WO2019229489A1 - Utilisation de mir-146a-5p et de mir-186 en tant que biomarqueurs de l'arthrose - Google Patents

Utilisation de mir-146a-5p et de mir-186 en tant que biomarqueurs de l'arthrose Download PDF

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WO2019229489A1
WO2019229489A1 PCT/IB2018/000676 IB2018000676W WO2019229489A1 WO 2019229489 A1 WO2019229489 A1 WO 2019229489A1 IB 2018000676 W IB2018000676 W IB 2018000676W WO 2019229489 A1 WO2019229489 A1 WO 2019229489A1
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mir
prevalent
mirna
osteoarthritis
incident
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PCT/IB2018/000676
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Roland CHAPURLAT
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université Claude Bernard - Lyon 1
Hospices Civils De Lyon
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Priority to PCT/IB2018/000676 priority Critical patent/WO2019229489A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to use of miR-l46a-5p and miR-l86 as biomarkers of osteoarthritis.
  • OA Osteoarthritis
  • OA Osteoarthritis
  • 3 is the most frequent chronic musculoskeletal disease affecting approximately 40% of adults aged 70 years and over. 1 Knee OA is usually detected only in late stage by radiography and poorly sensitive clinical symptoms. Sensitive and specific blood biomarkers to detect the initial stages of osteoarthritis (OA) and to predict the future development of the disease are not available in clinical routine. 2 Consequently, there is a considerable interest in the identification of new markers.
  • MicroRNAs miRs
  • miRs are small non coding RNAs of approximately 22 nucleotides in length that can silence gene expression by binding to target messenger RNA repressing the translation. Expression of miRs has been well documented in OA cartilage but data on circulating miRs is limited. 3
  • the present invention relates to use of miR-l46a-5p and miR-l86 as biomarkers of osteoarthritis.
  • the present invention is defined by the clOAs.
  • the inventors investigated the associations of prevalent and incident knee osteoarthritis (OA) with the expression levels of serum miRs in subjects with and without OA. With a next generation sequencing approach, they compared the miRom expression of 10 women with knee OA and 10 age-matched healthy subjects. By real-time PCR, they analyzed the expression levels of 19 miRs at baseline selecting 43 women with prevalent OA (Kellgren Lawrence score of 2/3), 23 women with incident OA over a 4 years follow-up and 67 healthy subjects without prevalent or incident OA matched for age and body mass index.
  • OA knee osteoarthritis
  • the inventors showed for the first time that miR-l46a-5p and miR-l86 are significantly associated with prevalent and incident OA respectively.
  • the first object of the present invention relates to a method of determining whether a subject has or is at risk of having osteoarthritis comprising i) determining the expression level of miR-l46a-5p or miR-l86 in sample obtained from the subject, and ii) comparing the expression level determined at step i) with a predetermined reference value wherein detecting differential between the expression level determined at step i) and the predetermined reference value is indicative of whether a subject has or is at risk of having osteoarthritis.
  • OA osteoarthritis
  • cartilage loss and subchondral bone degeneration/damage in any kind of (bone-) joints in the body of a mammal.
  • it refers to arthritic changes of the larger and the smaller joints of the body, including the hands, wrists, feet, back, hip, and knee.
  • risk in the context of the present invention, relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion.
  • "Risk evaluation,” or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk.
  • the present invention may be used so as to discriminate those at risk from normal.
  • the method of diagnosing described herein is applied to a subject who presents symptoms of OA without having undergone the routine screening to rule out all possible causes for OA.
  • the methods described herein can be part of the routine set of tests performed on a subject who presents symptoms of OA.
  • the method of the present invention can be carried out in addition of other diagnostic tools.
  • the sample is a blood sample.
  • blood sample means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art.
  • the blood sample is a serum sample or a plasma sample.
  • miR As used herein, the term“miR” or‘miRNA” has its general meaning in the art and refers to the miRNA sequence publicly available from the data base http://microma.sanger.ac.uk/sequences/ under the miRBase Accession number. miR-l46a-5p or miR- 186 pertaining to the invention are thus known per se.
  • measuring the expression level of miR-l46a-5p or miR- 186 in the sample obtained from the subject can be performed by a variety of techniques.
  • the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid- binding resins following the manufacturer's instructions.
