Circulating serum microrna biomarkers and methods for determining progression rate of parkinson's disease

Abstract

To provide serum-based microRNAs and methods for differentiating patients suffering from Parkinson's disease based on the rate of disease progression, and additionally for assisting clinicians in determining treatment protocols for such patients.SOLUTION: The invention provides biomarkers and methods for identifying, verifying and confirming circulating serum-based microRNAs. The microRNAs (PrognomiRs) can be used to differentiate patients suffering from rapidly progressing Parkinson's disease (PD) from slowly progressing PD patients.SELECTED DRAWING: None

Description

[Technical field]

[0001] 1. Field of the invention The present invention relates generally to serum-based microRNAs and methods for identifying patients suffering from Parkinson's disease based on the rate of disease progression as well as assisting clinicians in determining treatment protocols for such patients. [Background technology]

[0002] 2. Brief description of the background technology Parkinson's disease (PD) is a highly specific degeneration of dopamine-containing cells in the substantia nigra of the midbrain, leading to dopamine deficiency in the striatum. PD currently affects approximately 10 million people worldwide. PD patients can be effectively managed for the first 5-7 years of treatment, after which a series of often debilitating complications develop, collectively referred to as Late Motor Fluctuations (LMF). It is believed that treatment with the most effective anti-Parkinson's drug, levodopa ((-)-L-α-amino-β-(3,4-dihydroxybenzene)propanoic acid), or L-dopa, may predispose or even promote the appearance of LMF. Dopamine agonists are used as alternative treatments, but do not provide patients with the same degree of symptomatic relief as L-dopa.

[0003] Symptomatic treatment improves signs and symptoms without affecting the underlying disease state. Levodopa increases dopamine concentration in the striatum, especially when its peripheral metabolism is inhibited by peripheral decarboxylase inhibitors (PDIs). Levodopa / PDI therapies, such as combination with levodopa, combination with carbidopa ((-)-L-α-hydrazino-α-methyl-β-(3,4-dihydroxybenzene)propanoic acid monohydrate), levodopa and controlled-release carbidopa, levodopa and benserazide, and levodopa and controlled-release benserazide (2-amino-3-hydroxy-propionic acid N'-(2,3,4-trihydroxy-benzyl)-hydrazide), are widely used in the symptomatic treatment of Parkinson's disease.

[0004] Catechol-O-methyltransferase (COMT) inhibitors enhance levodopa treatment by inhibiting the metabolism of levodopa and increasing its bioavailability, making more of the drug available in the synaptic cleft for a longer period of time. Examples of COMT inhibitors include tolcapone (3,4-dihydroxy-4'-methyl-5-nitrobenzophenone) and entacapone ((E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-2-propenamide).

[0005] Dopamine agonists provide symptomatic benefit by directly stimulating postsynaptic striatal dopamine receptors. Examples include bromocriptine ((5α)-2-bromo-12'-hydroxy-2'-(1-methylethyl)-5'-(2-methylpropyl)ergotaman-3',6',18-trione), pergolide (8B-[(methylthio)methyl]-6-propylergoline), ropinirole (4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one), pramipexole ((S)-4,5,6,7-tetrahydro-N 6-propyl-2,6-benzothiazolediamine), lisuride (N'-[(8α)-9,10-didehydro-6-methylergolin-8-yl]-N,N-diethyl-urea), cabergoline ((8β)-N-[3-(dimethylamino)propyl]-N-[(ethylamino)carbonyl]-6-(2-propenyl)ergoline-8-carboxamide), apomorphine ((6aR)-5,6,6a,7-tetrahydro-6-methyl-4H-dibenzo[de,g]quinoline-10,11-diol), sumanirole (5-(methylamino)-5, Dopamine agonists include 6-dihydro-4H-imidazo{4,5,1-ij}quinolin-2(1H)-one), rotigotine ((-)(S)-5,6,7,8-tetrahydro-6-[propyl[2-(2-thienyl)ethyl]amino]-1-naphthol-), talipexole (5,6,7,8-tetrahydro-6-(2-propenyl)-4H-thiazolo[4,5-d]azepin-2-amine), and dihydroergocryptine (ergotaman-3',6',18-trione, 9,10-dihydro-12'-hydroxy-2'-methyl-5'-(phenylmethyl)(5' cc)). Dopamine agonists are effective as monotherapy early in the course of Parkinson's disease and as adjuncts to levodopa in more advanced stages. Unlike levodopa, dopamine agonists directly stimulate postsynaptic dopamine receptors, do not undergo oxidative metabolism, and are not thought to accelerate the disease process.

