WO2023033268A1 - Novel microrna biomarker for early diagnosis of mild cognitive impairment or alzheimer's dementia - Google Patents
Novel microrna biomarker for early diagnosis of mild cognitive impairment or alzheimer's dementia Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
Definitions
- the present invention relates to a novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia.
- Alzheimer's disease is diagnosed through neuropsychological testing and imaging based on the cognitive impairment and amyloid hypothesis. Efforts are actively being made.
- miRNA is a single-chain small RNA with about 20 nucleotide sequences. It is a short sequence that can bind to the 3'-UTR (3'-untranslated regions) of a specific target mRNA and regulate proteins by inhibiting mRNA translation. is the RNA of About 1,000 miRNAs have been identified in humans, but most miRNAs have unknown functions.
- RNA is used as a diagnostic indicator, it is a single-stranded RNA of 21 to 25 nucleotides present in all biological fluids, including blood, urine, and saliva, and plays a role in directly controlling gene expression by suppressing mRNA translation.
- Alzheimer's disease In the diagnosis of Alzheimer's disease, it is very important to take an accurate medical history through the report of the guardian who knows the patient best. The doctor checks whether there is a change in cognitive function, including memory, compared to the previous one, when and how it appeared, and biochemical tests such as physical examination, neurological examination, mental status examination, daily living function level examination, blood examination, etc.
- biochemical tests such as physical examination, neurological examination, mental status examination, daily living function level examination, blood examination, etc.
- there are still various limitations in diagnosing Alzheimer's disease making early diagnosis difficult. Mainly, Alzheimer's disease is diagnosed through neuropsychological tests and imaging diagnosis based on the cognitive impairment and amyloid hypothesis, and recently, efforts to develop indicators for diagnosing Alzheimer's dementia from blood, a non-invasive sample, have been actively pursued. It is being done.
- miRNA is a single-chain small RNA with a sequence of about 20 bases that binds to the 3'-UTR (3'-untranslated regions) of a specific target mRNA and regulates protein through inhibition of mRNA translation. It is a short sequence of RNA. About 1,000 human miRNAs have been identified so far, but most of them have not been identified.
- Dementia analysis using blood mainly relies on proteins, but has the disadvantage of severe variation among patients compared to structural stability and inability to fully reflect the pathology of Alzheimer's dementia in blood. You need a target.
- abnormal protein aggregation is not caused by genetic factors alone, but by various environmental stimuli. It is important to find
- miRNA when used as a diagnostic indicator, it is a single-stranded RNA of 21 to 25 nucleotides present in all biological fluids including blood, urine, and saliva, which inhibits mRNA translation and directly controls gene expression. In addition, it is resistant to degradation by RNase and stably exists in the blood. Defects in metabolism or function of miRNA are closely related to the onset of dementia, and it is known to have high specificity and sensitivity depending on the disease, so it can be used as the most optimal diagnostic index. there is.
- Korean Patent No. 1992539 discloses a composition and method for diagnosing a miRNA-based cognitive disorder disease
- Korean Patent No. 1718940 discloses welfare for Alzheimer's dementia or mild cognitive impairment.
- a composition for early diagnosis of genetics has been disclosed, but the novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia has not yet been disclosed.
- the present invention was derived from the above needs, and the present invention provides a novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia, and the novel miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 according to the present invention is expressed in the plasma of mild cognitive impairment or Alzheimer's dementia patient group and Alzheimer's dementia patient group for early diagnosis of mild cognitive impairment or Alzheimer's dementia, and confirmed that it can reduce the expression of Alzheimer's dementia related protein, thereby completing the present invention.
- the present invention provides a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia comprising novel microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.
- the present invention provides a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
- the present invention provides a kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia comprising the biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia as an active ingredient.
- the present invention comprises the steps of (1) collecting blood from an individual;
- step (3) checking whether the miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed in the plasma isolated in step (2); to provide.
- the present invention relates to a novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia. It has the effect of reducing the expression of dementia-related proteins. Therefore, the present invention has an effect of enabling early diagnosis before the symptoms of Alzheimer's disease appear by using novel miRNA present in relatively low-invasive plasma as a diagnostic index.
- Figure 1 shows the stem loop structure and sequence information of the novel miRNA2 identified in the present invention.
- Figure 2 shows the stem loop structure and sequence information of the novel miRNA3 identified in the present invention.
- Figure 3 shows mimic and inhibitor synthetic sequences of miRNA2 (A) and miRNA3 (B) to confirm the function of the novel miRNA of the present invention.
- Figure 4 is a Western blot result confirming changes in the expression levels of target gene proteins BDNF, NRG3, and NEFL of the novel miRNA2 of the present invention.
- *, **, *** indicates that there is a statistically significant difference in the target gene expression level between the control group treated with nothing and the group treated with the miRNA mimic or miRNA inhibitor of the present invention, * indicates p ⁇ 0.05, and ** is p ⁇ 0.01, *** is p ⁇ 0.001.
- Figure 5 is a Western blot result confirming the change in the expression level of the target gene protein HOOK3 of the novel miRNA3 of the present invention. *, *** indicates that there is a statistically significant difference in the target gene expression level between the control group treated with nothing and the group treated with the miRNA mimic or miRNA inhibitor of the present invention, * is p ⁇ 0.05, and *** is p ⁇ 0.001.
- the present invention relates to a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising novel microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.
- microRNA refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting their translation.
- miRNAs represented by specific nucleotide sequences, but also precursors (pre-miRNA, pri-miRNA) of the miRNAs, miRNAs with biological functions equivalent to these, for example, homologues (i.e., homologs or orthologs), variants such as polymorphisms, and derivatives.
- the novel microRNA is preferably derived from human plasma, but is not limited thereto.
- the novel microRNA of SEQ ID NO: 1 reduces the expression levels of Brain-Derived Neurotrophic Factor (BDNF), Neuregulin3 (NRG3), and Neurofilament Light Chain (NEFL) genes, and the novel microRNA of SEQ ID NO: 3 is HOOK3 (Protein Hook homolog 3) It is characterized by reducing the expression level of genes.
- BDNF Brain-Derived Neurotrophic Factor
- NRG3 Neuregulin3
- NEFL Neurofilament Light Chain
- 'Diagnosis' of the present invention is to determine the susceptibility of an object, that is, a test subject, to a specific disease or disorder, to determine whether an object currently has a specific disease or disorder, to a specific disease or disorder
- Concepts include determining the prognosis of an affected subject or therametrics (eg, monitoring the condition of a subject to provide information about treatment efficacy).
- the present invention relates to a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
- the agent may be an antisense oligonucleotide, primer or probe complementary to the microRNA, but is not limited thereto.
- Mild cognitive impairment refers to an intermediate stage between cognitive decline and dementia caused by normal aging. In other words, it refers to a state in which cognitive ability is lowered compared to those of the same age group, and it is different from dementia that daily life is possible. This mild cognitive impairment is an intermediate stage between normal and dementia, and is known as a high-risk group for dementia.
- the 'mild cognitive impairment or Alzheimer's dementia patient group' was selected as an experimental group for early diagnosis of mild cognitive impairment or Alzheimer's dementia.
- the present invention is a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia comprising as an active ingredient an agent capable of detecting a new microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, comprising as an active ingredient It relates to a kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia.
- the kit is preferably for RT-PCR, real-time PCR, isothermal PCR, Northern blotting, RNA protection assay or microarray chip, but is not limited thereto.
- the kit of the present invention may additionally include a nucleic acid capable of specifically binding to the microRNA of SEQ ID NO: 1 or SEQ ID NO: 3, which means that the nucleic acid includes both RNA, DNA, or RNA/DNA (chimera),
- the DNA may include all of cDNA, genomic DNA, and synthetic DNA.
- the RNA may include all of total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA.
- the kit of the present invention may include, in addition to the nucleic acid, a known nucleic acid or a nucleic acid that can be discovered in the future that enables early diagnosis of Alzheimer's disease, and an antibody for measuring a known marker for early diagnosis of Alzheimer's disease. can also be included.
- the nucleic acids included in the kit may be individually or arbitrarily combined and packaged in different containers.
- the kit of the present invention may include a fluorescent material for labeling, an enzyme and medium for nucleic acid amplification, instructions for use, and the like.
- the kit of the present invention is a device for early diagnosis of Alzheimer's disease in which the nucleic acid is bound or attached to, for example, a solid phase.
- the material of the solid phase include plastic, paper, glass, silicon, and the like, and a material of the solid phase that is preferable from ease of processing may be plastic.
- the shape of the solid phase is arbitrary, and may be, for example, square, circular, rectangular, or film-like.
- the kit of the present invention may be included in a high-throughput-screening (HTS)-based multiplexed POCT device that simultaneously processes a large amount of miRNA samples.
- the cartridge in the device can be composed of a thermostat, stirrer, disposable tip, sample injection container, etc., and the 'dementia risk index' is automatically derived when human blood is injected into the all-in-one device with a built-in ultra-small whole blood separator, fluorescence detector, and liquid handler. Algorithms and fully automatic detection systems may be included.
