Diagnosis and treatment of psoriasis arthritis and corresponding kit
阅读说明：本技术 干癣性关节炎的诊断及治疗及其相应的试剂盒 (Diagnosis and treatment of psoriasis arthritis and corresponding kit ) 是由 林尚宏 李志宏 萧长春 于 2019-07-11 设计创作，主要内容包括：用于在需要诊断或预测发展干癣性关节炎风险的受试者中诊断或预测发展干癣性关节炎之风险的方法及试剂盒,其包含在受试者的样品中测量一种或多种本文所公开的miRNA的表达量,并与在无干癣性关节炎样品的测试样品中的至少一种miRNA之表达量比较。亦提供藉由降低至少一种本文所公开的miRNA的表达量来治疗干癣性关节炎的方法。(Methods and kits for diagnosing or predicting the risk of developing psoriatic arthritis in a subject in need of diagnosis or prediction of the risk of developing psoriatic arthritis comprising measuring the expression level of one or more mirnas disclosed herein in a sample of the subject and comparing the expression level of at least one miRNA in a test sample without a psoriatic arthritis sample. Also provided are methods of treating rheumatoid arthritis by reducing the expression level of at least one miRNA disclosed herein.)
1. Use of a reagent that measures the expression level of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941 in the preparation of a kit for detecting or predicting the risk of developing psoriatic arthritis in a subject, wherein said detecting or predicting comprises:
a step of measuring the expression level of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941 in a test sample of the subject,
a higher expression level of at least one miRNA in the test sample relative to the expression level of at least one corresponding miRNA in a sample without psoriasis indicates that the subject has psoriasis or is at risk of developing psoriasis.
2. The use of claim 1, wherein the expression level of the miRNA is detected by real-time quantitative fluorescent PCR (real-time PCR).
3. The use of claim 1, wherein the expression level of the miRNA is determined by the nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO: 2 at least 90% identical oligonucleotide probes.
4. The use of claim 1, wherein the test sample is CD14+ monocytes (monocytes).
5. The use of claim 1, wherein the test sample is an osteoclast (osteoplast).
6. The use of claim 5, wherein the osteoclast is derived from a CD14+ monocyte.
7. A kit for detecting or predicting the risk of developing psoriatic arthritis in a subject, comprising:
at least one microbead for extracting CD14+ monocytes from the subject's sample; and
a reagent for sequencing or measuring the expression level of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941.
8. The kit of claim 7, further comprising a culture medium comprising M-CSF, RANKL, and TNF- α.
9. And SEQ ID NO: 1 miR-146a-5p inhibitor that is at least 90% identical, or a miR-146a-5p inhibitor that is at least 90% identical to SEQ ID NO: use of a miR-941 inhibitor that is at least 90% identical in the manufacture of a medicament for treating psoriatic arthritis in a subject.
10. The use of claim 9, wherein the miR-146a-5p inhibitor is SEQ ID NO: 5.
11. the use of claim 9, wherein the inhibitor of miR-941 is SEQ ID NO: 4.
12. use of an agent that measures the amount of expression of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941 in a first sample taken from the subject in the manufacture of a kit for determining the effect of treating rheumatoid arthritis in a subject, wherein determining the effect of treating rheumatoid arthritis in a subject comprises the steps of:
(a) measuring the expression level of at least one miRNA of the group consisting of miR-146a-5p and miR-941 in a first sample obtained from the subject prior to providing at least a portion of the treatment to the subject; and
(b) measuring the amount of expression of the at least one corresponding miRNA of step (a) in a second sample obtained from said subject after providing at least a portion of said treatment to said subject, wherein a decreased amount of expression of the at least one miRNA relative to the corresponding miRNA in said first sample in the second sample is indicative that the treatment is effective in treating rheumatoid arthritis.
13. The use of claim 12, wherein the first and second samples are CD14+ monocytes.
[ technical field ] A method for producing a semiconductor device
The present invention claims the benefits of U.S. application No. 62/702,551, filed 24/7/2018, which is incorporated herein by reference in its entirety.
