CN108823300B

CN108823300B - Application of circRNA in preparation of product for diagnosing osteoarthritis

Info

Publication number: CN108823300B
Application number: CN201810637203.XA
Authority: CN
Inventors: 肖刻; 翁习生; 边焱焱; 冯宾
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2018-06-20
Filing date: 2018-06-20
Publication date: 2021-03-05
Anticipated expiration: 2038-06-20
Also published as: CN108823300A

Abstract

The invention discloses application of hsa _ circ _0045714 and/or hsa _ circ _0005567 as molecular markers in preparation of products for diagnosing or treating osteoarthritis, and further proves that hsa _ circ _0045714 and hsa _ circ _0005567 are both up-regulated in osteoarthritis tissues. The invention further discloses the use of hsa _ circ _0045714 and/or hsa _ circ _0005567 in the preparation of a diagnostic kit for the detection of osteoarthritis, said kit comprising primers and instructions for the specific amplification of osteoarthritis-associated circRNA. The osteoarthritis detection method based on hsa _ circ _0045714 and/or hsa _ circ _0005567 can quickly and effectively achieve early detection, and provides treatment targets and important bases for clinical applications such as gene therapy, drug therapy and the like.

Description

Application of circRNA in preparation of product for diagnosing osteoarthritis

Technical Field

The invention relates to the field of biological medicines, in particular to application of hsa _ circ _0045714 and/or hsa _ circ _0005567 in preparation of products for diagnosing or treating osteoarthritis.

Background

Osteoarthritis (OA) is a chronic disease in which joint cartilage, which covers the ends of bones and forms the articular surface of a joint, gradually degrades over time. A number of factors have been reported to predispose patients to osteoarthritis, including genetic predisposition, obesity, accidental or athletic trauma, surgery, medications, and heavy physical demands. Osteoarthritis is believed to begin with damage to the articular cartilage. The two most common types of damage to joints are motion-related damage and long-term "repetitive use" joint damage. The joints most often affected by osteoarthritis are the knee, hip, and hand. In most cases, osteoarthritis in these joints is more likely to cause disability than osteoarthritis in the hands due to the necessary weight bearing functions of the knee and hip. As cartilage degradation progresses, secondary changes occur in the joint and other tissues surrounding the joint, including bone, muscle, ligaments, meniscus, and synovium. The net effect of primary failure of cartilage tissue and secondary damage to other tissues is that the patient experiences pain, swelling, weakness and loss of function of the affected joint. These symptoms often develop to the point of having serious effects, such as loss of labor or quality of life for the patient.

Articular cartilage is composed mainly of chondrocytes, type II collagen, proteoglycans and water. Articular cartilage has no blood or nerve distribution, and chondrocytes are the only cell type in this tissue. Chondrocytes are responsible for the production of type II collagen and proteoglycans that form the cartilage matrix. The matrix thereafter has physicochemical properties that allow saturation of the matrix with water. The net effect of this structural functional relationship is that the articular cartilage has special wear characteristics and that virtually frictionless movement occurs between the articular cartilage surfaces. In the absence of osteoarthritis, articular cartilage often provides a lifetime of painless weight bearing and unrestricted joint movement even under high demand physical conditions.

As with all living tissues, articular cartilage is constantly undergoing a renewal process in which the "old" cells and matrix components are removed (catabolically active) and "new" cells and molecules are produced (anabolically active). The turnover rate of anabolism or catabolism of articular cartilage is low relative to most tissues. Long-term maintenance of the structural integrity of mature cartilage depends on an appropriate balance between matrix synthesis and degradation. Chondrocytes maintain matrix balance by responding to chemical and mechanical stimuli from their environment. Appropriate and effective responses of chondrocytes to these stimuli are essential for cartilage homeostasis. Disruption of homeostasis by either insufficient anabolic or excessive catabolic activity can lead to cartilage degradation and osteoarthritis (Adams et al, 1995, Nature377Suppl: 3-174). Most tissues that are damaged and have increased catabolic activity are able to initiate an enhanced anabolic response, allowing tissue rejuvenation. Unfortunately, articular cartilage has a very limited ability to up-regulate its anabolic activity and increase the synthesis of proteoglycans and type II collagen in response to cartilage matrix damage or consumption.