- the present invention comprises the steps of (1) collecting blood from an individual;
- MCI Mild cognitive impairment
- AD Alzheimer's disease
- K-MMSE screening test tools
- SNSB imaging diagnosis
- PET imaging diagnosis
- RNA from the plasma of each of the above-obtained patient and normal groups was extracted using the QIAGEN miRNeasy Serum/Plasma Separation Kit, which can purify miRNA and other small RNAs mainly from small amounts of serum and plasma.
- RNA-seq library was created on the Illumina Platform using the SMARTer smRNA-Seq Kit, which can produce a high-quality miRNA library even with a small amount of RNA.
- Samples prepared with the NovaSeq 6000 sequencing platform were sequenced according to the SMARTer smRNA-Seq Kit manual.
- Contaminated or low-quantity data in plasma was removed in the order of adapter, quality, and length trimming from the raw data obtained by NGS analysis.
- miRNA Deep2* sequencing was performed with clean data from which unnecessary information was removed through this trimming process.
- miRNA Deep2* is a software that can identify new miRNAs from refined raw data.
- miRBase (Pre-miRNA) libraries cut short in mature, star, and loop sequences are arranged side by side to predict new miRNAs.
- the RNAfold algorithm can be derived by predicting the secondary structure of the RNA sequence with the minimum free energy using the most similar thermodynamic model.
- the RNA fold generation graphic includes the number of reads for each part of the hairpin, the score for the minimum free energy, the score for the randfold, and the score for the conserved seed sequence.
- the miRDeep2* system can detect previously known miRNAs and detect new miRNAs by mapping using an RNA database.
- miRNAs target genes
- SH-SY5Y cells used as a neural function and differentiation model were used, and a miRNA mimic showing the overexpression effect of the selected new miRNA, a miRNA inhibitor suppressing the expression of the new miRNA, and a negative control miRNA were transfected into SH-SY5Y After injection, the protein expression of the target gene was confirmed by Western blot.
- Example 1 Sample acquisition and total RNA extraction
- RNA in plasma collected from patients and normal groups was extracted using the QIAGEN miRNeasy Serum/Plasma Kit, which can purify miRNA and other small RNAs from small amounts of serum and plasma. Subsequently, it was confirmed that a sufficient amount of RNA was secured for NGS analysis by quantifying RNA using a Bioanalyzer RNA Small RNA chip to determine whether a sufficient amount of RNA was secured for library production for NGS.
- RNA-seq Library for sequencing on the Illumina Platform for NGS
- the SMARTer smRNA-Seq Kit which can produce a high-quality miRNA library even with a small amount of RNA
- Libraries for total RNA sequencing of patients and normal groups obtained from plasma were prepared, and raw data were derived by sequencing using the prepared libraries and the NovaSeq 6000 sequencing platform.
- the raw data obtained from NGS analysis was trimmed in the order of Adapter, Quality, and Length to remove contaminated or small amounts of data.
- miRNA Deep2* predicts new miRNAs by arranging short-cut miRBase (Pre-miRNA) libraries side by side in mature, star, and loop sequences according to the RNAfold algorithm of the software that can identify newly known miRNAs from the primary processed data.
- the RNAfold algorithm was derived by predicting the secondary structure with the minimum free energy of the RNA sequence using the most similar thermodynamic model.
- the RNA fold generation graphic contains the number of reads for each part of the hairpin, the score for the minimum free energy, the score for the randfold, and the conserved seed sequence score.
- the miRDeep2* system detected previously known miRNAs and new miRNAs by mapping using an RNA database.
- RNAcentral release 14.0 The reads finally processed for each sample were classified into piRNA, tRNA, snRNA, snoRNA, known miRNA, and novel miRNA through miRBase v22.1 and non-coding RNA database (RNAcentral release 14.0).
- Genome mapping was processed by Bowtie and STAR using RSEM. Bowtie was then used for miRDeep2 analysis using the genome sequence. Known/novel miRNAs predicted by miRDeep2 and other small RNAs matching RNAcentral were aligned using Bowtie (target small RNA, ⁇ 50nt) and Bowtie2 (target smRNA, ⁇ 50nt).
- RNA-Seq by Expectation-Maximization a tool to quantify RNA-Seq transcripts
- RNAfold function predicted the minimum free energy secondary structure of an RNA sequence using a nearest-neighbor thermodynamic model.
- graphics generated by RNAfold include real in silico-folded hairpins, read counts for each part of the hairpin, the minimum free energy score, and the randfold score, as well as the score of the conserved seed sequence.
- miRDeep2* derived novel miRNAs through a score of -10 to 10.
- the novel miRNAs derived above were expressed in both the mild cognitive impairment group and the Alzheimer's disease group.
- two miRNAs having a miRDeep2* score of 1 or more, an RNA fold of "yes", and a mature read number of 10 or more were selected.
- the stem loop structures of the selected novel miRNA2 and miRNA3 are shown in Figures 1 and 2, and sequence information is shown in Figure 3.
- miRDB software was used to screen the target gene by score based on the sequence of the miRNA.
- genes related to Alzheimer's dementia were selected.
- SH-SY5Y cells used as a model for neural function and differentiation were used, and after transfection of miRNA mimics that show the overexpression effect of selected new miRNAs, miRNA inhibitors that inhibit the expression of new miRNAs, and negative control miRNAs into SH-SY5Y , The protein expression of the target gene was confirmed by Western blot.
- the miRNA mimic is actually a miRNA that can function like a novel miRNA in vivo, and is designed with a double bond.
- the miRNA inhibitor is a complementary sequence of the derived miRNA, and is a miRNA that can inhibit the miRNA produced by single binding.
- the negative control miRNA is a miRNA composed of miRNAs that do not regulate genes and serves as a standard for verification. mimics of the newly produced miRNA2 and miRNA3; and miRNA inhibitors are disclosed in FIG. 3 .
- miRNA 2 mimic was based on sequence 5'-UAAAGAGGAAAGAGGAAAGAGG-3' (SEQ ID NO: 1)
- miRNA 3 mimic was sequence Based on 5'-CGGGAGGCAGCAGUGGGGC-3' (SEQ ID NO: 3)
- genes were screened by score using target prediction miRDB software.
- the miRNA inhibitor for the miRNA mimic of SEQ ID NO: 1 is 5'-CCUCUUUCCUCUUUCCUCUUUA-3' (SEQ ID NO: 2)
- the miRNA inhibitor for the miRNA mimic of SEQ ID NO: 3 is 5'-GCCCACUGCUGCCUCCG-3' (SEQ ID NO: 4).
- Light Chain NRG3 (Neuregulin3), which regulates the growth and differentiation of epithelial and glial cells and plays an important role in the development, maintenance, and restoration of the nervous system, was selected.
- a target gene HOOK3 Protein Hook homolog 3 related to the production of amyloid beta, a causative protein of Alzheimer's disease, was selected.
- Human neuroblastoma SH-SY5Y was transfected with 100 nM of each miRNA miRNA for overexpression and miRNA inhibitor for function suppression for 24 hours, and the protein was isolated and the expression level was confirmed by Western blotting.
- miRNA3 mimic for overexpression of novel miRNA3 and miRNA3 inhibitor for suppression of function were transfected into human neuroblastoma SH-SY5Y for 24 hours in a concentration-dependent manner, respectively, and the protein was isolated and Western A blot was performed.
- miRNA 3 mimic treatment which shows the effect of overexpression of the new miRNA 3
- treatment with miRNA 3 inhibitor for HOOK3 increased expression (Fig. 5).
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Abstract
The present invention relates to a novel microRNA biomarker for the early diagnosis of mild cognitive impairment or Alzheimer's disease, wherein the novel miRNA of SEQ ID No. 1 or 3 obtained by molecular-based analysis is expressed in the plasma of groups of mild cognitive impairment and Alzheimer's disease patients and has the effect of reducing the expression of proteins related to mild cognitive impairment and Alzheimer's disease. Therefore, the present invention has the effect of making it possible to achieve an early diagnosis of mild cognitive impairment or Alzheimer's dementia before the onset of symptoms by utilizing, as a molecular diagnostic indicator, a novel miRNA present in relatively low-invasive plasma.
Description
본 발명은 경도인지장애 또는 알츠하이머성 치매 조기 진단을 위한 신규 마이크로 RNA 바이오마커에 관한 것이다.The present invention relates to a novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia.