[ background of the invention ]
Psoriatic arthritis (PsA) is a chronic inflammatory disease involving progressive joint disease associated with psoriasis. Approximately 30% of patients with psoriasis are estimated to have psoriatic arthritis. Generally, the manifestations of the skin (manifestations) are about 10 years before the onset of PsA. Haroon et al, (Annals of the rhematic diseases 2015; 74 (6): 1045-50) reported poor radiographic and poor functional outcomes in patients with PsA due to a delay in diagnosis of psoriatic arthritis for more than 6 months. Early diagnosis and timely intervention are critical to avoid permanent joint deformity and destruction. Currently, diagnosis of PsA is mainly based on clinical phenotype. There is no standard and reliable assessment for early detection of PsA to date, resulting in many patients with psoriasis who have not been diagnosed with psoriatic arthritis with poor therapeutic outcome (Veale DJ et al, Lancet 2018; 391 (10136): 2273-84).
MicroRNAs (MicroRNAs, miRNAs) are evolutionarily conserved non-coding RNA molecules, typically 21-25 nucleotides in length, that inhibit protein translation or promote mRNA degradation by binding to The 3 '-untranslated region (3' -UTRs) of mRNA (see Griffiths-Jones S (2004). most of the miRNAs are now being studied at a basic level, and there is a need to further establish their use in psoriatic arthritis.
Thus, there is a need for a non-invasive and convenient method to diagnose and predict the risk of and/or treatment of psoriatic arthritis, and the present invention meets these and other needs.
[ summary of the invention ]
In one embodiment, the present invention provides a method for detecting or predicting the risk of developing psoriatic arthritis in a subject, comprising the step of measuring the expression level (expressonevel) of at least one miRNA selected from the group consisting of miR-146a-5p (SEQ ID NO: 1) and miR-941(SEQ ID NO: 2) in a test sample of the subject, wherein the miRNA is expressed in the test sample via extracted (extracted) CD14+ monocytes (monocyte) or osteoclasts (osteoplast), and a higher expression level of the at least one miRNA in the test sample relative to the expression level of the at least one corresponding miRNA in the psoriatic arthritis-free sample is indicative of the subject having or being at risk of developing psoriatic arthritis.
In another embodiment, the present invention provides a method for detecting and treating psoriatic arthritis in a subject, comprising the steps of: (a) measuring in a test sample of the subject the expression level of at least one miRNA selected from the group consisting of miR-146a-5p (SEQ ID NO: 1) and miR-941(SEQ ID NO: 2), wherein miRNA is extracted CD14+ monocyte or osteoclast expression in the test sample, and a higher expression level of the at least one miRNA in the test sample relative to the expression level of the at least one corresponding miRNA in the psoriatic arthritis-free sample indicates that the subject has psoriatic arthritis; and (b) administering a therapeutic agent to the subject to treat psoriatic arthritis.
The present invention also provides a kit for detecting or predicting the risk of developing psoriatic arthritis in a subject, comprising: (a) at least one microbead (microbead) for extracting CD14+ monocytes from a sample of a subject; and (b) a reagent for sequencing or measuring the expression level of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941.
In an exemplary embodiment, the kit further comprises a culture medium comprising M-CSF, RANKL, and TNF- α.
Also provided is a method for treating psoriatic arthritis, comprising: administering to a subject in need thereof an effective amount of a polypeptide at least as set forth in SEQ ID NO: 1, or administering an effective amount of a miR-146a-5p inhibitor that is 90% identical to at least one of SEQ ID NO: 2 has a step of 90% identity of the miR-941 inhibitor.
Also disclosed are methods of treating the effects of a treatment in a subject with psoriatic arthritis, the method comprising: (a) measuring the expression level of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941 of the extracted CD14+ monocytes in a first sample from the subject prior to providing at least a portion of treatment to the subject; and (b) measuring the expression level of the at least one corresponding miRNA of step (a) of extracting CD14+ monocytes in a second sample obtained from the subject after providing at least a portion of the treatment to the subject, wherein a decreased expression level of the at least one miRNA from extracted CD14+ monocytes in the second sample relative to the expression level of the corresponding miRNA from extracted CD14+ monocytes in the first sample represents an indication of effective treatment of rheumatoid arthritis.
[ brief description of drawings ]
Illustrative embodiments of the invention are described in detail below with reference to the following figures:
FIG. 1A is a combination of histograms showing the expression levels of miR-146a-5p and miR-941 in extracted CD14+ monocytes of a normal control group (normal control, NC) and patients with psoriatic arthritis (PsA). FIG. 1B is a combination of histograms showing Δ Ct (delta Ct) values for miR-146a-5p and miR-941. FIGS. 1C and 1D are histograms showing the expression of miR-146a-5p and miR-941 in extracted CD14+ monocytes from a normal control group (CD14+ NC) and a patient with rheumatoid arthritis (PsACD14+) and the expression in peripheral monocytes (peripheral mononarcercercells) from a normal control group (CD14-NC) and a patient with rheumatoid arthritis (PsACD 14-).