  • RNA isolation kit (Roche), Trizol (Invitrogen), Guanidinium thiocyanate-phenol-chloroform extraction, PureLinkTM miRNA isolation kit (Invitrogen), PureLink Micro-to- Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol-chloroform extraction, TCA/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, Pure YieldTM RNA Midiprep (Promega), PolyATtract System 1000 (Promega), Maxwell® 16 System (Promega), SV Total RNA Isolation (Promega), geneMAG-RNA / DNA kit (Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNATM Kit (Ambion), Poly
  • the expression level of miR-l46a-5p or miR- 186 in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of miRNA in a sample may be used.
  • miRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms).
  • Other exemplary techniques include ribonuclease protection assay (RP
  • R A is converted to DNA (cDNA) prior to analysis.
  • cDNA can be generated by reverse transcription of isolated miRNA using conventional techniques. miRNA reverse transcription kits are known and commercially available. Universal primers, or specific primers, including miRNA-specific stem-loop primers, are known and commercially available, for example, from Applied Biosystems.
  • miRNA is amplified prior to measurement.
  • the expression level of miRNA is measured during the amplification process.
  • the expression level of miRNA is not amplified prior to measurement.
  • amplification-based methods exist for detecting the expression level of miRNA nucleic acid sequences, including, but not limited to, PCR, RT-PCR, qPCR, and rolling circle amplification.
  • Other amplification-based techniques include, for example, ligase chain reaction (LCR), multiplex ligatable probe amplification, in vitro transcription (IVT), strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art.
  • LCR ligase chain reaction
  • IVTT in vitro transcription
  • SDA strand displacement amplification
  • TMA transcription-mediated amplification
  • NASBA nucleic acid sequence based amplification
  • RNA (Eberwine) amplification and other methods that are known to persons skilled in the art.
  • a typical PCR reaction includes multiple steps, or cycles, that selectively amplify target nucleic acid species: a denaturing step, in which a target nucleic acid is denatured; an annealing step, in which a set of PCR primers (i.e., forward and reverse primers) anneal to complementary DNA strands, and an elongation step, in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target sequence.
  • Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation.
  • a reverse transcription reaction (which produces a cDNA sequence having complementarity to a miRNA) may be performed prior to PCR amplification.
  • Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.
  • Kits for quantitative real time PCR of miRNA are known, and are commercially available. Examples of suitable kits include, but are not limited to, the TaqMan® miRNA Assay (Applied Biosystems) and the mirVanaTM qRT-PCR miRNA detection kit (Ambion).
  • the miRNA can be ligated to a single stranded oligonucleotide containing universal primer sequences, a polyadenylated sequence, or adaptor sequence prior to reverse transcriptase and amplified using a primer complementary to the universal primer sequence, poly(T) primer, or primer comprising a sequence that is complementary to the adaptor sequence.
  • custom qRT-PCR assays can be developed for determination of miRNA levels. Custom qRT-PCR assays to measure miRNAs in a sample can be developed using, for example, methods that involve an extended reverse transcription primer and locked nucleic acid modified PCR.
  • Custom miRNA assays can be tested by running the assay on a dilution series of chemically synthesized miRNA corresponding to the target sequence. This permits determination of the limit of detection and linear range of quantitation of each assay. Furthermore, when used as a standard curve, these data permit an estimate of the absolute abundance of miRNAs measured in the samples. Amplification curves may optionally be checked to verify that Ct values are assessed in the linear range of each amplification plot. Typically, the linear range spans several orders of magnitude. For each candidate miRNA assayed, a chemically synthesized version of the miRNA can be obtained and analyzed in a dilution series to determine the limit of sensitivity of the assay, and the linear range of quantitation.
  • Relative expression levels may be determined, for example, according to the 2(- DD C(T)) Method, as described by Fivak et ah, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-AA C(T)) Method. Methods (2001) Dec;25(4):402-8.
  • Rolling circle amplification is a DNA-polymerase driven reaction that can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (see, for example, Fizardi et al, Nat. Gen. (1998) l9(3):225-232; Gusev et al, Am. J. Pathol. (2001) 159(l):63-69; Nallur et al, Nucleic Acids Res. (2001) 29(23):El l8).
  • a complex pattern of strand displacement results in the generation of over 109 copies of each DNA molecule in 90 minutes or less.
  • Tandemly linked copies of a closed circle DNA molecule may be formed by using a single primer.
  • the process can also be performed using a matrix- associated DNA.