현재 인지기능의 손상과 아밀로이드 가설을 기반으로 신경심리검사 및 영상진단을 통해 알츠하이머성 치매를 진단하고 있지만 많은 한계점으로 인해, 최근 상대적으로 비침습적인 시료인 혈액으로부터 알츠하이머성 치매의 진단지표를 개발하려는 노력이 활발하게 이루어지고 있다.Currently, Alzheimer's disease is diagnosed through neuropsychological testing and imaging based on the cognitive impairment and amyloid hypothesis. Efforts are actively being made.
miRNA는 약 20개 정도의 염기서열을 가진 단일 사슬의 작은 RNA로서 특정 타겟이 되는 mRNA의 3'-UTR(3'-untranslated regions)과 결합하여 mRNA의 번역 억제를 통해 단백질을 조절할 수 있는 짧은 서열의 RNA이다. 현재까지 밝혀진 miRNA는 사람의 경우 1,000여개 정도의 miRNA가 밝혀졌으나 대부분의 miRNA는 기능이 밝혀지지 않은 상태이다.miRNA is a single-chain small RNA with about 20 nucleotide sequences. It is a short sequence that can bind to the 3'-UTR (3'-untranslated regions) of a specific target mRNA and regulate proteins by inhibiting mRNA translation. is the RNA of About 1,000 miRNAs have been identified in humans, but most miRNAs have unknown functions.
현재, 혈액을 이용한 치매 분석은 주로 단백질에 의존하고 있으나, 구조적 안정성에 비해 환자별 편차가 심하고, 알츠하이머성 치매 병리를 혈액에 온전히 반영하지 못하는 단점이 있다. 더 예민한 진단 지표 도출을 위해서는, 더 작은 사이즈의 타겟이 필요하다. 특히 알츠하이머성 치매의 경우, 비정상적 단백질의 응집 등이 유전적인 요인으로만 일어나는 것이 아닌, 다양한 환경적 자극으로 유발되기 때문에, 단백질이라는 결과보다, 단백질의 번역 이전인 유전자 발현 조절 변화 분석으로 근본적인 타겟을 찾는 것이 중요할 것으로 사료된다. miRNA를 진단 지표로 활용할 경우 혈액, 소변 및 타액을 포함한 모든 생체 유체에 존재하는 21~25 뉴클레오타이드의 단일 가닥 RNA로 mRNA의 번역을 억제하여 유전자 발현을 직접 제어하는 역할을 한다. 또한 RNase에 의한 분해에 강해 혈액에서 안정적으로 존재하며, miRNA의 대사 또는 기능의 결함은 치매의 발병과 밀접한 관련이 있고, 질병에 따라 높은 특이성 및 민감도를 가지는 것으로 알려져 가장 최적의 진단지표로 활용할 수 있다.Currently, dementia analysis using blood mainly relies on proteins, but there are disadvantages in that the variation between patients is severe compared to structural stability and the pathology of Alzheimer's dementia cannot be fully reflected in blood. In order to derive a more sensitive diagnostic index, a target with a smaller size is required. In particular, in the case of Alzheimer's disease, abnormal protein aggregation is not caused by genetic factors alone, but by various environmental stimuli. It is thought to be important to find. When miRNA is used as a diagnostic indicator, it is a single-stranded RNA of 21 to 25 nucleotides present in all biological fluids, including blood, urine, and saliva, and plays a role in directly controlling gene expression by suppressing mRNA translation. In addition, it is resistant to degradation by RNase and stably exists in the blood. Defects in metabolism or function of miRNA are closely related to the onset of dementia, and it is known to have high specificity and sensitivity depending on the disease, so it can be used as the most optimal diagnostic index. there is.
알츠하이머병의 진단은 환자에 대해 가장 잘 알고 있는 보호자의 보고를 통한 정확한 병력 청취가 매우 중요하다. 의사는 이전에 비해 기억력을 포함한 인지 기능의 변화가 있는지, 있다면 언제부터 어떠한 양상으로 나타났는지 확인하고, 신체검사와 신경학적 검사, 정신상태 검사, 일상생활 기능수준 검사, 혈액 검사 등의 생화학적 검사, 뇌영상학 검사, 신경심리 검사 등을 통해 진단하고 있지만, 아직까지는 알츠하이머병을 진단하는데는 여러가지 한계점이 존재하여, 조기진단이 어려운 상황이다. 주로, 인지기능의 손상과 아밀로이드 가설을 기반으로 신경심리검사 및 영상진단을 통해 알츠하이머성 치매를 진단하고 있고, 최근들어 비침습적 시료인 혈액으로부터 알츠하이머성 치매를 진단하는 지표를 개발하려는 노력이 활발하게 이루어지고 있다.In the diagnosis of Alzheimer's disease, it is very important to take an accurate medical history through the report of the guardian who knows the patient best. The doctor checks whether there is a change in cognitive function, including memory, compared to the previous one, when and how it appeared, and biochemical tests such as physical examination, neurological examination, mental status examination, daily living function level examination, blood examination, etc. However, there are still various limitations in diagnosing Alzheimer's disease, making early diagnosis difficult. Mainly, Alzheimer's disease is diagnosed through neuropsychological tests and imaging diagnosis based on the cognitive impairment and amyloid hypothesis, and recently, efforts to develop indicators for diagnosing Alzheimer's dementia from blood, a non-invasive sample, have been actively pursued. It is being done.
한편, miRNA는 약 20개 정도의 염기서열을 가진 단일 사슬의 작은 RNA로서 특정 타겟이 되는 mRNA의 3'-UTR(3'-untranslated regions)과 결합하여 mRNA의 번역 억제를 통해 단백질을 조절할 수 있는 짧은 서열의 RNA이다. 현재까지 밝혀진 인간에 대한 miRNA는 1000여개 정도가 밝혀졌으나, 대부분 기능이 밝혀지지 않은 상태이다. On the other hand, miRNA is a single-chain small RNA with a sequence of about 20 bases that binds to the 3'-UTR (3'-untranslated regions) of a specific target mRNA and regulates protein through inhibition of mRNA translation. It is a short sequence of RNA. About 1,000 human miRNAs have been identified so far, but most of them have not been identified.
혈액을 이용한 치매 분석은 주로 단백질에 의존하고 있으나, 구조적 안정성에 비해 환자별 편차가 심하고, 알츠하이머성 치매 병리를 혈액에 온전히 반영하지 못하는 단점이 있으므로, 특이적 진단 지표를 도출하기 위해서 더 작은 크기의 타겟이 필요하다. 특히 알츠하이머성 치매의 경우, 비정상적 단백질의 응집 등이 유전적인 요인으로만 일어나는 것이 아닌, 다양한 환경적 자극으로 유발되기 때문에, 단백질이라는 결과보다, 단백질의 번역 이전인 유전자 발현 조절 변화 분석으로 근본적인 타겟을 찾는 것이 중요하다. Dementia analysis using blood mainly relies on proteins, but has the disadvantage of severe variation among patients compared to structural stability and inability to fully reflect the pathology of Alzheimer's dementia in blood. You need a target. In particular, in the case of Alzheimer's disease, abnormal protein aggregation is not caused by genetic factors alone, but by various environmental stimuli. It is important to find
일례로, miRNA를 진단 지표로 활용할 경우, 혈액, 소변 및 타액을 포함한 모든 생체 유체에 존재하는 21~25 뉴클레오티드의 단일 가닥 RNA로 mRNA의 번역을 억제하여 유전자 발현을 직접 제어하는 역할을 한다. 또한 RNase에 의한 분해에 강해 혈액에서 안정적으로 존재하며, miRNA의 대사 또는 기능의 결함은 치매의 발병과 밀접한 관련이 있고, 질병에 따라 높은 특이성 및 민감도를 가지는 것으로 알려져 가장 최적의 진단지표로 활용할 수 있다.For example, when miRNA is used as a diagnostic indicator, it is a single-stranded RNA of 21 to 25 nucleotides present in all biological fluids including blood, urine, and saliva, which inhibits mRNA translation and directly controls gene expression. In addition, it is resistant to degradation by RNase and stably exists in the blood. Defects in metabolism or function of miRNA are closely related to the onset of dementia, and it is known to have high specificity and sensitivity depending on the disease, so it can be used as the most optimal diagnostic index. there is.
한편, 인지기능장애의 진단 관련 기술로는 한국등록특허 제1992539호에 miRNA 기반의 인지장애 질환 진단용 조성물 및 방법이 개시되어 있고, 한국등록특허 제1718940호에 알츠하이머성 치매 또는 경도인지장애를 위한 후생유전학 조기진단용 조성물이 개시되어 있으나, 아직까지는 본 발명의 경도인지장애 또는 알츠하이머성 치매 조기 진단을 위한 신규 마이크로 RNA 바이오마커에 대해서는 개시된 바 없다.On the other hand, as technology related to the diagnosis of cognitive dysfunction, Korean Patent No. 1992539 discloses a composition and method for diagnosing a miRNA-based cognitive disorder disease, and Korean Patent No. 1718940 discloses welfare for Alzheimer's dementia or mild cognitive impairment. A composition for early diagnosis of genetics has been disclosed, but the novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia has not yet been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명은 경도인지장애 또는 알츠하이머성 치매 조기 진단을 위한 신규 마이크로 RNA 바이오마커를 제공하고, 본 발명에 따른 서열번호 1 또는 서열번호 3의 신규 miRNA는 경도인지장애 또는 알츠하이머성 치매의 조기진단을 위해 경도인지장애 환자군 및 알츠하이머성 치매 환자군의 혈장에서 발현되며, 알츠하이머성 치매 관련 단백질의 발현을 감소시킬 수 있다는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs, and the present invention provides a novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia, and the novel miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 according to the present invention is expressed in the plasma of mild cognitive impairment or Alzheimer's dementia patient group and Alzheimer's dementia patient group for early diagnosis of mild cognitive impairment or Alzheimer's dementia, and confirmed that it can reduce the expression of Alzheimer's dementia related protein, thereby completing the present invention.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 신규 마이크로 RNA를 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물을 제공한다.In order to achieve the above object, the present invention provides a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia comprising novel microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.