Panels A and B of FIG. 2 show the number of tender joints and swollen joints (swollen joints) in 11 patients with PsA before and after 6 months of treatment. Panels C and D of FIG. 2 show expression of miR-146a-5p and miR-941 in extracted CD14+ monocytes in Normal Control (NC), 11 patients with PsA before treatment (PsA (0)), and 6 months after treatment (PsA (28 weeks)).
FIGS. 3A and 3B are bar graphs showing the expression of miR-146a-5p and miR-941 in CD14+ monocytes derived from osteoclasts in the normal control group (NC), before treatment of 11 patients with PsA (0)), and after 6 months of treatment (PsA (28 weeks))
FIGS. 4A to 4D are a combination of microscopic images showing the effect of miR-free transfection (transfection) (FIG. 4A), transfection with a negative control miRNA (FIG. 4B), miR-941 inhibitor (FIG. 4C), or miR-146a-5p inhibitor (FIG. 4D) on the number of CD14+ monocytes derived from osteoclasts. FIG. 4E is a bar graph showing the effect of miR-free transfection (PsA), transfection with a negative control miRNA, miR-941 inhibitor, or miR-146a-5p inhibitor on the number of CD14+ monocytes derived from osteoclasts.
Panels 5A and 5B show expression of miR-146a-5p and miR-941 in extracted CD14+ monocytes from Normal Control (NC), patients with psoriasis without arthritis (PsO), and patients with psoriatic arthritis.
[ embodiment ] A method for producing a semiconductor device
As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
The use of the term "or" in the claims means "and/or" in the claims.
When used in this specification and claims, the terms "comprising," "including," or "containing" are intended to mean either inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
As used herein, "patient" or "subject" refers to a mammalian subject diagnosed as having or suspected of having a risk of developing psoriatic arthritis. Exemplary subjects can be humans, apes, dogs, pigs, cows, cats, horses, sheep, rodents and other mammals that can develop psoriatic arthritis.
As used herein, alternatively, "microRNA" (microRNA), "microRNA" (miR), "or" microRNA "(miRNA)" refers to unprocessed (e.g., prepro), or processed (e.g., mature) RNA transcripts (transcript) from miR genes. microRNAs are endogenous non-coding single stranded RNAs that negatively regulate gene expression in eukaryotes and constitute novel gene regulators (regulators) (Chua, et al (2009) curr. Opin. mol. ther.11: 189-. Individual miRNAs were identified and sequenced in different organisms and given their names. Provided herein are names of miRNAs and their sequences.
All numbers herein are understood to be modified by "about" and are intended to encompass variations of ± 10% of a particular value when, for example, such variations are suitable for expression of miRNA amounts.
The expression of "higher" mirnas is a relative term and can be determined by comparing the amount of miRNA expression in a test sample to a reference pool (filtered pool) of healthy subjects known to be free of rheumatoid arthritis (i.e., a normal control group).
The term "sample" as used herein refers to a sample containing miRNA. The sample can be used to detect the presence and/or amount of expression of the miRNA of interest. Although biological fluids and organs containing mirnas that are differentially expressed compared to normal controls are predicted to be most appropriate, extracted CD14+ monocytes are used with the method of the claimed subject matter. In some embodiments, a sample, such as, for example, blood or a component thereof, is relatively easy to obtain. Non-limiting examples of samples include bodily fluids (e.g., peripheral blood), cells (e.g., CD14+ monocytes or osteoclasts), and tissues (e.g., biopsy specimens or synovial fluid).
The measurement of the expression amount of miRNA refers to quantifying the amount of miRNA present in a sample. Measurement of the expression level of a particular or any miRNA can be achieved using any of the methods described herein, as known to those of ordinary skill in the art or as by real-time quantitative fluorescence PCR (real-time PCR), Northern blot (Northern blot) analysis. measuring the expression level of the miRNA comprises measuring the expression of the mature form of the miRNA or a precursor form associated with the expression of the miRNA.