  • the template used for rolling circle amplification may be reverse transcribed. This method can be used as a highly sensitive indicator of miRNA sequence and expression level at very low miRNA concentrations (see, for example, Cheng et al., Angew Chem. Int. Ed. Engl. (2009) 48(l8):3268-72; Neubacher et al, Chembiochem. (2009) 10(8): 1289-91).
  • miRNA quantification may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe.
  • said method comprises a first step wherein the stem-loop primers are annealed to miRNA targets and extended in the presence of reverse transcriptase. Then miRNA-specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of miRNAs is estimated based on measured CT values. Many miRNA quantification assays are commercially available from Qiagen (S. A. Courtaboeuf, France), Exiqon (Vedbaek, Denmark) or Applied Biosystems (Foster City, USA).
  • Nucleic acids exhibiting sequence complementarity or homology to the miRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin). The probes and primers are“specific” to the miRNAs they hybridize to, i.e.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • miRNA may be detected using hybridization-based methods, including but not limited to hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, and in situ hybridization.
  • hybridization arrays e.g., microarrays
  • NanoString analysis e.g., NanoString analysis
  • Northern Blot analysis e.g., Northern Blot analysis
  • branched DNA (bDNA) signal amplification e.g., branched DNA (bDNA) signal amplification
  • in situ hybridization e.g., in situ hybridization.
  • Microarrays can be used to measure the expression levels of large numbers of miRNAs simultaneously.
  • Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre- made masks, photolithography using dynamic micromirror devices, inkjet printing, or electrochemistry on micro electrode arrays.
  • microfluidic TaqMan Low-Density Arrays which are based on an array of micro fluidic qRT-PCR reactions, as well as related micro fluidic qRT-PCR based methods.
  • oligonucleotides e.g., 200+ 5'- amino- modified-C6 oligos
  • human sense miRNA sequences are spotted on three- dimensional CodeLink slides (GE Health/ Amersham Biosciences) at a final concentration of about 20 mM and processed according to manufacturer's recommendations.
  • First strand cDNA synthesized from 20 pg TRIzol-purified total RNA is labeled with biotinylated ddUTP using the Enzo BioArray end labeling kit (Enzo Life Sciences Inc.).
  • Hybridization, staining, and washing can be performed according to a modified Affymetrix Antisense genome array protocol.
  • Axon B-4000 scanner and Gene-Pix Pro 4.0 software or other suitable software can be used to scan images. Non-positive spots after background subtraction, and outliers detected by the ESD procedure, are removed. The resulting signal intensity values are normalized to per-chip median values and then used to obtain geometric means and standard errors for each miRNA. Each miRNA signal can be transformed to log base 2, and a one-sample t test can be conducted. Independent hybridizations for each sample can be performed on chips with each miRNA spotted multiple times to increase the robustness of the data.
  • Microarrays can be used for the expression profiling of miRNAs.
  • RNA can be extracted from the sample and, optionally, the miRNAs are size- selected from total RNA.
  • Oligonucleotide linkers can be attached to the 5' and 3' ends of the miRNAs and the resulting ligation products are used as templates for an RT-PCR reaction.
  • the sense strand PCR primer can have a fluorophore attached to its 5' end, thereby labeling the sense strand of the PCR product.
  • the PCR product is denatured and then hybridized to the microarray.
  • a PCR product referred to as the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed.
  • the spot will then fluoresce when excited using a microarray laser scanner.
  • the fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA.
  • Total RNA containing the miRNA extracted from the sample can also be used directly without size-selection of the miRNAs.
  • the RNA can be 3' end labeled using T4 RNA ligase and a fluorophore-labeled short RNA linker.
  • Fluorophore-labeled miRNAs complementary to the corresponding miRNA capture probe sequences on the array hybridize, via base pairing, to the spot at which the capture probes are affixed.
  • the fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA.
  • microarrays can be employed including, but not limited to, spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays or spotted long oligonucleotide arrays.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • A“detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook- A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphtho fluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20l320l6, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos.
  • quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, Calif.).
  • thermocyclers typically involve a format of glass capillaries, plastics tubes, 96-well plates or 384-well plates.
  • the thermocylcer also involves software analysis.
  • miRNAs can also be detected without amplification using the nCounter Analysis System (NanoString Technologies, Seattle, WA).
  • This technology employs two nucleic acid-based probes that hybridize in solution (e.g., a reporter probe and a capture probe). After hybridization, excess probes are removed, and probe/target complexes are analyzed in accordance with the manufacturer's protocol.