Current osteoarthritis treatment methods include exercise, medication, rest and joint care, surgery, pain relief techniques, replacement therapy and weight control. Common drugs used in the treatment of osteoarthritis include non-steroidal anti-inflammatory drugs such as aspirin, ibuprofen, and the like; can be directly applied to skin for relieving topical pain such as cream, rubber, and spray. Surgery may be performed to resurface bone, reposition bone, and replace joints. Although various drug therapies have been used to treat diseases, they are not effective for long-term control and prevention. Furthermore, since osteoarthritis occurs insidiously and progresses slowly, osteoarthritis is often identified late in disease progression rather than early in disease progression where potential treatments may be more effective. Further progression in the prevention, alteration or treatment of osteoarthritis disease processes therefore relies heavily on the identification of early diagnostic markers of the disease, allowing early intervention.

Disclosure of Invention

The invention aims to provide the application of a novel marker in products for diagnosing or treating osteoarthritis.

To achieve the above objects, the present invention provides the use of hsa _ circ _0045714 and/or hsa _ circ _0005567 as molecular markers for the preparation of a product for the diagnosis or treatment of osteoarthritis.

Further, hsa _ circ _0045714 and hsa _ circ _0005567 are both up-regulated in osteoarthritis biological samples.

Still further, the marker comprises the nucleotide sequences shown as SEQ ID NO 1 and SEQ ID NO 2.

Preferably, the product is a product capable of quantitatively detecting the expression level of hsa _ circ _0045714 and/or hsa _ circ _ 0005567.

Still more preferably, the product is an osteoarthritis diagnostic kit.

Further, the kit includes primers and instructions for specifically amplifying hsa _ circ _0045714 and/or hsa _ circ _0005567 associated with osteoarthritis.

Still further, the primer sequences of hsa _ circ _0045714 are shown as SEQ ID NO.3 and SEQ ID NO. 4; the primer sequences of hsa _ circ _0005567 are shown in SEQ ID NO.5 and SEQ ID NO. 6.

Further, the primers are suitable for detection by SYBR Green, TaqMan probes and/or molecular hybridization probes.

Still further, the kit also comprises 10 xbuffer, dNTP and Mg²⁺Taq enzyme and fluorescent dye.

The invention has the following beneficial effects:

the invention discloses hsa _ circ _0045714 and hsa _ circ _0005567 related to osteoarthritis for the first time, and further proves that hsa _ circ _0045714 and hsa _ circ _0005567 are up-regulated in osteoarthritis tissues, and when the circRNA is used for detecting osteoarthritis, the invention not only can quickly and effectively realize early detection, but also provides a treatment target and an important basis for clinical application such as gene therapy, drug therapy and the like.

Drawings

FIG. 1 expression of hsa _ circ _0045714 and hsa _ circ _0005567 and their possible target genes in osteoarthritic tissue samples.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The invention is described in detail below with reference to the figures and examples.

The experimental procedures, for which specific conditions are not indicated in the examples, are generally conventional in the art, e.g. according to conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (third edition) (scientific Press, 2002), or according to conditions recommended by the reagent manufacturers.

The inventor of the invention carries out high-throughput transcriptome sequencing on a cartilage tissue sample of an OA patient and a normal cartilage tissue sample, carries out gene screening by a bioinformatics method, selects hsa _ circ _0045714 and hsa _ circ _0005567 with obvious differential expression, does not report that hsa _ circ _0045714 and hsa _ circ _0005567 are related to osteoarthritis in the existing research, and further carries out molecular biological method verification to confirm that hsa _ circ _0045714 and hsa _ circ _0005567 are up-regulated in osteoarthritis tissues.

Hsa _ circ _0045714 and hsa _ circ _0005567 of the invention are known circrnas prior to the invention, retrievable by circBase (http:// circBase. org), with the following basic information:

circbase ID: hsa _ circ _0045714, located on human chromosome 17, chr17:73808192 and 73809959; circbase ID: hsa _ circ _0005567, located on human chromosome 1, chr1: 51868106-.

The term "osteoarthritis" as used herein refers to a particular form of arthritis, particularly a chronic disease in which articular cartilage, which covers the ends of bones that reform the articular surfaces, gradually degrades over time.