[0006] Amantidine (1-aminotricyclo(3,3,1,1 3,7 Amantidine) is an antiviral agent that was discovered serendipitously to have antiparkinsonian activity. Its mechanism of action in PD has not been established, but it is thought to act by increasing dopamine release. Patients who take amantidine, either as monotherapy or in combination with levodopa, show improvement in akinesia, rigidity, and tremor.

[0007] Other medicines used to treat Parkinson's disease include MAO-B inhibitors. Inhibition of L-dopa metabolism via inactivation of monoamine oxidase type B (MAO-B) is an effective means of enhancing the availability of both endogenous residual dopamine and dopamine derived from its exogenous precursor, L-dopa. Selegiline (methyl-(1-methyl-2-phenyl-ethyl)-prop-2-ynyl-amine) is an MAO-B inhibitor. There is evidence that treatment with selegiline may slow disease progression in PD by blocking free radical formation derived from the oxidative metabolism of dopamine. Other examples of MAO B inhibitors include lazabemide (N-(2-aminoethyl)-5-chloro-2-pyridinecarboxamide), rasagiline (N-propargyl-1-(R)aminoindan), and caroxazone (2-oxo-2H-1,3-benzoxazine-3(4H)-acetamide).

[0008] In order to increase the effectiveness of therapeutic agents, it is essential to diagnose individuals with PD at an early stage, and it is also important to determine the prognosis of the disease.However, there are no objective tests or established biomarkers for diagnosing PD.In addition, the diversity, subtypes, and progression of this disease make it difficult to develop specific therapeutic candidates.

[0009] MicroRNAs ("miRNAs") are a class of non-coding RNAs that play an important role in regulating gene expression. miRNAs act at the post-transcriptional level to fine-tune the expression of as many as 30% of all mammalian protein-coding genes. Mature miRNAs are short, single-stranded RNA molecules, approximately 22 nucleotides in length. miRNAs can be encoded by multiple gene loci and can be organized into tandemly co-transcribed clusters. miRNA genes are transcribed by RNA polymerase II into large primary transcripts (pri-microRNAs) that are processed by a protein complex containing the ribonuclease III enzyme Drosha, DGCR8, and other cofactors to form ~70 nucleotide precursor microRNAs (pre-miRNAs) (Cathew RW, Cell, 2009; Kim VN, Nat Rev Mol Cel Biol, 2009; Siomi H, Mol Cel, 2010; Bartel DP, Cell, 2004; Lee Y, Nature 2003; Han J, Genes Dev, 2004). Pre-miRNAs are transported to the cytoplasm by exportin 5, where they are processed by a second ribonuclease III enzyme, DICER, along with TRBP, PACT, and Ago2 in the RNA-induced silencing complex, to double-stranded miRNAs (Kim VN, Nat Rev Mol Cel Biol, 2009; Gregory RI, Nature 2004; MAcRae IJ, PNAS, 2008). The guide strand of the double-stranded miRNA separates and associates with Ago2 to be incorporated into ribonucleophores to form the RNA-induced silencing complex, RISC, which mediates gene silencing. The mechanisms of miRNAs range from direct degradation or silencing of mRNAs and translational repression to post-transcriptional upregulation (MacRae IJ, PNAS, 2008).