In a particular embodiment, the expression level of at least one miRNA is quantified using northern blot analysis. For example, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. The nucleic acid is precipitated and the DNA is removed by treatment with a DNA digesting enzyme (DNase) and precipitation. The RNA molecules were then separated by colloidal electrophoresis (gel electrophoresis) on agarose gels (agarose gels) according to standard techniques and transferred to nitrocellulose filters. Thereafter, the RNA was immobilized (immobilized) on a filter by heating. Detection and quantification of specific RNA is accomplished using appropriate labeled DNA or RNA probes complementary to the RNA in question. See, e.g., Molecular Cloning: the disclosure of ALaboratory Manual, J.Sambrook et al, eds.,2nd edition, Coldspring Harbor Laboratory Press,1989,
In some embodiments, the use of a microarray is possible. Microarrays are microscopic, ordered arrays of nucleic acids, proteins, small molecules, cells, or other substances that can be analyzed in parallel on complex biological samples. The technique provides a number of oligonucleotides or polynucleotides having known sequence information as probes to find and hybridize with complementary strands in a sample, thus capturing the complementary strands by selective binding. The probe comprises an oligonucleotide or polynucleotide sequence that is complementary or substantially complementary to at least a portion of a target miRNA sequence. "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands. It is known that if the residue is thymine (thymine) or uracil (uracil), an adenine residue of a first nucleic acid region (adenine residue) can form a specific hydrogen bond ("base pair") with a residue of a second nucleic acid region that is antiparallel to the first region. Similarly, it is known that if the residue is guanine, cytosine residues of the first nucleic acid region can base pair with residues of the second nucleic acid strand that are antiparallel to the first strand. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid when at least one nucleotide residue of the first region can base-pair with a residue of the second region if the two regions are arranged in an antiparallel (washion) fashion. Preferably, the first region comprises a first portion and the second region comprises a second portion, such that when the first portion and the second portion are arranged in an antiparallel fashion, at least about 50% and preferably at least about 75%, at least about 90% or at least about 95% of the nucleotide residues of the first portion base pair with nucleotide residues in the second portion. More preferably, all of the nucleotide residues of the first portion are base-paired with nucleotide residues in the second portion.
For example, microarray analysis of miRNAs can be accomplished according to any method known in the art. In one example, RNA is extracted from cells of the sample, such as CD14+ monocytes, and size-selected (size-selected) small RNA (18-26 nucleotides) from all RNA using denaturing polyacrylamide gel electrophoresis (polyacrylamide gel electrophoresis). Oligonucleotide linkers are attached to the 5 'and 3' ends of the small RNAs, and the resulting ligation products serve as a template for RT-PCR reactions for 10 cycles of amplification. The sense strand (sense strand) PCR primer has a fluorophore attached to its 5' end, thus fluorescently labeling the sense strand of the PCR product. The PCR products were denatured and then hybridized to a microarray. The PCR product refers to the target nucleic acid as complementary to the corresponding miRNA capture probe sequence on the array, which will hybridize via base pairing to the point where the capture probe is immobilized. The spot will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then evaluated using several positive and negative controls and a normalization of the array data to assess the number of copies of the particular miRNA that will result in an assessment of the amount of expression of the particular miRNA. With respect to the mirnas disclosed herein, the probes may be 100% complementary to the target miRNA or polynucleotide sequence. However, since the probe can specifically bind to and capture the target polynucleotide from the sample, the probe need not necessarily be fully complementary to the target polynucleotide along its entire length. In some embodiments, the probe is a probe that hybridizes to SEQ ID NO: 1 or SEQ ID NO: 2 at least 90%, at least 95%, or 100% identical.
In some embodiments, the use of quantitative reverse transcription polymerase chain reaction (quantitative RT-PCR) is feasible. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a modification of polymerase chain used to rapidly determine the amount of product of the polymerase chain reaction. The purpose of qRT-PCR is typically to determine whether a gene sequence, such as a miRNA, is present in a sample, and if it is present, what the amount of replication is in the sample. Any method of PCR that can measure the expression of nucleic acid molecules comprising mirnas falls within the scope of the present disclosure. Several variations of qRT-PCR methods known in the art include, but are not limited to, via agarose gel electrophoresis analysis (agar gel electrophoresis), the use of SYBR Green (double-stranded DNA stain), and the use of fluorescent reporter probes (fluorescent reporter probes).
