnosis by measuring the expression level by combining miRNAs showing different expression levels under diabetic neuropathy.

It is also to provide a composition for prediction or diagnosis of diabetic neuropathy for this purpose.

In addition, it is an object to provide a biomarker capable of diagnosing or predicting diabetic neuropathy.

Another object is to provide a diagnostic kit for diabetic neuropathy using such biomarkers.

The problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

The composition for prediction or diagnosis of diabetic neuropathy according to an embodiment of the present invention for solving any of the above problems is for measuring one or more biomarkers selected from the group consisting of miR-122, miR-199b, and miR-8485 Includes reagents.

The reagent may include a nucleic acid sequence of each biomarker, a nucleic acid sequence complementary to the nucleic acid sequence, and a fragment of the nucleic acid sequence.

The nucleic acid sequence or fragment of the nucleic acid sequence may be a primer or a probe capable of specific binding to each of the markers, or a primer and a probe.

The reagent is a reverse transcription polymerase chain reaction of each biomarker, a polymerase chain reaction, a competitive polymerase chain reaction, a nuclease protection assay (RNase, S1 nuclease assay), an in situ hybridization method, a ligation-based polymerase chain reaction, a micro It may be a reagent used in an array or northern blot.

The method for detecting a diabetic neuropathy marker according to an embodiment of the present invention for solving any of the above other problems is a subject suspected of diabetic neuropathy in order to provide information necessary for diagnosing or predicting diabetic neuropathy. providing a sample derived from; measuring the expression level of one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485 in the sample; comparing the measurement result with the corresponding result of the corresponding marker in the control group; and when there is a change in the expression level of the subject sample compared to the control sample, determining it as diabetic neuropathy.

The detection may be by a hybridization or nucleic acid amplification method.

The hybridization may be a microarray analysis and the nucleic acid amplification may be reverse transcriptase polymerase chain reaction.

In addition, the biological sample may be whole blood, plasma, or serum from a diabetic neuropathy patient.

The method may be to use the average value of the expression levels of the miR-122, miR-199b, and miR-8485 markers.

One or more biomarkers selected from the group consisting of miR-122, miR-199b, and miR-8485 for predicting or diagnosing diabetic neuropathy according to an embodiment of the present invention for solving any of the above other problems Reagents for marker measurement are included.

Details of other embodiments are included in the detailed description.

According to embodiments of the present invention, diabetic neuropathy can be diagnosed simply by measuring the expression level of miRNAs showing a specific expression pattern under diabetic neuropathy and comparing it with a normal control group.

In addition, it is possible to increase the accuracy of diagnosis by measuring the expression level by combining miRNAs showing different expression levels under diabetic neuropathy.

Effects according to the embodiments of the present invention are not limited by the contents exemplified above, and more various effects are included in the present specification.

1 is a heat map analysis result between two groups using a next-generation genomic analysis method for discovering miRNA biomarkers in a normal group and a diabetic neuropathy group.

2 is a result of real-time RT-PCR analysis of three miRNAs showing differences in expression in the normal group and the diabetic neuropathy group.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the embodiments allow the disclosure of the present invention to be complete, and those of ordinary skill in the art to which the present invention pertains. It is provided to fully inform the person of the scope of the invention, and the present invention is only defined by the scope of the claims.

That is, various changes may be made to the embodiments presented by the present invention. It should be understood that the embodiments described below are not intended to limit the embodiments, and include all modifications, equivalents, and substitutes thereto.

The terminology used herein is for the purpose of describing the embodiments and is not intended to limit the present invention. In this specification, 'and/or' includes each and every combination of one or more of the recited items. The singular also includes the plural, unless the phrase specifically states otherwise.

As used herein, 'comprises' and/or 'comprising' does not exclude the presence or addition of one or more other components in addition to the stated components. Numerical ranges indicated using 'to' indicate numerical ranges including the values stated before and after them as lower and upper limits, respectively. 'About' or 'approximately' means a value or numerical range within 20% of the value or numerical range recited thereafter.