The term "upregulated expression" as used herein refers to a sequence corresponding to an expressed gene, wherein measurement of the amount of the sequence demonstrates an increased level of expression of the gene in a biological sample isolated from an individual having osteoarthritis or an osteoarthritis-identified disease state as determined by the osteoarthritis stage, as compared to the same gene in a biological sample isolated from a normal individual or from an individual having an identified disease state as distinct from osteoarthritis as determined by the osteoarthritis stage. According to the invention, "up-regulation of expression" means an increase in expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, e.g. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 1-fold, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more as measured by the hybridization intensity of the method of the invention.

The term "expression level" as used herein refers to the measurable quantity of a given nucleic acid or protein as determined by methods known to those skilled in the art and described herein. In relation to circRNA corresponding to the molecular marker of the invention, the expression level may be determined by hybridization or more quantitative measurements, such as including the use of SYBR Green, TaqMan and quantitative real-time RT-PCR.

Example 1 high throughput sequencing screening of differentially expressed genes

1. Sampling

19 cartilage tissue specimens (average age of 64.5 years, age range of 58-75 years, 2 males and 17 females) obtained in Beijing cooperative hospital osteoarthrosis surgery are taken, and all specimens are verified by pathological examination and diagnosed as knee osteoarthritis; the normal cartilage tissue is from Beijing cooperative hospital orthopedics and is articular cartilage tissue of a patient with trauma surgery. The obtained tissue is stored in a low-temperature refrigerator at-80 ℃ after being numbered. Clinical samples used in this study were informed and passed through the ethical committee of the hospital.

2. Total RNA extraction from tissue samples

By using

Reagent (Invitrogen, Carlsbad, Calif., USA) to extract sample RNA, the experimental operation was performed according to the product instruction, and the specific operation was as follows:

collecting a sample, freezing the sample in liquid nitrogen, taking out the sample, putting the tissue into a precooled mortar for grinding, and after the tissue sample is powdered:

adding Trizol, and preserving for 5 minutes at room temperature;

adding 0.2mL of chloroform, forcibly oscillating the centrifuge tube, fully mixing the materials, and standing the mixture at room temperature for 5 to 10 minutes;

③ centrifuging at 12000rpm for 15 minutes, sucking the upper water phase (sucking 70%) into another new centrifugal tube, and taking care not to suck the protein material between the two water phases. Moving into a new tube, adding equal volume of pre-cooled isopropanol at-20 ℃, fully reversing and uniformly mixing, and placing on ice for 10 minutes;

fourthly, after 15 minutes of high-speed separation at 12000rpm, the supernatant is carefully discarded, 75 percent DEPC ethanol is added according to the proportion of 1mL/mL Trizol to wash and precipitate (the precipitate is stored at 4 ℃), the precipitate is washed and mixed by shaking, and the mixture is centrifuged at 12000rpm at 4 ℃ for 5 minutes;

fifthly, removing the ethanol liquid, standing for 5 minutes at room temperature to fully air-dry the precipitate, and adding DEPC treated water to dissolve the precipitate;

new use of

Spectrophotometer (IMPLEN, CA, USA) for measuring RNA purity and use

RNA Assay Kit in

2.0 Fluorometer assay kit (Life Technologies, CA, USA) concentration, frozen at-80 ℃. By passing

Detecting the extraction condition of an RNA sample by a spectrophotometer, wherein the sample requirement of RNA-seq sequencing is as follows: OD₂₆₀/OD₂₈₀Is 1.8-2.2.

RNA quality determinationThe standard is as follows: OD of RNA sample₂₆₀/OD₂₈₀The value is between 1.7 and 2.2; the total RNA electrophoresis pattern has clear 28S and 18S bands; the electrophoresis pattern after the water bath heat preservation for 1 hour at 70 ℃ has no obvious difference with the pattern before the water bath heat preservation.

RNA integrity assessment RNA Nano 6000 detection kit was used as well as bioanalyzer 2100 system (Agilent Technologies, CA, USA). And (3) diluting the samples of the normal group and the osteoarthritis group to the same concentration, and respectively preparing the samples of the normal group and the osteoarthritis group for constructing and sequencing the library.