The identification of mirnas for differential expression in subjects with and without psoriasis allows these information to be used in a variety of ways. For example, a particular treatment regimen may be evaluated (e.g., to determine whether the treatment is effective in a subject with psoriatic arthritis). Diagnosis of psoriatic arthritis can be accomplished or confirmed by comparing miRNA expression levels of extracted CD14+ monocytes or osteoclasts of the test sample to known expression miRNA expression level profiles of extracted CD14+ monocytes or osteoclasts of non-psoriatic arthritis samples. Further, multiplex assays can be performed for one or more diagnostic or predictive miRNA amounts, and changes in the duration of expression can be used to determine, diagnose, prognosing (prognosis), or relapse (relapse). For example, a particular amount of miRNA can be determined at a starting time and again at a second time. In some embodiments, an increase in the amount of miRNA from the initial time to the second time can be diagnosed with psoriatic arthritis or give a prognosis (given prognosis). Similarly, a decrease in the amount of miRNA from the starting time to the second time may indicate psoriatic arthritis or give a prognosis. Further, the degree of change in the amount of one or more mirnas may be correlated with the severity of psoriatic arthritis.
In another embodiment, there is provided a method of determining the efficacy of a treatment for treating psoriatic arthritis in a subject, the method comprising the steps of comparing: (a) measuring the expression level of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941 of extracted CD14+ monocytes in a first sample from the subject prior to providing at least a portion of treatment to the subject; and (b) the expression amount of the at least one corresponding miRNA in step (a) of the extracted CD14+ monocytes in a second sample obtained from the subject after providing at least a portion of the treatment to the subject, wherein a decreased expression amount of the at least one miRNA in the second sample relative to the expression amount of the corresponding miRNA in the first sample is indicative of the treatment being effective to treat rheumatoid arthritis. In an exemplary embodiment, the sample is extracted CD14+ monocytes and the miRNA is miR-146a-5p, miR-941, or a combination thereof.
Referring to fig. 1C and 1D, without being bound by any particular theory, the expression levels of miR-146a-5p and miR-941 were assessed only in extracted CD14+ monocytes or osteoclasts from PsA patients, and not in peripheral monocytes of PsA subjects. Therefore, measuring the expression level of miR-146a-5p, miR-941 or a combination thereof in peripheral monocytes instead of CD14+ monocytes does not provide a diagnosis of PsA. The one or more osteoclasts may be differentiation (differentiated) from one or more CD14+ monocytes or osteoclasts from synovial fluid of PsA patients.
In one embodiment, the kit comprises at least one reagent for sequencing or measuring the expression level of at least one miRNA selected from the group consisting of miR-146a-5p and miR-941 in a sample of a subject in need of diagnosis or prediction of risk of developing psoriatic arthritis.
In yet another embodiment, the kit further comprises a medium comprising a medium of M-CSF, RANKL, and TNF- α, the medium to differentiate extracted CD14+ monocytes into osteoclasts.
In one exemplary embodiment, the reagents are RT-PCR. In another exemplary embodiment, the kit comprises at least one miRNA-specific oligonucleotide probe (miRNA-specific oligonucleotide probe) that hybridizes to SEQ ID NO: 1 or SEQ ID NO: 2 at least 90%, at least 95%, or 100% identical.
As used herein, "identity" refers to the degree of nucleotide sequence similarity between two regions of the same nucleic acid strand or between two regions of different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, the region at that position is homologous (homologus). The first region is homologous to the second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed by the ratio of nucleotide residue positions of the two regions occupied by the same nucleotide residue. As an example, the region having the nucleotide sequence 5'-ATTGCC-3'
Provided is a method for detecting psoriatic arthritis in a subject, comprising the steps of: measuring the expression level of at least one of the following mirnas in a test sample of a subject: miR-146a-5p and miR-941, wherein a higher expression level of at least one miRNA in the test sample relative to the expression level of at least one corresponding miRNA in the non-psoriatic arthritis sample indicates that the subject has, or is at risk for developing, psoriatic arthritis.