As used herein, the term 'diagnosis' refers to confirming the presence or characteristics of a pathological condition. That is, to determine the susceptibility of the test subject to the disease for a specific disease or disorder, to determine whether or not to currently have the specific disease or disorder, to determine the prognosis of the subject suffering from the specific disease or disorder adjudicating, monitoring the condition of the subject to provide information about whether the disease has relapsed after treatment or therametrics, such as efficacy of the treatment.

In the present invention, the diagnosis can be interpreted not only as confirming whether the progress or onset of diabetic neuropathy, but also as confirming the possibility of the onset of diabetic neuropathy.

As used herein, the term 'diagnostic subject' refers to a specimen or biological sample such as blood, body fluid, secretion, tissue, or sample isolated from a patient, individual, or these for diagnosing a corresponding indication. The expression level of miRNA of the present invention can be measured by collecting and detecting blood, body fluid, tissue, nerve cell or nerve tissue of a patient or individual.

Biological samples are taken from investigators expected to have diabetic neuropathy. In addition, control samples may be taken from investigators already aware of the disease state to corroborate the data obtained.

The biological sample refers to an organ, tissue, cell or body fluid derived from an organism. Examples of biological samples include, but are not limited to, tissue sections, whole blood, plasma, serum, urine or blood-derived white blood cells, red blood cells, or platelets, or tissue or cell cultures. Also, one or more samples may be mixed and used.

The biological sample may be obtained directly from a subject by a routine sample obtaining method from a body having or suspected of having diabetic neuropathy, or may be previously isolated and stored. In addition, if necessary, the sample may be used as a biological sample after purification from the obtained liquid. According to the present invention, the expression level of the nucleic acid marker in the present invention is determined in a biological sample derived from a subject.

The sample used for detection in the in vitro method of the present invention should generally be collected in a clinically acceptable manner, and is preferably preserved in a nucleic acid (especially RNA) or protein method. A sample awaiting analysis is usually taken from blood.

As used herein, the term 'normal control' refers to a patient, individual, or blood, body fluid, secretion, sample, etc. isolated from a patient or individual who does not have the relevant indication, and the miRNA related to the indication is expressed at a normal level to be diagnosed It can be a criterion for evaluating the miRNA expression level of

As used herein, the term 'biomarker' or 'diagnosis marker' refers to a tissue or cell in which diabetic neuropathy has occurred can be diagnosed by distinguishing it from normal cells or tissues or cells that have received appropriate diabetic neuropathy treatment. It contains non-coding nucleic acids that show an increase or decrease in the diseased tissue or site compared to a normal sample (control).

The biomarker according to the present disclosure may be used as one or a combination of two or more, for example, two or three combinations, and may be used together with an existing marker and/or a diagnostic method.

As used herein, the term 'miRNA (micro-RNA)', or 'miR', or 'miRNA', or 'micro RNA', or 'microRNA' refers to a target RNA that promotes degradation. or 21 to 23 non-coding RNAs that post-transcriptionally regulate gene expression by inhibiting their translation. The mature sequence of the miRNA used herein can be obtained from the miRNA database (http://www.mirbase.org).

In general, microRNA is transcribed into a precursor of about 70-80 nt (nucleotide) in length with a hairpin structure called pre-miRNA, and then cut by the RNAse III enzyme Dicer to produce a mature form. MicroRNA forms a ribonucleo complex called miRNP to cleave a target gene through complementary binding to a target site or inhibit translation. More than 30% of human miRNA exists as a cluster, and after being transcribed into a single precursor, it undergoes a cleavage process to form a final mature miRNA.

Those of ordinary skill in the art may also use miRNAs modified to have a sequence that maintains 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more homology to the miRNA of the present invention. It will be readily understood that it is equivalent to miRNA.

As used herein, the term 'nucleic acid' includes polynucleotides, oligonucleotides, DNA, RNA, and analogs and derivatives thereof, and includes, for example, peptide nucleic acids (PNA) or mixtures thereof. In addition, the nucleic acid may be single or double-stranded, and may encode a molecule including a polypeptide, mRNA, microRNA, or siRNA.