  • nCounter miRNA assay kits are available from NanoString Technologies, which are capable of distinguishing between highly similar miRNAs with great specificity.
  • the basis of the nCounter® Analysis system is the unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO 08/124847, U.S. Patent No.
  • the code is composed of an ordered series of colored fluorescent spots which create a unique barcode for each target to be assayed.
  • a pair of probes is designed for each oligonucleotide target, a biotinylated capture probe and a reporter probe carrying the fluorescent barcode. This system is also referred to, herein, as the nanoreporter code system. Specific reporter and capture probes are synthesized for each target.
  • the reporter probe can comprise at a least a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; at least a second label attachment region, which is non-over- lapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and a first target- specific sequence.
  • each sequence specific reporter probe comprises a target specific sequence capable of hybridizing to no more than one gene and optionally comprises at least three, or at least four label attachment regions, said attachment regions comprising one or more label monomers that emit light, constituting at least a third signal, or at least a fourth signal, respectively.
  • the capture probe can comprise a second target-specific sequence; and a first affinity tag.
  • the capture probe can also comprise one or more label attachment regions.
  • the first target-specific sequence of the reporter probe and the second target- specific sequence of the capture probe hybridize to different regions of the same gene to be detected.
  • Reporter and capture probes are all pooled into a single hybridization mixture, the "probe library".
  • the relative abundance of each target is measured in a single multiplexed hybridization reaction.
  • the method comprises contacting the sample with a probe library, such that the presence of the target in the sample creates a probe pair - target complex.
  • the complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution.
  • the tripartite hybridized complexes (probe pairs and target) are purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to universal sequences present on the capture and reporter probes.
  • This dual purification process allows the hybridization reaction to be driven to completion with a large excess of target- specific probes, as they are ultimately removed, and, thus, do not interfere with binding and imaging of the sample. All post hybridization steps are handled robotically on a custom liquid handling robot (Prep Station, NanoString Technologies). Purified reactions are typically deposited by the Prep Station into individual flow cells of a sample cartridge, bound to a streptavidin-coated surface via the capture probe, electrophoresed to elongate the reporter probes, and immobilized.
  • the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technologies).
  • the expression level of a target is measured by imaging each sample and counting the number of times the code for that target is detected. For each sample, typically 600 fields-of-view (FOV) are imaged (1376 X 1024 pixels) representing approximately 10 mm2 of the binding surface.
  • Typical imaging density is 100- 1200 counted reporters per field of view depending on the degree of multiplexing, the amount of sample input, and overall target abundance.
  • Data is output in simple spreadsheet format listing the number of counts per target, per sample. This system can be used along with nanoreporters. Additional disclosure regarding nanoreporters can be found in International Publication No.
  • nucleic acid probes and nanoreporters can include the rationally designed (e.g. synthetic sequences) described in International Publication No. WO 2010/019826 and US Patent Publication No. 2010/0047924, incorporated herein by reference in its entirety.
  • RNA endonucleases RNases
  • MS/MS tandem MS
  • the first approach developed utilized the on-line chromatographic separation of endonuclease digests by reversed phase HPLC coupled directly to ESTMS. The presence of post-transcriptional modifications can be revealed by mass shifts from those expected based upon the RNA sequence.
  • MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry
  • MALDI-MS has also been used as an analytical approach for obtaining information about post-transcriptionally modified nucleosides.
  • MALDI- based approaches can be differentiated from EST-based approaches by the separation step.
  • the mass spectrometer is used to separate the miRNA.
  • a system of capillary LC coupled with nanoESI-MS can be employed, by using a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) or a tandem-quadrupole time-of- flight mass spectrometer (QSTAR® XL, Applied Biosystems) equipped with a custom-made nanospray ion source, a Nanovolume Valve (Valeo Instruments), and a splitless nano HPLC system (DiNa, KYA Technologies). Analyte/TEAA is loaded onto a nano-LC trap column, desalted, and then concentrated.
  • LTQ Orbitrap XL linear ion trap-orbitrap hybrid mass spectrometer
  • QSTAR® XL tandem-quadrupole time-of- flight mass spectrometer
  • Analyte/TEAA is loaded onto a nano-LC trap column, desalted, and then concentrated.
  • Intact miRNAs are eluted from the trap column and directly injected into a Cl 8 capillary column, and chromatographed by RP-HPLC using a gradient of solvents of increasing polarity.