[0010] miRNAs have been reported to be present at detectable levels in body fluids including blood, cerebrospinal fluid (CSF), plasma, serum, and saliva. The tissue specificity of miRNAs suggests their crucial and essential role in various physiological processes. Their tissue enrichment promises new, less explored roles as diagnostic biomarkers and potential therapeutic targets. It is understood that miRNAs in blood originate from passive leakage from damaged tissues as a result of cell lysis or apoptosis, active transport from cells via microvesicles such as exosomes, or binding within RISC protein complexes (Etheridge et al., 2011). Exosome- and osmotic pump-mediated delivery of small RNA molecules to the brain and CNS, respectively, offers a solution to overcome the limitations of miRNA-based therapeutics (Alvarez-Erviti et al., 2011; Koval et al., 2013, Hum. Mol. Gen). miRNAs are extremely stable and therefore have proven to be strong candidates for potential biomarkers ( Chen et al., 2008 ; Grasso, 2014 ). [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Cathew RW, Cell, 2009 [Non-Patent Document 2] Kim VN, Nat Rev Mol Cel Biol, 2009 [Non-Patent Document 3] Siomi H, Mol Cel, 2010 [Non-Patent Document 4] Bartel D.P., Cell, 2004 [Non-Patent Document 5] Lee Y, Nature 2003 [Non-Patent Document 6] Han J, Genes Dev, 2004 [Non-Patent Document 7] Gregory RI, Nature 2004

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[0012] It is an object of the present invention to identify miRNAs that are associated with patients suffering from Parkinson's disease.

[0013] It is another object of the present invention to provide a method for determining the rate of disease progression in a patient suffering from Parkinson's disease.

[0014] It is another object of the present invention to determine a method for determining treatment options for a patient suffering from Parkinson's disease based on the rate of disease progression. [Means for solving the problem]

[0015] These and other objects are achieved by the present invention, which provides miRNA biomarkers that can be used alone, or in pairs or combinations, to determine which patients suffer from Parkinson's disease. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the mean fold change of five PrognomiRNAs between fast and slow progressing PD patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] method Serum sample handling and classification All patients and controls participated in the Norwegian ParkWest project, an ongoing population-based prospective longitudinal cohort study investigating the incidence, neurobiology, and prognosis of PD. The Norwegian ParkWest study is a prospective longitudinal multicenter cohort study of patients with Parkinson's disease (PD) events in Western and Southern Norway. Between November 1, 2004 and August 31, 2006, we sought to recruit all new cases of Parkinson's disease in the study area. Since the start of the study, 212 of 265 (80%) of these patients and age / sex-matched controls have been followed up. Further information on this study is available at http: / / www.parkvest.no.

[0018] Every effort was made to establish an unselected and population-representative cohort of PD patients. Patients who provided serum at study entry and met the National Institute of Neurological Disorders and Stroke (http: / / www.ninds.nih.gov / disorders / parkinsons_disease / parkinsons_disease.htm) and UK Brain Bank (http: / / www.ncbi.nlm.nih.gov / projects / gap / cgi-bin / GetPdf.cgi?id=phd000042) diagnostic criteria for PD at most recent follow-up were included. Patients with secondary parkinsonism at study entry were excluded from the study. Control subjects were recruited from multiple sources, including friends, spouses, and, for the elderly, public organizations, and were included in the study if they provided serum. All patients and controls were Caucasian. Study participants were followed for 8 years to establish disease progression rates.

[0019] In this study of potential biomarkers for PD, a two-stage procedure was applied. For the first discovery stage, sera from 8 fast and 8 slow PD progressors were randomly selected. For validation purposes, the remaining 164 PD patients eligible for the study were selected.

[0020] Serum samples were collected on the same day as clinical testing and then stored frozen at −70°C until shipped on dry ice to the New York facility.

[0021] Example 1 Analysis of differentially expressed human miRNAs by qPCR RNA isolation and QC from serum samples After thawing on ice, 24 serum samples (16 from PD) were centrifuged at 3000xg for 5 min to remove debris. The supernatant was used to perform small RNA isolation using the miRCURY RNA Isolation Kit - Biofluids (Exiqon, MA). Prior to RNA isolation, lysis buffer was spiked with 0.267 fmol / ul of spike-in control cel-miR-39-3p (Qiagen, CA). The remainder of the RNA isolation was performed according to the manufacturer's protocol and isolated RNA was quantified with Nanodrop 2000 (Thermo Scientific, MA). This RNA was used to run Affymetrix v4 microRNA microarray chips for subsequent cDNA synthesis and qPCR. RNA was isolated from 180 serum samples (from PD patients from the ParkWest project) as described above and, although they were not quantified by Nanodrop, qPCR data from these samples were normalized by the reference small RNA, scaRNA17.