As used herein, the term 'primer' is a nucleic acid sequence having a short free 3' hydroxyl group, which can form a complementary template and base pair, and is a start for template strand copying. It refers to a short nucleic acid sequence that functions as a point. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures.

As used herein, the term "complementary" means that under certain hybridization (hybridization) or annealing conditions, preferably under physiological conditions, the antisense oligonucleotide is sufficiently complementary to selectively hybridize to the target of the microRNA according to the present application, , has a meaning encompassing both partially or partially substantially complementary and perfectly complementary, and preferably means completely complementary. Substantially complementary refers to a degree of complementarity that is not completely complementary, but is sufficient to bind to a target sequence and produce an effect sufficient to interfere with the effect according to the present application, that is, microRNA activity.

As used herein, the term 'probe' refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to a gene or mRNA, oligonucleotide It may be manufactured in the form of a probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.

Hereinafter, the present invention will be described in detail.

The present invention is based on the discovery of a non-coding RNA marker such as miRNA that can be used as a biomarker that enables the prediction and accurate early diagnosis of diabetic neuropathy.

In one aspect, the present invention relates to a composition for predicting and diagnosing diabetic neuropathy, comprising a reagent for detecting one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485.

A person skilled in the art can select a combination of markers satisfying the desired sensitivity and specificity through a method such as an analysis using a biological sample from a subject, including a normal person and a patient, and/or a logistic regression analysis, such as the method described in the Examples herein. will be. In one embodiment according to the present application, a combination of miR-122, miR-199b, and miR-8485 is used.

In one embodiment according to the present application, a blood sample such as whole blood, serum or plasma is used.

In one embodiment, urine, whole blood, serum and/or plasma may be used. In another embodiment, a tissue/cell or an in vitro cell culture obtained from a subject having, suspected, or likely to develop diabetic neuropathy may be used, but is not limited thereto. It also includes fractions or derivatives of the blood, cells or tissues. In the case of using cells or tissues, the cells themselves or a lysate of the cells or tissues may be used. A subject herein includes a mammal suspected of having a disease, a mammal who has been treated after having a disease but is suspected of relapse, particularly a human.

The composition according to the present application detects the expression level of the one or more miRNAs in a biological sample, and compares it with a control or reference group, and can diagnose or predict diabetic neuropathy according to the degree of increase or change in the expression level.

Accordingly, the composition according to the present application can measure/detect one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485 using the following method, and thus used in such method It may contain reagents that are

The measurement of miRNA according to the present disclosure includes methods for qualitative, quantitative and semi-quantitative detection of a desired miRNA. Any known method related to nucleic acid detection, for example, a nucleic acid hybridization and/or polymerization and/or amplification method described below and/or a hybridization-based ligation method may be used. The biomarker according to the present disclosure can be detected at the level of the presence or absence of a nucleic acid, particularly miRNA, and/or its expression level itself, change in expression level, and difference in expression level through quantitative or qualitative analysis.

Nucleic acid hybridization can be performed using a nucleic acid biochip array (microarray) or in situ hybridization. miRNA microarray technology enables the analysis of multiple miRNAs simultaneously. Nucleotides complementary to the miRNA according to the present disclosure may be spotted on a coated carrier or may be spotted on a carrier by an in situ synthesis method. In one embodiment, miRNA isolated from a biological sample may be detected by incorporation of a label (eg, biotin, fluorescent dye) detected by an enzymatic reaction after hybridization with a complementary sequence on the carrier. In another embodiment, the miRNA isolated from the biological sample is labeled with a fluorescent material to bind to the corresponding sequence, and the resulting fluorescent signal is indicative of the presence of a specific miRNA. Microarray fabrication techniques are described, for example, in Schena et al., 1996, Proc Natl Acad Sci USA. 93(20):10614-9; Schena et al., 1995, Science 270(5235):467-70; and U.S. Pat. Nos. 5,599,695, 5,556,752 or 5,631,734.