The present invention further provides methods for detecting and treating psoriatic arthritis in a subject comprising the steps of (a) measuring the expression level of at least one miRNA, miR-146a-5p and miR-941, in a test sample from the subject, wherein a higher expression level of the at least one miRNA in the test sample relative to the expression level of the at least one corresponding miRNA in the psoriatic arthritis-free sample indicates that the subject has or is at risk of developing psoriatic arthritis, and (b) administering a therapeutic agent to treat psoriatic arthritis non-limiting examples of the therapeutic agent include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), immunosuppressive agents (immunosupressants), TNF- α inhibitors, aspirin inhibitors (aspirin), anti-rheumatic drugs (aspirin), a inhibitors, or a
In some embodiments, there is provided a method of treating osteoarthritis in a subject in need thereof, the method comprising administering to the subject an antibody that binds to SEQ ID NO: 1a miR-146a-5p inhibitor that is at least 90%, at least 95%, or 100% identical to a polynucleotide that is complementary to SEQ ID NO: 2 a miR-941 inhibitor that is at least 90%, at least 95%, or 100% identical to the polynucleotide complement.
Embodiments of the present invention are illustrated by the following examples, which should not be construed as imposing limitations upon the scope thereof in any way. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention. In the studies describing the following examples, conventional procedures were followed, unless otherwise indicated. Some procedures will be described below for illustrative purposes.
Example 1
Study participation: case control studies were performed to investigate the expression of various miRNAs in patients with psoriatic arthritis (cases) and healthy Normal Controls (NCs) without PsA.
16 healthy Normal Controls (NC) and 31 patients with PsA (based on the classified on the Classification Criteria for Psoriatic Arthritis), CASPAR, Taylor W et al, Arthritis and rheumatism 2006; 54 (8): 2665-73) were enrolled into the study.
Next-generation sequencing (NGS) with subsequent validation was analyzed by quantitative pcr (qpcr) to explore potential PsA biomarkers. RNA samples from 2 NCs and 4 PsA were used as initial validation of candidate mirnas from NGS studies.
CD14+ monocytes in peripheral blood of enrolled subjects were extracted and isolated by CD14+ MicroBeads (MicroBeads) (Miltenyi Biotec, taiwan). RNA samples were extracted from isolated CD14+ monocytes and subsequently subjected to a quality test using an Agilent Bioanalyzer 2100(Agilent, USA) to select RNA samples having an RNA integer (integer number value) ≧ 8.0. Selected RNA samples were prepared using TruSeq Small RNA preparation protocol (Illumina, USA).
RNA pools from 2 pooled male NCs, 2 pooled female NCs, two pooled male patients with PsA and 2 pooled female patients with PsA were sequenced with the Illumina NGS platform followed by profiling the expression of micro (mi) -RNA by miRSeq8 analysis. The prepared amplicons (amplics) were sequenced on MiSeq fality (Illumina) with V3150-cycle sequencing reagents to generate 51-nt single-ended sequencing (single-endiads). Group analysis (Clusterd analysis) was performed to examine miRNA expression profiles of PsA and NC samples.
There were approximately 27.7 million original reads (raw reads) in total among the RNA samples examined with NGS, and an average of 6.9 million reads per total sample. The resulting NGS data were analyzed by miRSeq (Pan CT. et al., Biomed Res Int 2014: 462135) for miRNA quantification and assessment of sequencing quality. miRSeq expresses miRNA expression in parts per million (TPM) of transcription. As shown in table 1, 13 miRNAs had TPM values above 1000 and average expression rates (normal versus disease or disease versus normal) of at least 1.5 in at least one sample.
TABLE 1 List of miRNAs with higher expression levels in PsA patients
Among the 13 miRNAs, expression of miR-941 was increased in patients with PsA.
qPCR was performed on 2 NC samples and 4 PsA samples to further validate NGS data. The qPCR analysis showing expression of miR-941 and miR-146a-5p is increased in patients with PsA compared to those from NCs (see FIG. 1A).
SVM is a Machine learning algorithm (Machine learning) used to solve the problem of binary classification, such as treatment versus (versus) control, or disease versus normal. Shown in FIG. 1B, DCt values for miR-941 and miR-146a-5p in NC and PsA patients were used to train SVM classification with 5-fold cross-validation to eliminate overfitting and low-degree fitting.
FIGS. 1C and 1D show elevated expression of miR-941 and miR-146a-5p in extracted CD14+ monocytes (PsACD14+) from patients with psoriatic arthritis, but not in peripheral monocytes (PsACD14-) from patients with psoriatic arthritis.
Table 2 shows that the combination of miR-146a-5p and miR-941 elicits an unexpected synergy in area (auROC) values under sensitivity, specificity and receiver operating characteristics in the diagnosis of PsA.