또한, 본 발명은 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 마이크로 RNA를 검출할 수 있는 제제를 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물을 제공한다.In addition, the present invention provides a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
또한, 본 발명은 상기 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물을 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트를 제공한다.In addition, the present invention provides a kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia comprising the biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia as an active ingredient.
또한, 본 발명은 (1) 개체로부터 혈액을 채취하는 단계;In addition, the present invention comprises the steps of (1) collecting blood from an individual;
(2) 상기 단계 (1)의 혈액으로부터 혈장을 분리하는 단계; 및(2) separating plasma from the blood of step (1); and
(3) 상기 단계 (2)에서 분리한 혈장 내 서열번호 1 또는 서열번호 3의 miRNA 발현여부를 확인하는 단계;를 포함하는 경도인지장애 또는 알츠하이머성 치매의 조기 진단을 위한 정보를 제공하는 방법을 제공한다.(3) checking whether the miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed in the plasma isolated in step (2); to provide.
본 발명은 경도인지장애 또는 알츠하이머성 치매 조기 진단을 위한 신규 마이크로 RNA 바이오마커에 관한 것으로, 본 발명에 따른 서열번호 1 또는 서열번호 3의 신규 miRNA가 경도인지장애 환자군의 혈장에서 발현되며, 알츠하이머성 치매 관련 단백질의 발현을 감소시키는 효과가 있는 것이다. 따라서 본 발명은 상대적으로 침습성이 낮은 혈장에 존재하는 신규 miRNA를 진단지표로 활용함으로써, 알츠하이머성 치매의 증상이 나타나기 전 조기 진단이 가능한 효과가 있다.The present invention relates to a novel microRNA biomarker for early diagnosis of mild cognitive impairment or Alzheimer's dementia. It has the effect of reducing the expression of dementia-related proteins. Therefore, the present invention has an effect of enabling early diagnosis before the symptoms of Alzheimer's disease appear by using novel miRNA present in relatively low-invasive plasma as a diagnostic index.
도 1은 본 발명에서 확인한 신규 miRNA2의 스템 루프(stem loop) 구조 및 서열 정보를 나타낸 것이다.Figure 1 shows the stem loop structure and sequence information of the novel miRNA2 identified in the present invention.
도 2는 본 발명에서 확인한 신규 miRNA3의 스템 루프(stem loop) 구조 및 서열 정보를 나타낸 것이다.Figure 2 shows the stem loop structure and sequence information of the novel miRNA3 identified in the present invention.
도 3은 본 발명의 신규 miRNA 기능을 확인하기 위한 miRNA2(A) 및 miRNA3(B)의 mimic 및 저해자 합성서열을 나타낸 것이다.Figure 3 shows mimic and inhibitor synthetic sequences of miRNA2 (A) and miRNA3 (B) to confirm the function of the novel miRNA of the present invention.
도 4는 본 발명의 신규 miRNA2의 타겟 유전자 단백질 BDNF, NRG3 및 NEFL의 발현량 변화를 확인한 웨스턴블랏 결과이다. *, **, ***은 아무것도 처리하지 않은 대조군과 본 발명의 miRNA mimic 또는 miRNA 저해자를 처리한 군에서의 타겟 유전자 발현량이 통계적으로 유의미한 차이가 있다는 것으로, *은 p<0.05이고, **은 p<0.01이며, ***은 p<0.001이다. Figure 4 is a Western blot result confirming changes in the expression levels of target gene proteins BDNF, NRG3, and NEFL of the novel miRNA2 of the present invention. *, **, *** indicates that there is a statistically significant difference in the target gene expression level between the control group treated with nothing and the group treated with the miRNA mimic or miRNA inhibitor of the present invention, * indicates p<0.05, and ** is p<0.01, *** is p<0.001.
도 5는 본 발명의 신규 miRNA3의 타겟 유전자 단백질 HOOK3의 발현량 변화를 확인한 웨스턴블랏 결과이다. *, ***은 아무것도 처리하지 않은 대조군과 본 발명의 miRNA mimic 또는 miRNA 저해자를 처리한 군에서의 타겟 유전자 발현량이 통계적으로 유의미한 차이가 있다는 것으로, *은 p<0.05이고, ***은 p<0.001이다. Figure 5 is a Western blot result confirming the change in the expression level of the target gene protein HOOK3 of the novel miRNA3 of the present invention. *, *** indicates that there is a statistically significant difference in the target gene expression level between the control group treated with nothing and the group treated with the miRNA mimic or miRNA inhibitor of the present invention, * is p <0.05, and *** is p <0.001.
도 6은 본 발명의 신규 miRNA3 타겟 유전자 단백질 HOOK3의 발현량 변화를 면역세포화학법으로 확인한 결과이다.6 is a result of confirming the change in the expression level of the novel miRNA3 target gene protein HOOK3 of the present invention by immunocytochemistry.
본 발명은 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 신규 마이크로 RNA를 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물에 관한 것이다.The present invention relates to a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising novel microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.
본 발명에서 마이크로 RNA(miRNA)는 표적 RNA의 분해(degradation)를 촉진시키거나 또는 그들의 번역을 억제시킴으로써 유전자 발현을 전사 후에 조절하는 21~23개의 비코딩 RNA를 말한다. 특정 염기서열로 나타내는 miRNA뿐만 아니라 상기 miRNA의 전구체(pre-miRNA, pri-miRNA), 이들과 생물학적 기능이 동등한 miRNA, 예를 들면 동족체(즉, 호몰로그 또는 오솔로그), 유전자다형 등의 변이체, 및 유도체도 포함한다. In the present invention, microRNA (miRNA) refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting their translation. Not only miRNAs represented by specific nucleotide sequences, but also precursors (pre-miRNA, pri-miRNA) of the miRNAs, miRNAs with biological functions equivalent to these, for example, homologues (i.e., homologs or orthologs), variants such as polymorphisms, and derivatives.
상기 신규 마이크로 RNA는 인간의 혈장 유래인 것이 바람직하지만 이에 한정하지 않는다.The novel microRNA is preferably derived from human plasma, but is not limited thereto.
상기 서열번호 1의 신규 마이크로 RNA는 BDNF(Brain-Derived Neurotrophic Factor), NRG3(Neuregulin3) 및 NEFL(Neurofilament Light Chain) 유전자의 발현량을 감소시키며, 서열번호 3의 신규 마이크로 RNA는 HOOK3(Protein Hook homolog 3) 유전자의 발현량을 감소시키는 것이 특징이다. The novel microRNA of SEQ ID NO: 1 reduces the expression levels of Brain-Derived Neurotrophic Factor (BDNF), Neuregulin3 (NRG3), and Neurofilament Light Chain (NEFL) genes, and the novel microRNA of SEQ ID NO: 3 is HOOK3 (Protein Hook homolog 3) It is characterized by reducing the expression level of genes.
본 발명의 '진단'은 특정 질병 또는 질환에 대한 한 객체 즉 검사 대상자의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)를 판정하는 것 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링하는 것)을 포함하는 개념이다.'Diagnosis' of the present invention is to determine the susceptibility of an object, that is, a test subject, to a specific disease or disorder, to determine whether an object currently has a specific disease or disorder, to a specific disease or disorder Concepts include determining the prognosis of an affected subject or therametrics (eg, monitoring the condition of a subject to provide information about treatment efficacy).
또한, 본 발명은 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 마이크로 RNA를 검출할 수 있는 제제를 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물에 관한 것이다.In addition, the present invention relates to a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
상기 제제는 상기 마이크로 RNA에 상보적인 안티센스 올리고뉴클레오티드, 프라이머 또는 프로브일 수 있으나, 이에 제한되지 않는다.The agent may be an antisense oligonucleotide, primer or probe complementary to the microRNA, but is not limited thereto.
경도인지장애는 정상적인 노화현상으로 인한 인지능력의 감퇴와 치매의 중간 단계를 의미한다. 즉, 동일한 연령대에 비해 인지 능력이 저하되어 있는 상태를 말하며, 일상생활이 가능한 것이 치매와 다른 점이다. 이러한 경도인지장애는 정상과 치매의 중간단계이며, 치매의 고위험군으로 알려져 있다. Mild cognitive impairment refers to an intermediate stage between cognitive decline and dementia caused by normal aging. In other words, it refers to a state in which cognitive ability is lowered compared to those of the same age group, and it is different from dementia that daily life is possible. This mild cognitive impairment is an intermediate stage between normal and dementia, and is known as a high-risk group for dementia.