Nucleic acid polymerization or amplification methods may also be used for the detection of miRNAs according to the present application, and are particularly suitable for detecting miRNAs present in trace amounts. Various known nucleic acid amplification or synthesis methods can be used, for example, reverse transcription reaction, reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, PCR, real-time PCR, quantitative RT-PCR, quantitative PCR, NASBA ( Nucleic Acid Sequence-Base Amplification), LCR (Ligase Chain Reaction), Multiple ligatable probe amplification, Invader Technology (Third Wave), SDA (Strand Displacement Amplification), TMA (Transcription Mediated Amplification), and Eberwine RNA It may include, but is not limited to, amplification.

In one embodiment, a real-time quantitative PCR method is used after the reverse transcription reaction, which may be performed with reference to, for example, Chen et al., Nucleic Acids Research, 33(20):e170, 2005. RT-PCR is a method to isolate a sample RNA, specifically miRNA, synthesize cDNA therefrom, and then use a specific primer or a combination of a primer and a probe to detect a specific gene in the sample, and the presence/ It is a method that can determine the absence or expression level. Such methods are described, for example, in (Han, H. et al, 2002. Cancer Res. 62: 2890-6).

A typical PCR method consists of three steps for amplification of a specific target sequence, consisting of denaturation of the template, annealing in which forward and reverse primers bind to the target sequence, and elongation by thermostable polymerase, in several cycles, e.g., usually 20 More than one time is performed. Alternatively, annealing and stretching may be performed in the same step. Since mature miRNA is single-stranded, reverse transcription reaction can be performed first before PCR. The reverse transcription reaction requires the use of a primer and a reverse transcriptase. In PCR and quantitative PCR, one set of primers, forward and reverse, are used.

The length of the primer is determined according to various factors such as the hybridization temperature, the composition of the target sequence, and the complexity of the target sequence. In one embodiment, the length of the primer is about 10-35 nucleotides, for example 15, 20, 25, 30 or 35 nucleotides. The forward primer includes at least one sequence capable of specifically binding to the biomarker miRNA, and may further include a non-complementary sequence on the 5' side. The sequence of the reverse primer may be independent of the sequence of the biomarker, and a plurality of miRNA biomarkers may be amplified with one type of reverse primer, or may include one or more sequences specific for the biomarker.

In one embodiment, two or more miRNAs are amplified in one reaction using multiple quantitative PCR and multiple quantitative RT-PCR methods. In this case, one or more pairs of primers and/or probes are used, for example, each pair of primers specifically amplifies a specific miRNA, and the probe is used to distinguish each amplified miRNA to enable multiple amplification.

Reverse transcription and PCR can be performed together in reverse transcription quantitative PCR. In this case, the reaction includes both reverse transcriptase and thermostable polymerase, and a "hot start" reaction method that controls the activity of thermostable polymerase by chemical or thermal method (See, eg, US Pat. Nos. 5,411,876, 5,550,044, etc.) may be used.

The amplified product has a sequence corresponding to the molecule used as a template, and can be analyzed by various methods known in the art. Such methods are known in the art, for example, gel electrophoresis, real-time PCR analysis, single strand conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), capillary zone electrophoresis (CZE), WAVE (HPLC-based nucleic acid) analyzing technology), including, but not limited to, microchips.

In addition, hybridization-based ligation techniques can be used for quantitative analysis of miRNAs. Such methods are known in the art and do not bind a detectable probe that binds to a target nucleic acid sequence, such as, for example, oligonucleotide ligation (OLA) and methods using HARP-like probes described in US Publication 2006-0078894. It includes, but is not limited to, a method for isolating from a non-existing probe.

Another technique using ligation is Multiplex Ligation-dependent Probe Amplification (MLPA) (Schouten et al., Nucleic Acids Research 30:e57 (2002)). The above technique binds in such a way that ligation occurs only when a pair of probes bind side by side to the target sequence, and the ligated probe includes a primer binding site so that it can be amplified by PCR.