3. Sequencing preparation and high throughput sequencing

Mu.g of each RNA sample was input. First using Epicentre Ribo-zero^TMrRNA Removal kit (Epicentre, USA) removes rRNA from the total RNA extracted in the previous step, and then removes free rRNA residues by ethanol precipitation. Reuse of

Ultra^TMThe direct RNA Library Prep kit (NEB, USA) prepares sequencing libraries according to the instruction, and adds index codes to the attribute sequences of each sample. The sequencing library was cleaved in NEBNext's First Strand Synthesis Reaction Buffer (First Strand Synthesis Reaction Buffer, 5 ×) using divalent cations at high temperature. First strand cDNA was synthesized using random primer (random hexamer primer) and M-MuLV reverse transcriptase. Second strand cDNA synthesis was then performed with DNA polymerase I and RNase H in reaction buffer, dUTP was used instead of dTTP in dNTPS. The cDNA fragments are then converted to blunt ends using a polymerase and an exonuclease. After the 3' end of the DNA fragment is phosphorylated, it is ligated to NEBNext adapter with a hairpin structure for hybridization. To select cDNA fragments of approximately 150-200 bp in length, the library fragments were purified using the ApHealthXP system (Beckman Coulter, Beverly, USA). Then, 3. mu.L of USER Enzyme (NEB, USA) was used for size selection, and cDNA ligation was performed at 37 ℃ for 15 minutes and at 95 ℃ for 5 minutes. PCR was performed using Phusion high fidelity DNA polymerase. Index coding was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS Kit (Illumia) according to the instructionsAnd performing high-throughput sequencing on the clustering by using an Illumina Hiseq 4000 platform to obtain a Sequenced Reads, and then performing biological information analysis under the condition of referring to a reference sequence or genome of a related species.

4. Analysis of test data

4.1 Reads quality control

The data in the Fastq form obtained in the previous step is firstly programmed through an internal script of the original data, reads with a connector, reads containing poly N and low-quality reads are removed, and clean data is obtained. Meanwhile, Q20, Q30 and GC contents of clean data are calculated. All subsequent analyses were based on high quality clean data.

4.2 Reads alignment of reference genes

The genome and genome model annotation files are downloaded directly from the genome site. The reference genome index was constructed by bowtie2v2.2.8 and the clear reads at both ends were matched to the reference genome by HISAT2v2.0.4. HISAT2.

4.4 identification of circRNA

The circRNA was identified using find _ circ (Sebastian Memczak et al, 2013). The basic principle of find _ circ is to extract 20-nt anchormer sequences from each end of reads that are not aligned to the reference sequence, align each pair of anchormer sequences to the reference sequence again, and if the 5 'end of the anchormer sequence is aligned to the reference sequence (the start and stop sites are designated as A3, a4, respectively) while the 3' end of the anchormer sequence is aligned upstream of this site (the start and stop sites are designated as a1, a2, respectively), and there is a splice site (GT-AG) between a2 and A3 of the reference sequence, then take this read as a candidate circRNA. And finally, using the candidate circRNA with the read count more than or equal to 2 as the identified circRNA.

4.5 expression level analysis of circRNA

The expression amount of the known circRNA and the new circRNA in each sample is counted, and expression amount normalization processing is performed by using TPM (Zhou et al, 2010) to obtain readcount of the sample.

4.6 results of differential expression analysis of circRNA

The input data for the differential expression of circRNA is readcount data obtained in the analysis of the expression level of circRNA. For samples with biological replicates, analysis we performed analysis using DESeq2(Michael et al, 2014) based on a negative binomial distribution; for samples without biological replicates, readcount data was normalized using TMM prior to differential analysis using DEGseq (Wang et al, 2010).

4.7 further analysis of differentially expressed circRNA

To better understand the function of differentially expressed genes, we performed Gene Ontology (GO) analysis, signaling pathway (KEGG) analysis, and miRNA binding site analysis of circrnas (for animals and plants, binding sites of mirnas of sheared circrnas were predicted using miRanda and psRobot software, respectively) on differentially expressed genes and functionally annotated, and in view of the results of the above data analysis, we screened differentially expressed hsa _ circ _0045714 and hsa _ circ _0005567 for the studies of the present application, which were up-regulated in cartilage tissue samples from OA patients.

Example 2RT-PCR validation of cartilage tissue hsa _ circ _0045714 and hsa _ circ _0005567 expression in OA patients

1. Material

17 samples of osteoarthritic cartilage tissue were obtained from Beijing cooperative Hospital osteoarthritis surgery (mean age 66 years), grouped and numbered. All samples were confirmed by pathological examination, and the control group was patients treated by arthroscopic surgery for meniscal injury and cruciate ligament injury. All samples are numbered and then stored in a low-temperature refrigerator at the temperature of minus 80 ℃.