  • the chromatographic eluent is sprayed from a sprayer tip attached to the capillary column, using an ionization voltage that allows ions to be scanned in the negative polarity mode.
  • Additional methods for miRNA detection and measurement include, for example, strand invasion assay (Third Wave Technologies, Inc.), surface plasmon resonance (SPR), cDNA, MTDNA (metallic DNA; Advance Technologies, Saskatoon, SK), and single-molecule methods such as the one developed by US Genomics.
  • miRNAs can be detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle- amplified SPR imaging (SPRI).
  • SPRI nanoparticle- amplified SPR imaging
  • the surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI.
  • This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attamole levels.
  • miRNAs can also be detected using branched DNA (bDNA) signal amplification (see, for example, Urdea, Nature Biotechnology (1994), 12:926-928).
  • bDNA branched DNA
  • miRNA assays based on bDNA signal amplification are commercially available.
  • One such assay is the QuantiGene® 2.0 miRNA Assay (Affymetrix, Santa Clara, CA).
  • Northern Blot and in situ hybridization may also be used to detect miRNAs. Suitable methods for performing Northern Blot and in situ hybridization are known in the art. Advanced sequencing methods can likewise be used as available.
  • miRNAs can be detected using Illumina ® Next Generation Sequencing (e.g. Sequencing-By-Synthesis or TruSeq methods, using, for example, the HiSeq, HiScan, GenomeAnalyzer, or MiSeq systems (Illumina, Inc., San Diego, CA)).
  • miRNAs can also be detected using Ion Torrent Sequencing (Ion Torrent Systems, Inc., Gulliford, CT), or other suitable methods of semiconductor sequencing.
  • the expression level of miR-l46a-5p or miR-l86 may be expressed as absolute expression levels or normalized expression levels.
  • expression levels are normalized by correcting the absolute expression level of miRNAs by comparing its expression to the expression of a mRNA that is not a relevant marker for determining whether a subject suffering from acute severe colitis (ASC) will be a responder or a non-responder to a corticosteroid, infliximab and cyclosporine, e.g., a housekeeping mRNA that is constitutively expressed.
  • ASC acute severe colitis
  • This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, or between samples from different sources.
  • expression levels are normalized by correcting the absolute expression level of miRNAs by comparing its expression to the expression of a reference mRNA.
  • the predetermined reference value is a threshold value or a cut off value.
  • a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of expression level of the gene in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate.
  • the predetermined reference value is the level of the miRNA determined in a population of healthy individuals.
  • subject the has or is at risk of having OA when the expression level of the miRNA is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 fold higher than the expression level determined in a population of healthy individuals.
  • treatment options may be prescribed. Typical treatment position include weight management, physical activity, medications, and joint replacement surgery. Medicines for osteoarthritis are available as pills, syrups, creams or lotions, or they are injected into a joint.
  • Corticosteroids include, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone).
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • corticosteroids include, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomet
  • FIGURES are a diagrammatic representation of FIGURES.
  • Prevalent OA percentage of women with osteoarthritis under and upper the median of miR-l46a-5p respectively (number of women with prevalent OA-total number of women).
  • Incident OA percentage of women with osteoarthritis in the first three quartiles vs the upper quartile (number of women with incident OA-total number of women).
  • Cl Confidence Interval.
  • RNA isolation was performed from 400 m ⁇ of serum followed by the uRNA sequencing (Illumina platform). Measurements were expressed as Tags per million (TPM).
  • cel-miR-39-3p was added as exogenous control before total RNA extraction from 200 m ⁇ of serum using miRCURY RNA isolation kit for biofluids according to the manufacturer’s protocol (EXIQON, Danemark).
  • MiRs were reverse-transcribed with an Advanced miRNA assays kit according to the manufacturer’s protocol (Applied BioSystems, CA, USA).
  • Amplified cDNAs (15m1) were mixed with 75 m ⁇ of TaqMan Fast Advanced Mastermix (Ref. 4444963, Applied BioSystems) and water (60m1) and 100 m ⁇ of this mixture were added to the reservoir.
  • the TaqMan array microRNA cards were designed for the quantification in duplicate of 19 miRs by real-time PCR reaction on a QuantStudio 7 flex (Applied Biosystems) according to the manufacturer’s protocol.