[0022] miRNA microarray and data analysis RNA isolated from serum samples of 24 patients was quantified and used for Affymetrix GeneChip® miRNA 4.0 Array by Yale Center for Genome Analysis (http: / / medicine.yale.edu / keck / ycga / index.aspx). Normalized CEL files obtained from Affymetrix Expression Console software were loaded into Partek Genomics Suite version 6.6 Copyright (C) 2012 (Partek, MO) for analysis. The "microRNA expression workflow" was used to detect differentially expressed miRNAs, and ANOVA was used to obtain a list of miRNAs that were significantly (p<0.05) expressed between fast and slow progressing PD cohorts. The detected miRNAs were used for further validation by qPCR.

[0023] Quantitative polymerase chain reaction cDNA for miRNA-specific qPCR was synthesized using qScript™ microRNA cDNA Synthesis kit (Quanta Biosciences, MD) following the manufacturer's protocol, and subsequent qPCR was performed using miRNA-specific forward primers (Table #) and PerfeCTa® Universal PCR primer (Quanta Biosciences, MD). scaRNA17 and U6 were used as reference small RNAs to normalize qPCR Cq values, whereas cel-miR-39-3p was used as a spike-in control. PerfeCTa® SYBR® GREEN SuperMix for IQ™ (Quanta Biosciences, MD) was used for all qPCR in the MyiQ™ Single color Real-Time PCR Detection System (Bio-Rad, CA). In MS Excel, R 2A standard curve for cel-miR-39-3p was analyzed with a PCR yield of 92.96% and a PCR efficiency of 0.97882. No template controls (NTCs) were included where appropriate.

[0024] Human miRNAs that were differentially expressed in serum samples of Parkinson's disease patients from the Norwegian ParkWest study were determined using miRNA microarrays. Presented below are miRNAs with greater than 1.2-fold differential expression.

[0025] 52 differentially expressed human pre-miRNAs and mature miRNAs with fold changes greater than 1.2 hsa-miR-6865-3p, hsa-miR-663a, hsa-miR-92b-3p, hsa-miR-455-3p, hsa-miR-937-5p, hsa-let-7b-3p, hsa-miR-6730-3p, hsa-miR-5010-3p, hsa-miR-1825, hsa-mir-4487, hsa-miR-4783-5p, hsa-miR-2117, hsa-mir-5090, hsa-mir-4484, hsa-miR-5094, hsa-mir-611, hsa-miR-4738-3p, hsa-miR-6894-5p, hsa-mir-8072, hsa-mir-762, hsa-let-7e, hsa-miR-6768-5p, hsa-mir-3917, hsa-mir-3673, hsa-mir-4431, hsa-miR-216a-3p, hsa-miR-635, hsa-miR-490-3p, hsa-mir-601, hsa-miR-636, hsa-miR-466, hsa-miR-1271-5p, hsa-miR-548u, hsa-miR-3606-5p, hsa-miR-510-5p, hsa-miR-4306, hsa-mir-4753, hsa-mir-6128, hsa-mir-4251, hsa-miR-1306-5p, hsa-miR-8052, hsa-mir-4310, hsa-mir-3128, hsa-miR-628-5p, hsa-miR-3660, hsa-miR-3156-3p, hsa-miR-548aj-3p, hsa-miR-4791, hsa-mir-532, hsa-miR-202-5p, hsa-miR-3613-3p, hsa-miR-8075