The miRNA after hybridization, amplification and/or hybridization-based ligation reaction as described above can detect hybridization or amplified miRNA products through, for example, staining or labeling of a target, staining or labeling of a primer or probe. For detection, a technique known in the art may be used, and a person skilled in the art will be able to select an appropriate method in consideration of the sensitivity of detection and/or the amount of the target. Depending on the sensitivity of the detection method and/or the amount of target, amplification may not be necessary prior to detection.

In addition, miRNAs can be detected by direct or indirect methods. In the direct method, miRNA is labeled with a detectable label bound thereto, and then bound to a probe connected to a solid support such as a bead, and then detected by screening the labeled miRNA. Alternatively, a labeled probe may be used for direct detection, and is detected through screening of the labeled probe after specific binding to the miRNA. In one embodiment, the amplified miRNA is detected using a bead conjugated to a probe capable of capturing a desired nucleic acid. In other embodiments, the probe may be labeled with a fluorescent material.

Labels for detection include, but are not limited to, compounds capable of generating or canceling a detectable fluorescence, chemiluminescent or bioluminescent signal such as a light emitting, light scattering, light absorbing material, for example, Garman Reference may be made to A., Non-Radioactive Labeling, Academic Press 1997. Fluorescent materials include, but are not limited to, fluorescein (eg, US Pat. No. 6,020,481), rhodamine (eg, US Pat. No. 6,191,278), benzophenoxazine (eg, US Pat. No. 6,140,500), donors and acceptors. Energy transfer fluorescent dyes including (eg US Pat. No. 5,945,526) and cyanine (eg WO1997-45539), lysamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5,Cy7 , FluorX (Amersham), Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPYR6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, 6-FAM, Fluorescein Isothiocyanate, HEX, Generates 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, Tetramethylrhodamine, and/or Texas Red as well as other detectable signals any fluorescent moiety capable of Fluorescent dyes include 6-carboxyfluorescein; 2',4',1,4,-tetrachlorofluorescein; and 2',4',5',7',1,4-hexachlorofluorescein. In one embodiment, SYBR-Green, 6-carboxyfluorescein (“FAM”), TET, ROX, VIC™, or JOE is used as the fluorescent label. In one embodiment, a probe labeled with two fluorescent substances, a reporter fluorescent material and an erasing fluorescent material, is used. In this case, a fluorescent material emitting a spectrum with a distinguishable wavelength is used as the fluorescent material. In addition, the marker is a compound capable of enhancing, stabilizing, or affecting the binding of a nucleic acid, for example, an intercalator including ethyl bromide and SYBR-Green, a minor groove binder, and a crosslinkable functional group can be used, but is not limited thereto, and is described in Blackburn et al., eds. See "DNA and RNA Structure" in Nucleic Acids in Chemistry and Biology (1996).

Accordingly, the composition according to the present disclosure may include reagents used in any one or more of the methods described above.

In one embodiment, including reagents required for a method for measuring the presence or absence of the miRNA and the amount or pattern by RT-PCR, for example, a probe and/or primer pair specific for the mRNA of the present marker is included. "Primer" or "probe" means a nucleic acid sequence having a free 3' hydroxyl group capable of complementary binding to a template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template do. As used herein, the reagent may be labeled with a chromogenic, luminescent or fluorescent substance as described above for detection of the amplified product. In one embodiment, reverse transcription PCR (polymerase chain reaction) is used for miRNA detection.

In another embodiment, the detection reagent may be provided in the form of an array or chip including a microarray. Detection reagents may be labeled directly or indirectly in a sandwich form for detection. In the case of the direct labeling method, serum samples used for arrays and the like are labeled with a fluorescent label such as Cy3 or Cy5.

The biomarker according to the present disclosure or a composition comprising the same may be usefully used for diagnosis, prediction and/or prognosis measurement of diabetic neuropathy.

In this context it relates to a composition herein, a kit comprising said composition, or a method.