TABLE 2 sensitivity, specificity and auROC of PsA diagnostics based on a combination of miR-146a-5p and miR-941
Expression of miR-941 and miR-146a-5p in extracted CD14+ monocytes was measured from 16 NC and 11 PsA patients before PsA treatment (including adalimumab, etanercept, golimumab, ustekinumab, or secukinumab) and after 6 months of treatment.
11 patients with PsA had significant relief from symptoms (decreased tender and swollen joints) after 28 weeks of treatment (see panels a and B of figure 2). Expression of miR-941 and miR-146a-5p in extracted CD14+ monocytes was significantly reduced in 11 PsA patients after 6 months of treatment (panels C and D of figure 2). The decreased expression of miR-941 and miR-146a-5p in extracted CD14+ monocytes was associated with remission in PsA patients.
Expression levels of miR-941 and miR-146a-5p in osteoclasts differentiated from extracted CD14+ monocytes.
CD14+ monocyte line was extracted from peripheral blood of 2 NC and two PsA patients before and 6 months after PsA treatment the extracted CD14+ monocytes were cultured with M-CSF 50ng/ml for 3 days, followed by 100ng/ml RANKL and 100ng/ml TNF- α for 9 days to differentiate osteoclasts, which are known to cause joint damage in PsA (joindescription). As shown in panels 3A and 3B, miR-941 and miR-146a-5p expression was higher in CD14+ monocyte-derived osteoclasts of PsA patients before and after successful treatment than NC (p <0.05 and p < 0.05). results show that miR-941 and miR-146a-5p expression was higher in CD14+ monocyte-derived osteoclasts regardless of disease activity (diseases activity).
Example 2
In vitro (in vitro) assessment of the effect of miR-941 inhibitors and miR-146a-5p inhibitors on CD14+ monocyte derived osteoclasts from patients with PsA was performed.
The CD14+ monocyte cell line was extracted from peripheral blood of PsA patients and cultured with M-CSF for 72 hours. Cultured CD14+ monocytes were treated in the following protocol:
(1) no miRNA transfection CD14+ monocytes were cultured with TNF- α and RANKL every 3 days for 9 days to differentiate into osteoclasts.
(2) Transfection of negative control miRNA CD14+ monocytes were transfected with negative control miRNA (SEQ ID NO: 3) for 6 hours, and then CD14+ monocytes were cultured every 3 days with TNF- α and RANKL for 9 days to differentiate into osteoclasts.
(3) Transfection of miR-941 inhibitor CD14+ monocytes were transfected with miR-941 inhibitor (SEQ ID NO: 4) for 6 hours, and then CD14+ monocytes were cultured every 3 days with TNF- α and RANKL for 9 days to differentiate into osteoclasts.
(4) Transfection of miR-146a-5p inhibitor CD14+ monocytes were transfected with miR-146a-5p inhibitor (SEQ ID NO: 5) for 6 hours, and then CD14+ monocytes were cultured every 3 days with TNF- α and RANKL for 9 days to differentiate into osteoclasts.
As depicted in figures 4A-4E, miR-941 inhibitors (figure 4C) or miR-941 inhibitors (figure 4D) significantly reduced the number of CD14+ monocyte-derived osteoclasts compared to the negative control miR transfection (figure 4B) and the miR-free transfection group (figure 4A). Therefore, inhibitors of miR-146a-5p or miR-941 decrease osteoclastogenesis (osteoplastogenesis) in PsA patients and can be used to treat PsA.
Example 3
The included case-control study was conducted to examine the expression of miR146a-5p and miR941 in 40 healthy Normal Controls (NC), 40 patients with psoriasis without arthritis (PsO) and 40 patients with psoriatic arthritis (PsA).
Diagnosis of PsA and expression of miRNAs were measured according to the guidelines and the procedure in example 1. FIGS. 5A and 5B show that the expression of miR146a-5p and miR941 is significantly higher than the expression levels of NC and PsO. This result shows that increased expression of miR146a-5p and miR941 is predictive of the risk of developing arthritis in patients with psoriasis.
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<220>
<223> negative control miRNA
<400>3
ucacaaccuc cuagaaagag uaga 24
<210>4
<211>23
<212>RNA
<213> Intelligent (Homo sapiens)
<220>
<223> miR-941 inhibitors
<400>4
gugggccgac acacguguac acg 23
<210>5
<211>22
<212>RNA
<213> Intelligent (Homo sapiens)
<220>
<223> miR-146a-5p inhibitor
<400>5
acucuugacu uaagguaccc aa 22
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