[0026] 36 differentially expressed mature miRNAs with fold changes greater than 1.2 hsa-miR-6865-3p, hsa-miR-663a, hsa-miR-92b-3p, hsa-miR-455-3p, hsa-miR-937-5p, hsa-let-7b-3p, hsa-miR-6730-3p, hsa-miR-5010-3p, hsa-miR-1825, hsa-miR-4783-5p, hsa-miR-2117, hsa-miR-5094, hsa-miR-4738-3p, hsa-miR-6894-5p, hsa-let-7e, hsa-miR-6768-5p, hsa-miR-216a-3p, hsa-miR-635, hsa-miR-490-3p, hsa-miR-636, hsa-miR-466, hsa-miR-1271-5p, hsa-miR-548u, hsa-miR-3606-5p, hsa-miR-510-5p, hsa-miR-4306, hsa-miR-1306-5p, hsa-miR-8052, hsa-miR-628-5p, hsa-miR-3660, hsa-miR-3156-3p, hsa-miR-548aj-3p, hsa-miR-4791, hsa-miR-202-5p, hsa-miR-3613-3p, hsa-miR-8075

[0027] 16 differentially expressed precursor miRNAs with fold changes greater than 1.2 hsa-mir-4487, hsa-mir-5090, hsa-mir-4484, hsa-mir-611, hsa-mir-8072, hsa-mir-762, hsa-mir-3917, hsa-mir-3673, hsa-mir-4431, hsa-mir-601, hsa-mir-4753, hsa-mir-6128, hsa-mir-4251, hsa-mir-4310, hsa-mir-3128, hsa-mir-532. These differentially expressed miRNA sequences are illustrated in Table 1 below, along with the reference / housekeeping small RNAs cel-miR-39-3p, U6, and ScaRNA17 used as controls. cel-miR-39-3p is a spike-in control to demonstrate the stability of the RNA samples. U6 and ScaRNA17 are used as internal controls to normalize the measurements of the remaining miRNAs or candidate miRNAs.

[0028] Example 1 [Table 1A]

[0029] [Table 1B]

[0030] [Table 1C]

[0031] Example 2 Validation of human mature miRNAs by qPCR in a cohort of eight fast and eight slow progressor samples The mean fold changes of hsa-let7b-3p, hsa-miR-3613-3p, hsa-miR-6865-3p, hsa-pre-miR-532, hsa-miR-663a, and PrognomiR between fast- and slow-progressing PD patients are shown in Table 2 below and illustrated in Figure 1.

[0032] [Table 2]

[0033] Example 3 Measuring the levels of a combination of two or more miRNAs in serum from a patient can potentially help clearly distinguish between fast and slow PD patients. A serum sample is obtained from blood drawn from a patient suspected of PD. The serum is used to isolate and enrich total microRNAs. The RNA is then tested to measure the levels of any two or more of the 52 miRNAs described in Example 1 or any one of the five miRNAs described in Example 2 using qPCR. A detectable level of one or more of the 52 miRNAs with a fold change of more than 1.2 or any one or more of these five miRNAs determines the progression rate of the PD patient. If desired, other sample body fluids may be utilized, including plasma, venous or arterial blood, or CSF samples obtained by lumbar puncture. Such plasma, blood, or CSF samples are processed as discussed above for serum to provide samples for processing and evaluation, for example, outside the human or animal body. It will be appreciated that measuring more than two miRNAs in the combination or set of combinations used in the test matrix may desirably increase the accuracy of predicting PD progression. After diagnosis, the results are communicated to the patient.

[0034] As discussed and exemplified above, detecting the miRNA with a fold change of more than 1.2 may be used to distinguish potentially fast-growing PD from potentially slow-growing PD in affected patients. However, as will be readily understood by those skilled in the art, other thresholds including fold changes of more than 1.0, 1.1, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 may be appropriately used as desired, with possible attendant variations in accuracy. In addition, it will be equally understood that these various fold change thresholds may be more appropriately used with certain miRNAs without affecting accuracy. Example 4