Reagents that may be included in the kit and detection using the same may be referred to as described above. In one embodiment according to the present application, the kit of the present application is used for nucleic acid amplification, in particular for amplification using RT-PCR. In this case, the kit contains the necessary buffer for the reaction of RT-PCR, reverse transcriptase, Taq polymerase and MgCl2. Various buffers known in the art may be used, for example, Tris-HCl, pH 9.0 buffer may be used, but is not limited thereto. Reverse transcriptase and Taq polymerase are commercially available, for example, AmpliTaq Gold (Applied Biosystems, USA) capable of hot start reaction with polymerase may be used, and an appropriate concentration, for example, 1.5 mM to 2.5 mM MgCl2 may be included.

The kit according to the present application further comprises a positive control group, a negative control group and instructions for use. The negative control group may include a sample that does not contain miRNA, and the positive control group may include one or more of the detection target miRNAs.

In this aspect, the present application also provides a sample derived from a subject suspected of diabetic neuropathy in order to provide information necessary for the diagnosis or prognosis of diabetic neuropathy; measuring the expression level of one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485 in the sample; comparing the measurement result with the corresponding result of the corresponding marker in the control group; and when there is a change in the expression level of the subject sample compared to the control sample, determining it as diabetic neuropathy.

Reagents and the like used in the method of the present application may be referred to as previously described.

In the method according to the present disclosure, as a control group, a sample from a diabetic patient may be used for prediction or early diagnosis of diabetic neuropathy.

The method may be performed over a specific period of time, for example, several times over a year or once a year, and may be used to monitor changes in expression patterns. Depending on the type of marker, an increase or decrease in expression can be associated with a diabetic neuropathy state. It can be used to determine the onset, progression, exacerbation, etc. of diabetic neuropathy by comparison with a previous test value for the same subject or a control value. Prophylactic measures can be taken to prevent the progression or onset of diabetic neuropathy based on changes in diabetic neuropathy markers over time. In addition, for the diagnosis of diabetic neuropathy, biomarkers can be used as an auxiliary means, and other diagnostic methods such as biopsy, ultrasound, computerized axial tomography (CT scan) or magnetic resonance imaging (magnetic resonance) imaging (MRI)). Hereinafter, the present invention will be described in detail through examples, but these examples are merely illustrative and do not limit the scope of the present application in any way.

Unless otherwise specified, the present invention can be practiced using conventional techniques within the skill level of those skilled in the art in cell biology, cell culture, molecular biology, gene transformation technology, microbiology, and DNA recombination technology.

Hereinafter, embodiments of the present invention will be described in more detail through specific experimental examples. However, the following experimental examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Collection of test subject specimens and miRNA extraction

Subjects of this experiment, with the approval of the Clinical Research Ethics Committee (IRB), received serum samples from the normal group and 3 patients (total of 6) diagnosed with diabetic neuropathy at Chungbuk National University Hospital and Keimyung University Dongsan Hospital as human resources. Obtained through the bank.

Then, total RNA was isolated from the obtained serum using the Qiagen RNA extraction kit according to the manufacturer's method, and the quality of the isolated total RNA was checked using an Agilent 2100 Bioanalyzer according to the manufacturer's method.

Example 2 miRNA biomarker selection using next-generation genome analysis

Vitinylated cDNA was synthesized from the total RNA isolated in Example 1 using SMARTer smRNA seq kit for Illumina (Takara). The cDNA was then hybridized to Affymetrix GeneChip ^® miRNA 4.0 according to the manufacturer's standard method. Measurement and analysis of fluorescence intensity after hybridization were performed using Genechip operating software (Affymetrix) according to the manufacturer's method. A total of 6658 human miRNAs were compared and analyzed in 3 cases of normal persons and diabetic neuropathy, respectively, and as shown in FIG. 1 , a heat map for miRNAs related to diabetic neuropathy (yellow: overexpressed miRNAs more than twice, blue) : Two-fold or more underexpressed miRNAs).

As a result, it was found that there were significant differences in miR-122, miR-199b, and miR-8485 between the diabetic neuropathy and normal groups in the two groups. Statistical analysis was performed using Student's t-test, and statistical significance was <0.05.