  • a miRs with a p value ⁇ 0.05 and a false discovery rate of 5% [Benjamini-Hochberg False Discovery Rate (FDR) correction for NGS approach] was considered as differentially expressed.
  • Wilcoxon tests were used to compare miR levels between women with and without OA.
  • ORs odds ratios
  • CIs 95% confidence intervals
  • the second step of our study combines the validation of our NGS results and the first attempt to replicate previously published results, a process that is lacking in the miR research field.
  • Our study can be compared to that of Borgonio et al, 2014. 7 Only two of the 10 miRs in common showed significant overexpression (l46a-5p and 186) emphasizing the challenges faced with patient heterogeneity. The reasons for this discrepancy could be the ethnic origin, Mexican vs European hencing the differences in exposure to environmental factors; the sex of the participants, men and women vs women only; the biological fluid used-plasma vs serum or the mean age of participants-55 vs 68 years.
  • miR-l46a-5p expression was significantly increased in prevalent OA independently of age and BMI. miR- 146a was found as expressed differently in joint tissues and in the circulation. 7 10 12 However, the interpretation of this increase is difficult because of the dual role of miR-l46a-5p in the pathophysiology of osteoarthritis. It may promote OA by increasing chondrocyte apoptosis and decreasing TGF-b responsiveness and cartilage anabolism. 13 14 . In contrast, miR-l46a-5p reduces inflammation and increases autophagy. 15 16 Taken together, these results suggest that miR-l46a-5p could be a new biological marker for knee OA.
  • miR- 186 In silico analysis has suggested that potential pathways regulated by miR- 186 could be signaling by PDGF, developmental biology, membrane trafficking and collagen formation. 7 However, miR- 186 has been essentially studied in oncology. Potential targets that may be related to OA included GFUT1 (glycolysis regulation) 18 and TWISTl (osteoblast differentiation). 19 Collectively, our results show that miR-l46a-5p is increased in women suffering from mild to moderate OA compared to healthy women. Importantly, miR-l86 is also increased in those women who will develop radiographic knee OA over the next 4 years, therefore with the potential to detect preclinical knee OA.
  • GFUT1 glycolysis regulation
  • TWISTl osteoblast differentiation
  • IQR Inter-Quartile Range

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Abstract

L'arthrose (OA) est la maladie musculo-squelettique chronique la plus fréquente affectant environ 40 % des adultes âgés de 70 ans et plus. Les inventeurs ont étudié les associations d'une arthrose (OA) du genou prévalente et incidente avec les taux d'expression de miR sériques chez des sujets avec et sans OA. Au moyen d'une réaction en chaîne de la polymérase (PCR) en temps réel, ils ont analysé les taux d'expression de 19 miR au point de départ en sélectionnant 43 femmes ayant une OA prévalente (score de Kellgren-Lawrence de 2/3), 23 femmes ayant une OA incidente pendant un suivi de 4 ans, et 67 sujets sains sans OA prévalente ou incidente assortis en termes d'âge et d'indice de masse corporelle. miR-146a-5p a été significativement accru dans le groupe de OA prévalente par comparaison avec les témoins (RQ : quantification relative ; médiane [intervalle interquartile] : 1,12 [0,73 ; 1,46] par rapport à 0,85 [0,62 ; 1,03], p = 0,015). La probabilité d'une OA prévalente a été significativement accrue (rapport des cotes [intervalle de confiance (CI) de 95 %] : 1,83 [1,21-2,77], p = 0,004) pour chaque augmentation quartile de miR-146a-5p sérique. Il y a eu une association significative entre les taux de miR-186 de ligne de base et le risque d'OA incidente (Q4 par rapport à Q1-3 ; rapport des cotes [CI de 95 %] : 6,13 [1,14-32,9], p = 0,034). En conclusion, les inventeurs ont montré pour la première fois que miR-146a-5p et miR-186 sont associés de manière significative à une OA prévalente et incidente, respectivement.
PCT/IB2018/000676 2018-05-31 2018-05-31 Utilisation de mir-146a-5p et de mir-186 en tant que biomarqueurs de l'arthrose WO2019229489A1 (fr)

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CN111621562A (zh) * 2020-06-17 2020-09-04 湖州市第一人民医院 非编码rna在制备骨关节炎早期诊断检测产品中的应用
CN111621562B (zh) * 2020-06-17 2023-09-22 湖州市第一人民医院 非编码rna在制备骨关节炎早期诊断检测产品中的应用

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