[0035] Since the combination of miRNAs may be used to predict prognosis, it would be prudent to test all candidates to eliminate any cohort-based variation. It is understood that any detectable amount of the relevant miRNA would be indicative of PD pathology. However, those skilled in the art recognize that it may be clinically useful to use values ​​from slow-progressing (8) samples versus fast-progressing (8) samples to set an artificial threshold for determining the rate of disease progression. In addition to enriching patient cohorts in clinical trials, differential miRNA levels may be used to develop prognostic biomarker kits that can be used by clinicians to infer disease prognosis. In this study, the presence and quantification of miRNAs from serum was determined by qRT-PCR to amplify and quantify the RNA in question. As an alternative, other suitable techniques known to those skilled in the art may be utilized herein, including the use of labeled antisense sequences and labeled antibodies. Typically, a suitable antibody that indicates a binding reaction between two molecules that exceeds background molecular association by 10 to 100 times under the measurement conditions is preferentially selected. Thus, under designated immunoassay conditions, a given antibody will bind to a particular miRNA sequence, thereby identifying its presence. Specific binding to an antibody under such conditions requires that the antibody be selected for its specificity for a particular miRNA. For example, antibodies raised against a particular miRNA can be selected by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies having specific immunoreactivity against a particular miRNA, including solid-phase ELISA immunoassays (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988), for a review of immunoassay formats and conditions that may be used to determine specific immunoreactivity).Methods for determining whether two molecules specifically interact are published therein, and methods for determining binding affinity and specificity are known in the art (see, e.g., Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Friefelder, "Physical Biochemistry: Applications to biochemistry and molecular biology" (WH Freeman and Co., 1976)). As used herein, the term "antibody" includes naturally occurring as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies, and humanized antibodies, as well as antigen-binding fragments thereof (e.g., Fab', F(ab')2, Fab, Fv, and rIgG). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL). See also, e.g., Kuby, J., Immunology, 3rd ed., WH Freeman & Co., New York (1998). Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, for example, as described in Huse et al., Science, Vol. 246 (1989) pp. 1275-81.These and other methods of generating, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those of skill in the art (Winter and Harris, Immunol. Today, Vol. 14 (1993) pp. 243-46; Ward et al., Nature, Vol. 341 (1989) pp. 544-46; Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995)). Methods for producing both monoclonal and polyclonal antibodies from identified RNA sequences are known in the art.

[0036] Example 5 In terms of symptoms as well as pathological markers, many neurodegenerative diseases are closely related to each other. A blood prognostic marker for one neurodegenerative disease may be useful for the diagnosis / prognosis of other diseases. Methods for diagnosing / prognosing other neurodegenerative diseases such as dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), multiple system atrophy (MSA), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) can also be developed using similar miRNA measurements of the above candidates. Disease-specific kits can be developed similarly to those described in

[0025] using various combinations of miRNAs described in

[0019] .

[0037] Example 6 The miRNAs detected in one or more combinations can regulate some proteins in cells. New protein targets for PD can be discovered using these microRNAs and their combinations. The involvement of these proteins in the pathogenesis of PD can be further established and targeted for treatment.

[0038] Example 7 Small nucleic acid molecules derived from the miRNAs described in

[0019] may be designed to therapeutically intervene by specifically targeting genes in the PD brain to achieve complete or partial improvement.

Claims

1. 1. A method for determining the expression levels of at least two miRNAs, comprising: determining the expression level of the miRNA of SEQ ID NO: 12 and at least one miRNA selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 45, and optionally further determining the expression level of at least one miRNA selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 46, 47, 48, 49, 50, 51, and 52 in a sample provided from a human patient suffering from Parkinson's disease. The method includes:

2. The method of claim 1, wherein a sample is prepared for determining said miRNA outside the human body and the results of said determination are communicated to the patient.

3. The method of claim 1 or 2, wherein the sample is serum, plasma, or whole blood.

4. The method of claim 1 , wherein the expression level of the miRNA is determined using qRT-PCR.

5. 4. The method of claim 1, wherein the expression level of the miRNA is determined using a labeled antisense nucleotide sequence.

6. The method of claim 1 , wherein the expression level of the miRNA is determined using a labeled antibody.

7. The method of claim 6, wherein the labeled antibody is monoclonal.

8. The method of any one of claims 1 to 3, wherein the expression levels of the miRNAs are determined using microarray profiling.

9. 4. The method of claim 1, wherein the expression level of the miRNA is determined using high-throughput NGS sequencing.

10. The method of claim 1, wherein the expression level of the miRNA according to SEQ ID NO: 33 is also determined.