Example 3 Real-time quantitative PCR analysis of miRNAs selected from clinical samples

Primers and fluorescent probes (SYBR GreenI) for three miRNAs (miR-122, miR-199b, and miR-8485) showing differences in expression in the normal group and diabetic neuropathy patient group selected in Example 2 MiRNA analysis was performed using RT-PCR in the sera of 200 people using the method as described.

Specifically, it was carried out using Bio-rad's CFX-96. Specifically, 10 ng RNA sample, 10 pM stem-loop RT primer, 10X RT buffer, 5 U/uL poly A polymerase, 2 mM of each dNTP, 200 U/uL M-MLV, 10mM ATP, and 5 U/uL RNase Inhibitor (enzynomics, Korea) was used to synthesize cDNA by reverse transcription. Then cDNA was denatured for 5 min at 95 °C using respective miRNA primers and SYBR greenI. Amplification was performed by 40 cycles of 10 seconds at 95 degrees and 40 seconds at 60 degrees, and the signals were collected and analyzed in real time with Bio-Rad CFX-Dx 3.1.

And the result is shown in FIG. As shown in Figure 2, the difference in the expression ages of miR-122, miR-199b, and miR-8485 was found to have a significant correlation between the normal group and the diabetic neuropathy group, which It shows that miR-199b and miR-8485 can be usefully used for the prediction and diagnosis of diabetic neuropathy.

In the above, the embodiment of the present invention has been mainly described, but this is only an example and does not limit the present invention. It will be appreciated that various modifications and applications not exemplified above are possible.

Therefore, it should be understood that the scope of the present invention includes changes, equivalents or substitutes of the technical idea exemplified above. For example, each component specifically shown in the embodiment of the present invention may be implemented by modification. And differences related to such modifications and applications should be construed as being included in the scope of the present invention defined in the appended claims.

Claims (10)

1. A composition for prediction or diagnosis of diabetic neuropathy, comprising a reagent for measuring one or more biomarkers selected from the group consisting of miR-122, miR-199b, and miR-8485.
2. According to claim 1,

   The reagent comprises a nucleic acid sequence of each biomarker, a nucleic acid sequence complementary to the nucleic acid sequence, and a fragment of the nucleic acid sequence, a composition for predicting or diagnosing diabetic neuropathy.
3. 3. The method of claim 2,

   The nucleic acid sequence or fragment of the nucleic acid sequence is a primer, or a probe, or a primer and a probe capable of specifically binding each of the markers, a composition for predicting or diagnosing diabetic neuropathy.
4. According to claim 1,

   The reagent is a reverse transcription polymerase chain reaction of each biomarker, a polymerase chain reaction, a competitive polymerase chain reaction, a nuclease protection assay (RNase, S1 nuclease assay), an in situ hybridization method, a ligation-based polymerase chain reaction, micro A composition for predicting or diagnosing diabetic neuropathy, which is a reagent used in an array or northern blot.
5. To provide information necessary for diagnosing or predicting diabetic neuropathy,

   providing a sample derived from a subject suspected of having diabetic neuropathy;

   measuring the expression level of one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485 in the sample;

   comparing the measurement result with the corresponding result of the corresponding marker in the control group; and

   Comparing with the control sample, when there is a change in the expression level of the subject sample, the method comprising the step of determining the diabetic neuropathy marker, detecting a diabetic neuropathy marker.
6. 6. The method of claim 5,

   Wherein the detection is by a hybridization or nucleic acid amplification method.
7. 7. The method of claim 6,

   The hybridization is a microarray analysis, the nucleic acid amplification is a reverse transcriptase polymerase chain reaction method, the method.
8. 6. The method of claim 5,

   The method of claim 1, wherein the biological sample is whole blood, plasma or serum from a diabetic neuropathy patient.
9. 6. The method of claim 5,

   The method is to use the average value of the expression levels of the miR-122, miR-199b, and miR-8485 markers.
10. A composition for prediction or diagnosis of diabetic neuropathy comprising miR-122, miR-199b, and miR-8485.

Images