따라서 본 발명에서는 경도인지장애 또는 알츠하이머성 치매의 조기 진단을 위한 실험군으로, '경도인지장애 또는 알츠하이머성 치매 환자군'을 채택하였다. Therefore, in the present invention, the 'mild cognitive impairment or Alzheimer's dementia patient group' was selected as an experimental group for early diagnosis of mild cognitive impairment or Alzheimer's dementia.
또한, 본 발명은 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 신규 마이크로 RNA를 검출할 수 있는 제제를 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물을 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트에 관한 것이다.In addition, the present invention is a biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia comprising as an active ingredient an agent capable of detecting a new microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, comprising as an active ingredient It relates to a kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia.
상기 키트는 RT-PCR, 실시간(real-time) PCR, 등온 PCR, 노던 블랏팅, RNA 보호분석법 또는 마이크로어레이 칩용인 것이 바람직하지만 이에 한정하는 것은 아니다.The kit is preferably for RT-PCR, real-time PCR, isothermal PCR, Northern blotting, RNA protection assay or microarray chip, but is not limited thereto.
본 발명의 키트는 서열번호 1 또는 서열번호 3의 마이크로 RNA와 특이적으로 결합 가능한 핵산을 추가적으로 포함할 수 있고, 상기 핵산은 RNA, DNA, 또는 RNA/DNA(키메라) 모두 포함하는 것을 의미하고, 상기 DNA에는 cDNA, 게놈 DNA, 및 합성 DNA 모두가 포함될 수 있다. The kit of the present invention may additionally include a nucleic acid capable of specifically binding to the microRNA of SEQ ID NO: 1 or SEQ ID NO: 3, which means that the nucleic acid includes both RNA, DNA, or RNA/DNA (chimera), The DNA may include all of cDNA, genomic DNA, and synthetic DNA.
또한, 상기 RNA에는 total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, 비코딩(non-coding)RNA 및 합성 RNA 모두가 포함될 수 있다. 본 발명의 키트는 상기 핵산에 추가하여 알츠하이머성 치매의 조기 진단을 가능하게 하는 기지의 핵산 또는 장래 발견될 수 있는 핵산을 포함할 수 있으며, 공지의 알츠하이머성 치매의 조기 진단용 마커를 측정하기 위한 항체도 포함시킬 수 있다. 상기 키트에 포함되는 상기 핵산은 개별적으로 또는 임의로 조합하여 다른 용기에 포장될 수 있다. In addition, the RNA may include all of total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA. The kit of the present invention may include, in addition to the nucleic acid, a known nucleic acid or a nucleic acid that can be discovered in the future that enables early diagnosis of Alzheimer's disease, and an antibody for measuring a known marker for early diagnosis of Alzheimer's disease. can also be included. The nucleic acids included in the kit may be individually or arbitrarily combined and packaged in different containers.
본 발명의 키트에는 표지용 형광물질, 핵산 증폭용 효소 및 배지, 사용 설명서 등을 포함시킬 수 있다. 본 발명의 키트는 상기 핵산이 예를 들면 고상에 결합 또는 부착된 알츠하이머성 치매의 조기 진단를 위한 장치이다. 고상의 재질의 예는 플라스틱, 종이, 유리, 실리콘 등이며, 가공의 용이함으로부터 바람직한 고상의 재질은 플라스틱일 수 있다. 고상의 형상은 임의이며, 예를 들면 사각형, 원형, 직사각형, 필름형 등일 수 있다.The kit of the present invention may include a fluorescent material for labeling, an enzyme and medium for nucleic acid amplification, instructions for use, and the like. The kit of the present invention is a device for early diagnosis of Alzheimer's disease in which the nucleic acid is bound or attached to, for example, a solid phase. Examples of the material of the solid phase include plastic, paper, glass, silicon, and the like, and a material of the solid phase that is preferable from ease of processing may be plastic. The shape of the solid phase is arbitrary, and may be, for example, square, circular, rectangular, or film-like.
본 발명의 키트는 다량의 miRNA 시료를 동시에 처리하는 HTS(high-throughput-screening) 기반 다중 현장진단(multiplexed POCT)용 장치에 포함될 수 있다. 장치 내 카트릿지에는 항온장치, 교반기, 일회용 팁, 검체 주입용기 등으로 구성될 수 있고, 초소형 전혈분리기, 형광검출기, 리퀴드핸들러 내장 올인원 장치로 사람의 혈액을 주입하면 자동으로 '치매 위험 지수'가 도출되는 알고리즘 및 전자동 검출 시스템이 포함될 수 있다.The kit of the present invention may be included in a high-throughput-screening (HTS)-based multiplexed POCT device that simultaneously processes a large amount of miRNA samples. The cartridge in the device can be composed of a thermostat, stirrer, disposable tip, sample injection container, etc., and the 'dementia risk index' is automatically derived when human blood is injected into the all-in-one device with a built-in ultra-small whole blood separator, fluorescence detector, and liquid handler. Algorithms and fully automatic detection systems may be included.
또한, 본 발명은 (1) 개체로부터 혈액을 채취하는 단계;In addition, the present invention comprises the steps of (1) collecting blood from an individual;
(2) 상기 단계 (1)의 혈액으로부터 혈장을 분리하는 단계; 및(2) separating plasma from the blood of step (1); and
(3) 상기 단계 (2)에서 분리한 혈장 내 서열번호 1 또는 서열번호 3의 miRNA 발현여부를 확인하는 단계;를 포함하는 경도인지장애 또는 알츠하이머성 치매의 조기 진단을 위한 정보를 제공하는 방법에 관한 것이다.(3) checking whether the miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed in the plasma isolated in step (2); it's about
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail using examples. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.
1. 알츠하이머성 치매 환자의 시료 확보1. Obtaining samples from patients with Alzheimer's disease
선별검사도구(K-MMSE, SNSB) 점수와 영상진단(PET, fMRI, CT)을 통해 알츠하이머성 치매 중증도에 따라 분류된 환자군 중에서 경도인지장애(MCI) 및 알츠하이머성 치매(Alzheimer's disease; AD)로 분류된 환자 그룹당 10인의 혈액을 채취하여 혈장을 분리하였다. 분리한 혈장은 사용하기 전까지, -70℃에서 냉동보관하였다. 한편, 대조군으로서, 정상인 10인의 혈액을 채취하여 혈장을 분리하였고, 상기 환자군과 동일하게 사용하기 전까지 -70℃에서 냉동보관하였다.Mild cognitive impairment (MCI) and Alzheimer's disease (AD) were classified among patients classified according to the severity of Alzheimer's dementia through screening test tools (K-MMSE, SNSB) scores and imaging diagnosis (PET, fMRI, CT). Blood was collected from 10 patients per group of patients classified and plasma separated. The separated plasma was stored frozen at -70°C until use. On the other hand, as a control group, blood was collected from 10 normal people, plasma was separated, and stored frozen at -70 ° C until use in the same way as the patient group.
2. Total miRNA 추출2. Total miRNA extraction
소량의 혈청 및 혈장에서 주로 miRNA 및 기타 small RNA를 정제할 수 있는 QIAGEN miRNeasy 혈청/혈장 분리 키트를 사용하여 상기 확보한 환자군 및 정상군의 각 혈장 내 있는 Total RNA를 추출하였다. Total RNA from the plasma of each of the above-obtained patient and normal groups was extracted using the QIAGEN miRNeasy Serum/Plasma Separation Kit, which can purify miRNA and other small RNAs mainly from small amounts of serum and plasma.
이후, Bioanalyzer RNA Small RNA chip을 이용하여 NGS 분석을 위한 라이브러리 제작에 충분한 RNA 양이 확보되었는지를 확인하였다.Then, it was confirmed whether sufficient amount of RNA was secured for library construction for NGS analysis using a Bioanalyzer RNA Small RNA chip.
3. NGS(Next Generation Sequencing) 분석3. NGS (Next Generation Sequencing) analysis
소량의 RNA에서도 고품질 miRNA 라이브러리 제작이 가능한 SMARTer smRNA-Seq Kit를 사용하여 Illumina Platform에서 small RNA-seq 라이브러리를 생성하였다. SMARTer smRNA-Seq Kit 매뉴얼에 따라 NovaSeq 6000 시퀀싱 플랫폼으로 준비된 시료를 시퀀싱하였다.A small RNA-seq library was created on the Illumina Platform using the SMARTer smRNA-Seq Kit, which can produce a high-quality miRNA library even with a small amount of RNA. Samples prepared with the NovaSeq 6000 sequencing platform were sequenced according to the SMARTer smRNA-Seq Kit manual.
4. 가공하지 않은 데이터 분석을 통한 miRNA 데이터 선별 4. Screening miRNA data through raw data analysis
NGS 분석으로 획득한 가공하지 않은 데이터를 Adapter, Quality 및 Length의 trimming 순으로 혈장 속에 존재하는 오염되거나 양이 적은 데이터를 제거하였다. 이러한 trimming 과정을 거쳐 불필요한 정보가 제거된 clean 데이터로 miRNA Deep2* 시퀀싱을 실시하였다.Contaminated or low-quantity data in plasma was removed in the order of adapter, quality, and length trimming from the raw data obtained by NGS analysis. miRNA Deep2* sequencing was performed with clean data from which unnecessary information was removed through this trimming process.