2. Expression profile analysis method

2.1 Total RNA extraction was performed on tissue samples in the same manner as in example 1.

2.2 Synthesis of cDNA by reverse transcription

By using

III Reverse transcription of cDNA by Reverse transcription of Transcriptase (Invitrogen, cat # 18080-044), the experimental procedures were performed according to the product instructions, and the specific procedures were as follows:

using a reverse transcription kit, one μ g of total RNA was subjected to reverse transcription with a reverse transcription buffer to synthesize cDNA. Using a 25. mu.L reaction system, 1. mu.g of total RNA was taken for each sample as template RNA. The obtained cDNA sample was diluted 10 times and stored in a refrigerator at-20 ℃ for further use.

2.3Real-Time PCR

2.3.1 Instrument and analytical method

Using ABI 7500 type fluorescent quantitative PCR instrument, adopting 2^-ΔΔCtThe method carries out relative quantitative analysis of data.

2.3.2 primer design

On-line primer design software is adopted, and the gene sequence refers to the sequence of Circbase ID: hsa _ circ _0045714 and hsa _ circ _0005567, GAPDH as internal reference, primer design and synthesis by invitrogen. The specific primer sequences are as follows:

TABLE 1 primer sequences

The operation process is as follows:

TABLE 2 Real Time reaction System

Components	Amount of addition
		2×mix	10μL
Upstream primer (10. mu.M)	0.5μL
		Downstream primer (10. mu.M)	0.5μL
Form panel	2μL
		Adding sterilized distilled water	To 25 μ L

By Power

Green PCR Master Mix (Invitrogen, cat # 4367659) amplified the target gene primers and the reference gene primers, respectively. The experimental operation was carried out according to the product instructions. The amplification procedure was: 95 ℃ for 5min, (95 ℃ 15sec, 60 ℃ 45sec, 72 ℃ 35 sec). times.40 cycles. At the same time, the dissolution curve analysis is carried out at 60-95 ℃. After the reaction, 5. mu.l of the PCR product was subjected to 2% agarose electrophoresis, and fragments of 252bp and 147bp in size were recovered by cutting with a quick gel recovery kit (Invitrogen Co.) and sequenced, and as a result, homology analysis was performed with blast software.

3. Results of the experiment

The inflection point of the real-time quantitative PCR amplification curve is clear, the overall parallelism of the amplification curve is good, the amplification efficiency of each reaction tube is similar, the base line is flat without raising phenomenon, the slope of the exponential phase of the curve is larger, and the amplification efficiency is higher; the dissolution curves of the sample amplification products are all unimodal, which indicates that only one amplification product is specifically amplified; according to the relative quantitative formula of qRT-PCR: 2^-ΔΔCtX 100%, comparing the expression levels of hsa _ circ _0045714 and hsa _ circ _0005567 in osteoarthritic tissue and normal tissue, and predicting the action networks of circRNA-miRNA-mRNA of hsa _ circ _0045714 and hsa _ circ _0005567 and the expression of the corresponding target gene mRNA by CircNET, predicting that hsa _ circ _0005567 acts on the hetr 5B gene, and hsa _ circ _0045714 acts on the UNK gene. The above results demonstrate that integrated analysis of high throughput transcriptome expression data results for the upregulation of hsa _ circ _0045714 and hsa _ circ _0005567 in osteoarthritis patients, approximately 128-Fold and 512-Fold of normal tissue, log2Fold change log2, respectively (normal tissue samples/bone)Arthritic tissue samples), see fig. 1 for specific results; after the RT-PCR product is recovered, sequencing is carried out on an ABI3730 full-automatic sequencer, and the nucleotide sequences of the 252bp and 147bp fragments are shown as SEQ ID NO.1 and SEQ ID NO. 2. The sequence was aligned with the entire circRNA sequences hsa _ circ _0045714 and hsa _ circ _0005567 using Vector NTI advance 10 software (Invitrogen corporation), and the alignment showed that the nucleotide sequence shown in SEQ ID NO:1 was a partial sequence of the hsa _ circ _0045714 gene with a 100% percent identity, and the nucleotide sequence shown in SEQ ID NO:2 was a partial sequence of the hsa _ circ _0005567 gene with a 100% identity.