5. miRNA Deep2* 시퀀싱5. miRNA Deep2* sequencing
miRNA Deep2*는 정제된 가공하지 않은 데이터에서 신규 miRNA를 식별할 수 있는 소프트웨어로, RNAfold 알고리즘에 따라, mature, star 및 loop 서열에 짧게 잘려진 miRBase(Pre-miRNA) 라이브러리를 나란히 배열하여 신규 miRNA를 예측할 수 있으며, RNAfold 알고리즘은 가장 비슷한 열역학 모델을 이용해 RNA 서열이 최소 자유에너지를 갖는 2차 구조를 예측하는 방식으로 도출할 수 있다. RNA fold 생성 그래픽에는 헤어핀(Hairpin)의 각 부분에 대한 읽기 수, 최소자유에너지에 대한 점수, randfold에 대한 점수 및 conserved seed sequence 점수가 포함되어 있다. miRNA Deep2* is a software that can identify new miRNAs from refined raw data. According to the RNAfold algorithm, miRBase (Pre-miRNA) libraries cut short in mature, star, and loop sequences are arranged side by side to predict new miRNAs. The RNAfold algorithm can be derived by predicting the secondary structure of the RNA sequence with the minimum free energy using the most similar thermodynamic model. The RNA fold generation graphic includes the number of reads for each part of the hairpin, the score for the minimum free energy, the score for the randfold, and the score for the conserved seed sequence.
miRDeep2* 시스템은 RNA 데이터베이스를 활용한 맵핑으로 기존에 알려진 miRNA를 탐지하고 신규 miRNA을 탐지할 수 있다. The miRDeep2* system can detect previously known miRNAs and detect new miRNAs by mapping using an RNA database.
6. 혈액에 반영되는 알츠하이머성 치매 관련 신규 miRNA의 검증6. Verification of novel miRNAs related to Alzheimer's disease reflected in blood
신규 miRNA가 알츠하이머성 치매와 어떤 연관이 있는지에 대한 기능 검증을 위해 miRNA의 서열기반으로 타겟 유전자(miRNA)를 스코어링하는 miRDB 소프트웨어를 사용하여 스크리닝하였다. 높은 유전자 스코어를 가진 타겟 유전자 중 알츠하이머성 치매와 관련된 유전자에 대한 발현량 분석을 통해 검증하였다. To verify the function of novel miRNAs and how they are associated with Alzheimer's disease, screening was performed using miRDB software that scores target genes (miRNAs) based on miRNA sequences. Among the target genes with high gene scores, it was verified through expression analysis of genes related to Alzheimer's disease.
신경기능 및 분화 모델로 사용되는 SH-SY5Y 세포를 사용하였으며, 선별된 신규 miRNA의 과발현 효과를 나타내는 miRNA mimic, 신규 miRNA의 발현을 억제하는 miRNA 억제제(inhibitor) 및 음성대조군 miRNA를 SH-SY5Y에 형질주입 후, 타겟 유전자의 단백질 발현을 웨스턴블랏으로 확인하였다.SH-SY5Y cells used as a neural function and differentiation model were used, and a miRNA mimic showing the overexpression effect of the selected new miRNA, a miRNA inhibitor suppressing the expression of the new miRNA, and a negative control miRNA were transfected into SH-SY5Y After injection, the protein expression of the target gene was confirmed by Western blot.
실시예 1. 시료 확보 및 Total RNA 추출Example 1. Sample acquisition and total RNA extraction
소량의 혈청 및 혈장에서 miRNA 및 기타 small RNA를 정제할 수 있는 QIAGEN miRNeasy Serum/Plasma Kit를 사용하여, 환자군 및 정상군에서 채취한 각 혈장 내에 있는 Total RNA를 추출하였다. 이후 NGS를 위한 라이브러리 제작에 충분한 RNA 양이 확보되었는지 Bioanalyzer RNA Small RNA chip를 이용하여 RNA를 정량하여 NGS 분석에 충분한 양이 확보되었다는 것을 확인하였다.Total RNA in plasma collected from patients and normal groups was extracted using the QIAGEN miRNeasy Serum/Plasma Kit, which can purify miRNA and other small RNAs from small amounts of serum and plasma. Subsequently, it was confirmed that a sufficient amount of RNA was secured for NGS analysis by quantifying RNA using a Bioanalyzer RNA Small RNA chip to determine whether a sufficient amount of RNA was secured for library production for NGS.
실시예 2. Next Generation Sequencing(NGS)Example 2. Next Generation Sequencing (NGS)
(1) 라이브러리 구축 및 가공하지 않은 데이터 도출(1) Building a library and deriving raw data
NGS를 위한 Illumina Platform에서 시퀀싱을 위한 작은 RNA-seq 라이브러리를 생성하는데 있어, 소량의 RNA에서도 고품질 miRNA 라이브러리 제작이 가능한 SMARTer smRNA-Seq Kit를 사용하였다. 혈장에서 획득한 환자군 및 정상군의 총 RNA 시퀀싱을 위한 라이브러리를 제작하였고, 제작된 라이브러리 및 NovaSeq 6000 시퀀싱 플랫폼을 이용하여 시퀀싱하여 가공하지 않은 데이터를 도출하였다. In generating a small RNA-seq library for sequencing on the Illumina Platform for NGS, the SMARTer smRNA-Seq Kit, which can produce a high-quality miRNA library even with a small amount of RNA, was used. Libraries for total RNA sequencing of patients and normal groups obtained from plasma were prepared, and raw data were derived by sequencing using the prepared libraries and the NovaSeq 6000 sequencing platform.
(2) 가공하지 않은 데이터(Raw data)로부터 miRNA의 데이터 선별(2) Selection of miRNA data from raw data
NGS 분석에서 획득한 가공하지 않은 데이터를 Adapter, Quality, Length 순서로 trimming 하여 오염되거나 적은 양의 데이터를 제거하였다. The raw data obtained from NGS analysis was trimmed in the order of Adapter, Quality, and Length to remove contaminated or small amounts of data.
시퀀싱하여 획득한 가공하지 않은 데이터인 Total Read Count에서 Adapter 유무(nonAdapter Read Count), 적당한 길이(Short Read Count), 낮은 품질(Low Quality Read Count)의 정도를 확인하였으며, 이러한 trimming 선별과정을 통해 불필요한 정보가 제거된 1차 가공 데이터를 이용하여 miRNA Deep2* 시퀀싱을 수행하였다.The degree of adapter presence (nonAdapter Read Count), appropriate length (Short Read Count), and low quality (Low Quality Read Count) were confirmed in Total Read Count, which is raw data obtained by sequencing. miRNA Deep2* sequencing was performed using the primary processing data from which information was removed.
(3) miRNA Deep2* sequencing(3) miRNA Deep2* sequencing
miRNA Deep2*는 상기 1차 가공된 데이터로부터 새롭게 알려진 miRNA를 식별할 수 있는 소프트웨어의 RNAfold 알고리즘에 따라, mature, star, loop 서열에 짧게 잘려진 miRBase(Pre-miRNA) 라이브러리를 나란히 배열하여 신규 miRNA를 예측하였으며, RNAfold 알고리즘은 가장 비슷한 열역학 모델을 이용해 RNA 서열이 최소 자유에너지를 갖는 2차 구조를 예측하는 방식으로 도출하였다. miRNA Deep2* predicts new miRNAs by arranging short-cut miRBase (Pre-miRNA) libraries side by side in mature, star, and loop sequences according to the RNAfold algorithm of the software that can identify newly known miRNAs from the primary processed data. The RNAfold algorithm was derived by predicting the secondary structure with the minimum free energy of the RNA sequence using the most similar thermodynamic model.
RNA fold 생성 그래픽에는 Hairpin의 각 부분에 대한 읽기 수, 최소 자유 에너지에 대한 점수, randfold에 대한 점수 및 conserved seed sequence 점수가 포함되어 있다. miRDeep2* 시스템은 RNA 데이터베이스를 활용한 맵핑으로 기존 알려진 miRNA 및 신규 miRNA을 탐지하였다.The RNA fold generation graphic contains the number of reads for each part of the hairpin, the score for the minimum free energy, the score for the randfold, and the conserved seed sequence score. The miRDeep2* system detected previously known miRNAs and new miRNAs by mapping using an RNA database.
상기 각각의 시료에 대해 최종 처리된 리드는 miRBase v22.1과 non-coding RNA database(RNAcentral release 14.0)을 통해 piRNA, tRNA, snRNA, snoRNA, 이미 알려진 miRNA 및 신규 miRNA를 분류하였다. The reads finally processed for each sample were classified into piRNA, tRNA, snRNA, snoRNA, known miRNA, and novel miRNA through miRBase v22.1 and non-coding RNA database (RNAcentral release 14.0).
Genome 맵핑은 RSEM을 사용하여 Bowtie와 STAR에 의해 처리되었다. Bowtie는 이후 Genome 서열을 사용하여 miRDeep2 분석에 이용하였다. miRDeep2에 의해 예측된 알려진/신규 miRNA 및 RNAcentral과 일치하는 다른 small RNA는 Bowtie(표적 small RNA, <50nt) 및 Bowtie2(표적 smRNA, ≥50nt)를 사용하여 정렬하였다.Genome mapping was processed by Bowtie and STAR using RSEM. Bowtie was then used for miRDeep2 analysis using the genome sequence. Known/novel miRNAs predicted by miRDeep2 and other small RNAs matching RNAcentral were aligned using Bowtie (target small RNA, <50nt) and Bowtie2 (target smRNA, ≥50nt).
- RSEM(RNA-Seq by Expectation-Maximization): RNA-Seq 전사체를 정량화하는 도구- RSEM (RNA-Seq by Expectation-Maximization): a tool to quantify RNA-Seq transcripts
- Bowtie: 서열 정렬 및 서열 분석에 일반적으로 사용되는 소프트웨어- Bowtie: software commonly used for sequence alignment and sequence analysis
- STAR(Spliced Transcripts Alignment to a Reference): 빠른 RNA-seq read mapper- STAR (Spliced Transcripts Alignment to a Reference): fast RNA-seq read mapper
신규 miRNA를 예측하기 위해 고유 클러스터링된 read는 reference genome 및 전구체 miRNA에 대해 별도로 정렬되었다. miRNA는 miRNA Deep2*를 사용하는 RNAfold 알고리즘에 따라 성숙한 star 및 loop 서열을 예측할 수 있다. RNAfold 함수는 가장 근접한 이웃 열역학 모델을 사용하여 RNA 서열의 최소 자유 에너지 2차 구조를 예측하였다. RNAfold에서 생성된 그래픽에는 실제 in silico-folded 헤어핀이 포함되어 있으며, 헤어핀의 각 부분에 대한 판독 횟수, 최소 자유 에너지 점수, randfold 점수 보존된 seed 서열의 점수도 포함되어 있다. To predict novel miRNAs, unique clustered reads were separately aligned to the reference genome and precursor miRNAs. miRNAs can predict mature star and loop sequences according to the RNAfold algorithm using miRNA Deep2*. The RNAfold function predicted the minimum free energy secondary structure of an RNA sequence using a nearest-neighbor thermodynamic model. The graphics generated by RNAfold include real in silico-folded hairpins, read counts for each part of the hairpin, the minimum free energy score, and the randfold score, as well as the score of the conserved seed sequence.
miRDeep2*는 -10~10의 점수를 통해 신규 miRNA를 도출하였다. 상기 도출된 신규 miRNA는 경도인지장애 군 및 알츠하이머병 군에서 모두 발현하는 것으로, 그 중 miRDeep2* 점수는 1이상, RNA fold는 "yes"이고, mature read의 수가 10 이상인 miRNA 2종을 선별하였다. miRDeep2* derived novel miRNAs through a score of -10 to 10. The novel miRNAs derived above were expressed in both the mild cognitive impairment group and the Alzheimer's disease group. Among them, two miRNAs having a miRDeep2* score of 1 or more, an RNA fold of "yes", and a mature read number of 10 or more were selected.
선별된 신규 miRNA2 및 miRNA3의 stem loop 구조를 도 1 및 도 2에 개시하였으며, 서열 정보는 도 3에 개시하였다. The stem loop structures of the selected novel miRNA2 and miRNA3 are shown in Figures 1 and 2, and sequence information is shown in Figure 3.
실시예 3. 신규 miRNA의 알츠하이머성 치매 관련 유전자 발현량 조절 확인Example 3. Confirmation of regulation of gene expression level related to Alzheimer's disease by novel miRNA
신규 miRNA가 알츠하이머성 치매와 어떤 연관이 있는지를 검증하기 위해 miRNA의 서열 기반으로 타겟이 되는 유전자를 스코어별로 스크리닝하는 miRDB 소프트웨어를 사용하였다. 높은 유전자 스코어를 가진 타겟 유전자 중 알츠하이머성 치매와 관련된 유전자를 선별하였다. In order to verify the association of the new miRNA with Alzheimer's disease, miRDB software was used to screen the target gene by score based on the sequence of the miRNA. Among the target genes having high gene scores, genes related to Alzheimer's dementia were selected.
신경 기능 및 분화에 모델로 사용되는 SH-SY5Y 세포를 사용하였으며, 선별된 신규 miRNA의 과발현 효과를 나타내는 miRNA mimic, 신규 miRNA의 발현을 억제하는 miRNA inhibitor, 음성대조군 miRNA를 SH-SY5Y에 형질주입 후, 타겟 유전자의 단백질 발현을 웨스턴블랏으로 확인하였다. SH-SY5Y cells used as a model for neural function and differentiation were used, and after transfection of miRNA mimics that show the overexpression effect of selected new miRNAs, miRNA inhibitors that inhibit the expression of new miRNAs, and negative control miRNAs into SH-SY5Y , The protein expression of the target gene was confirmed by Western blot.
miRNA mimic은 실제로 생체 내에 있는 신규 miRNA처럼 기능할 수 있는 miRNA로 이중결합으로 제작되었다. miRNA inhibitor는 도출된 해당 miRNA의 상보적인 서열로, 단일 결합으로 제작되어 해당 miRNA를 억제할 수 있는 miRNA이다. 한편, 음성대조군 miRNA는 유전자를 조절하지 않는 miRNA로 이루어진 miRNA로 검증의 기준이 된다. 제작한 신규 miRNA2, 및 miRNA3의 mimic; 및 miRNA inhibitor는 도 3에 개시하였다.The miRNA mimic is actually a miRNA that can function like a novel miRNA in vivo, and is designed with a double bond. The miRNA inhibitor is a complementary sequence of the derived miRNA, and is a miRNA that can inhibit the miRNA produced by single binding. On the other hand, the negative control miRNA is a miRNA composed of miRNAs that do not regulate genes and serves as a standard for verification. mimics of the newly produced miRNA2 and miRNA3; and miRNA inhibitors are disclosed in FIG. 3 .
[ miRDB 소프트웨어를 이용한 유전자 스크리닝 ][ Gene screening using miRDB software ]
신규 miRNA가 제어하는 유전자 중, 알츠하이머성 치매와 관련성이 높은 타겟을 선별하기 위해, 신규 miRNA 중 miRNA 2 mimic은 서열 5'-UAAAGAGGAAAGAGGAAAGAGG-3'(서열번호 1)를 기반으로, miRNA 3 mimic은 서열 5'-CGGGAGGCAGCAGUGGGC-3'(서열번호 3)를 기반으로, 타겟 예측 miRDB 소프트웨어를 이용해 유전자를 스코어별로 스크리닝하였다. 서열번호 1의 miRNA mimic에 대한 miRNA inhibitor는 5'-CCUCUUUCCUCUUUCCUCUUUA-3'(서열번호 2)이고, 서열번호 3의 miRNA mimic에 대한 miRNA inhibitor는 5'-GCCCACUGCUGCCUCCCG-3'(서열번호 4)이다.Among the genes controlled by novel miRNAs, in order to select targets highly related to Alzheimer's disease, among new miRNAs, miRNA 2 mimic was based on sequence 5'-UAAAGAGGAAAGAGGAAAGAGG-3' (SEQ ID NO: 1), miRNA 3 mimic was sequence Based on 5'-CGGGAGGCAGCAGUGGGGC-3' (SEQ ID NO: 3), genes were screened by score using target prediction miRDB software. The miRNA inhibitor for the miRNA mimic of SEQ ID NO: 1 is 5'-CCUCUUUCCUCUUUCCUCUUUA-3' (SEQ ID NO: 2), and the miRNA inhibitor for the miRNA mimic of SEQ ID NO: 3 is 5'-GCCCACUGCUGCCUCCG-3' (SEQ ID NO: 4).
해당 miRNA의 유전자 스코어의 컷-오프 값을 80으로 설정한 후, 신경 및 시냅스 기능과 관계되는 miRNA2 타겟 유전자로, 신경영양인자(neurotrophins)로서 중추신경계에서 신경세포 생존과 분화뿐만 아니라 시냅스 전달, 시냅스 가소성의 분자 기전에 필수적인 BDNF(Brain-Derived Neurotrophic Factor), 신경세포질에서 신경 필라멘트를 구성해 뉴런의 구조적 안정성을 증가시키는 역할을 하고 축삭의 손상과 관련되어 최근 퇴행성 뇌질환의 바이오마커로 밝혀진 NEFL(Neurofilament Light Chain), 상피 및 신경아교 세포의 성장과 분화를 조절하고 신경계의 발달, 유지 및 복구에 중요한 역할을 하는 NRG3(Neuregulin3)를 선별하였고, 신규 miRNA3의 타겟 유전자로는 뉴런에서 엔도좀 수송과 관련된 세포질 링커 유전자로 알츠하이머성 치매의 원인 단백질인 아밀로이드 베타의 생성과 관련 있는 타겟 유전자 HOOK3(Protein Hook homolog 3)를 선별하였다. After setting the cut-off value of the gene score of the corresponding miRNA to 80, miRNA2 target gene related to neuron and synaptic function, neurotrophins, as well as neuronal survival and differentiation in the central nervous system, as well as synaptic transmission and synaptic plasticity BDNF (Brain-Derived Neurotrophic Factor), which is essential for the molecular mechanism of neuronal cytoplasm, plays a role in increasing the structural stability of neurons by constructing nerve filaments in the neuronal cytoplasm, and is related to axonal damage. Light Chain), NRG3 (Neuregulin3), which regulates the growth and differentiation of epithelial and glial cells and plays an important role in the development, maintenance, and restoration of the nervous system, was selected. As a cytoplasmic linker gene, a target gene HOOK3 (Protein Hook homolog 3) related to the production of amyloid beta, a causative protein of Alzheimer's disease, was selected.
신규 miRNA의 과발현을 위한 miRNA mimic, 기능 억제를 위한 miRNA inhibitor를 인간 신경모세포종 SH-SY5Y에 각각의 miRNA 100nM를 24시간 동안 형질 주입하였고, 단백질을 분리하여 웨스턴 블랏으로 발현 정도를 확인하였다. Human neuroblastoma SH-SY5Y was transfected with 100 nM of each miRNA miRNA for overexpression and miRNA inhibitor for function suppression for 24 hours, and the protein was isolated and the expression level was confirmed by Western blotting.
신규 miRNA2의 과발현으로 3'-UTR에 결합하는 miRNA2가 많아져, 후보 타겟 유전자 단백질인 BDNF, NRG3 및 NEFL의 발현이 유의적으로 감소하는 것을 확인하였고, miRNA inhibitor로 3'에 결합하는 miRNA2를 제거함으로써, 후보 타겟 유전자 단백질 BDNF, NRG3 및 NEFL의 발현이 농도 의존적으로 증가하는 것을 확인하였다(도 4). Overexpression of the new miRNA2 increased the number of miRNA2 binding to 3'-UTR, and it was confirmed that the expression of candidate target gene proteins, BDNF, NRG3, and NEFL, was significantly reduced, and miRNA inhibitor was used to remove miRNA2 binding to 3'. By doing so, it was confirmed that the expression of the candidate target gene proteins BDNF, NRG3, and NEFL increased in a concentration-dependent manner (FIG. 4).
한편, 신규 miRNA3의 과발현을 위한 miRNA3 mimic, 기능 억제를 위한 miRNA3 inhibitor를 인간 신경모세포종 SH-SY5Y에 각각 miRNA3의 mimic과, inhibitor를 농도 의존적으로 24시간 동안 형질주입을 진행하고, 단백질을 분리하여 웨스턴 블랏을 실시하였다. 신규 miRNA 3의 과발현의 효과를 나타내는 miRNA 3 mimic 처리 시, 후보 타겟 유전자 단백질인 HOOK3의 발현이 유의미하게 감소한 것을 확인하였고, miRNA 3 inhibitor로 타겟 유전자의 3'-UTR에 결합하는 miRNA 3의 제거를 위한 miRNA 3 inhibitor 처리는 HOOK3의 발현이 증가한 것을 확인하였다(도 5). On the other hand, miRNA3 mimic for overexpression of novel miRNA3 and miRNA3 inhibitor for suppression of function were transfected into human neuroblastoma SH-SY5Y for 24 hours in a concentration-dependent manner, respectively, and the protein was isolated and Western A blot was performed. When miRNA 3 mimic treatment, which shows the effect of overexpression of the new miRNA 3, it was confirmed that the expression of HOOK3, a candidate target gene protein, was significantly reduced. It was confirmed that treatment with miRNA 3 inhibitor for HOOK3 increased expression (Fig. 5).
또한, miRNA mimic와 miRNA inhibitor 농도를 1nM로 고정한 후, 타겟 유전자의 HOOK3 단백질의 발현을 면역세포화학법으로 비교하였다. 그 결과, 웨스턴 블랏의 발현 패턴과 일치하는 것을 확인하였다(도 6).In addition, after fixing the miRNA mimic and miRNA inhibitor concentrations to 1 nM, the expression of the HOOK3 protein of the target gene was compared by immunocytochemistry. As a result, it was confirmed to be consistent with the expression pattern of Western blot (FIG. 6).
Claims (12)
- 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 신규 마이크로 RNA를 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오 마커 조성물.A biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia comprising novel microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as an active ingredient.
- 제1항에 있어서, 상기 신규 마이크로 RNA는 인간의 혈장 유래인 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물.The biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia according to claim 1, wherein the novel microRNA is derived from human plasma.
- 제1항에 있어서, 상기 서열번호 1의 신규 마이크로 RNA는 BDNF(Brain-Derived Neurotrophic Factor), NRG3(Neuregulin3) 및 NEFL(Neurofilament Light Chain) 유전자의 발현량을 감소시키며, 서열번호 3의 신규 마이크로 RNA는 HOOK3(Protein Hook homolog 3) 유전자의 발현량을 감소시키는 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물.The novel microRNA of SEQ ID NO: 1 according to claim 1, wherein the novel microRNA of SEQ ID NO: 1 reduces the expression levels of Brain-Derived Neurotrophic Factor (BDNF), Neuregulin3 (NRG3), and Neurofilament Light Chain (NEFL) genes, and the novel microRNA of SEQ ID NO: 3 A biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, characterized by reducing the expression level of HOOK3 (Protein Hook homolog 3) gene.
- 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 마이크로 RNA를 검출할 수 있는 제제를 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물.A biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising as an active ingredient an agent capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
- 제4항에 있어서, 상기 제제는 상기 마이크로 RNA에 상보적인 안티센스 올리고뉴클레오티드, 프라이머 또는 프로브인 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 바이오마커 조성물.The biomarker composition for early diagnosis of mild cognitive impairment or Alzheimer's dementia according to claim 4, wherein the agent is an antisense oligonucleotide, primer or probe complementary to the microRNA.
- 제4항의 조성물을 유효성분으로 포함하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트.A kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia, comprising the composition of claim 4 as an active ingredient.
- 제6항에 있어서, 상기 키트는 RT-PCR, 실시간(real-time) PCR, 등온 PCR, 노던 블랏팅, RNA 보호분석법 또는 마이크로어레이 칩용인 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트.The kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia according to claim 6, wherein the kit is for RT-PCR, real-time PCR, isothermal PCR, Northern blotting, RNA protection assay, or microarray chip. .
- 제6항에 있어서, 상기 키트는 HTS(high-throughput-screening) 기반 다중 현장진단(multiplexed POCT) 장치용인 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트.The kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia according to claim 6, wherein the kit is for a high-throughput-screening (HTS) based multiplexed POCT device.
- 제8항에 있어서, 상기 다중 현장진단(multiplexed POCT)용 장치는 항온장치, 교반기, 일회용 팁 및 검체 주입 용기를 포함하는 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트.The kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia according to claim 8, wherein the device for multiplexed POCT includes a thermostat, a stirrer, a disposable tip, and a sample injection container.
- 제8항에 있어서, 상기 다중 현장진단(multiplexed POCT) 장치는 초소형 전혈분리기, 형광검출기 및 리퀴드 핸들러가 내장된 올인원 장치인 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트.The kit for early diagnosis of mild cognitive impairment or Alzheimer's dementia according to claim 8, wherein the multiplexed POCT device is an all-in-one device equipped with a microscopic whole blood separator, a fluorescence detector, and a liquid handler.
- 제10항에 있어서, 상기 초소형 전혈분리기, 형광검출기 및 리퀴드 핸들러가 내장된 올인원 장치는 인간 혈액으로부터 치매 위험지수를 계산하는 알고리즘 및 검출시스템이 포함된 것을 특징으로 하는 경도인지장애 또는 알츠하이머성 치매 조기 진단용 키트.The method of claim 10, wherein the all-in-one device with a built-in microscopic whole blood separator, fluorescence detector, and liquid handler includes an algorithm and detection system for calculating a risk index of dementia from human blood, characterized in that mild cognitive impairment or early Alzheimer's dementia diagnostic kit.
- (1) 개체로부터 혈액을 채취하는 단계;(1) collecting blood from the subject;(2) 상기 단계 (1)의 혈액으로부터 혈장을 분리하는 단계; 및(2) separating plasma from the blood of step (1); and(3) 상기 단계 (2)에서 분리한 혈장 내 서열번호 1 또는 서열번호 3의 miRNA 발현 여부를 확인하는 단계;를 포함하는 경도인지장애 또는 알츠하이머성 치매의 조기 진단을 위한 정보를 제공하는 방법.(3) checking whether the miRNA of SEQ ID NO: 1 or SEQ ID NO: 3 is expressed in the plasma isolated in step (2); a method for providing information for early diagnosis of mild cognitive impairment or Alzheimer's dementia.
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