EXAMPLE 3 osteoarthritis diagnostic kit

The kit for osteoarthritis diagnosis according to the present invention, which includes primer sets for specific amplification of hsa _ circ _0045714 and hsa _ circ _0005567 as shown in table 1, was assembled based on the primer set obtained in example 2, and specifically:

1. the kit comprises specific amplification hsa _ circ _ 0045714: SEQ ID NO.3 and SEQ ID NO. 4;

2. the kit comprises specific amplification hsa _ circ _ 0005567: SEQ ID NO.5 and SEQ ID NO. 6;

3. the kit comprises specific amplification hsa _ circ _0045714 and hsa _ circ _ 0005567: SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO. 6.

And a primer pair for specifically amplifying the housekeeping Gene (GAPDH) is shown as SEQ ID NO. 7 and SEQ ID NO. 8; it also comprises SYBR Green polymerase chain reaction system, such as PCR buffer solution, SYBR Green fluorescent dye, dNTPs. The PCR buffer solution comprises 25mM KCl and 2.5mM MgCl₂，200mM(NH₄)₂SO₄(ii) a The kit also comprises bone joint cartilage normal tissue cDNA: as a negative control, the PCR assay was quantified together with the cDNA of the test sample, and the same amount of cDNA as the test sample was used for each reaction system.

The optimal reaction system was determined by optimizing the primer concentration and annealing temperature as shown in Table 5:

TABLE 5 PCR reaction System

Components	Amount of addition
		SYBR Green polymerase chain reaction system	12.5μL
Upstream primer (10. mu.M)	0.5μL
		Downstream primer (10. mu.M)	0.5μL
Template cDNA	2.0μL
		Adding sterilized distilled water	To 25 μ L

The optimal reaction conditions are as follows:

pre-denaturation at 95 ℃ for 5min, (denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 45sec, extension at 72 ℃ for 35 sec). times.40 cycles, extension at 72 ℃ for 15 min.

Example 4 osteoarthritis diagnostic kit test Effect

A small amount of cartilage tissue cells of 30 patients to be examined for osteoarthritis are obtained from Beijing coordination hospital orthopedics. All clinical samples from this study were informed and passed through the ethical committee of this hospital. Extracting RNA from cartilage tissue cells by using a conventional method (or using a specific kit), carrying out PCR reaction by using reagents in the kit according to an optimal reaction system and conditions, using normal cartilage tissue cDNA in the kit as control cDNA in Real-Time PCR quantitative detection, detecting the expression quantity change of hsa _ circ _0045714 and/or hsa _ circ _0005567 of a tissue sample relative to normal cartilage tissue, analyzing a detection result, comparing the sample with the control by adopting a t test, wherein the difference is obvious when P <0.05 is positive, and judging the detection sample to be positive.

1. Detection effect of kit with hsa _ circ _0045714 as molecular marker

The test results showed that in 30 patients tested, 24 patients had hsa _ circ _0045714 expression levels of osteoarthritic chondrocytes 40-100 times higher than those of normal tissues, and in 6 patients no difference was observed in the expression level. Through further clinical detection, the result is basically consistent with the detection result of the kit prepared by the invention. Therefore, the diagnosis kit can clearly distinguish osteoarthritis patients and provide diagnosis clues for clinic.

2. Detection effect of kit with hsa _ circ _0005567 as molecular marker

The test results showed that in 30 patients tested, hsa _ circ _0005567 in 22 patients had 300 times higher osteoarthritic chondrocyte expression level than in normal tissue, and in 8 patients no difference was observed in expression level. Through further clinical detection, the result is basically consistent with the detection result of the kit prepared by the invention. Therefore, the diagnosis kit can clearly distinguish osteoarthritis patients and provide diagnosis clues for clinic.

3. Detection Effect of the kit with hsa _ circ _0045714 and hsa _ circ _0005567 as molecular markers

The test results showed that of 30 patients tested, 26 patients had hsa _ circ _0005567 and hsa _ circ _0005567 expression levels of osteoarthritic chondrocytes 50-250 times higher than those of normal tissues, and no difference was observed in the expression levels of 4 patients. Through further clinical detection, the result is basically consistent with the detection result of the kit prepared by the invention. Therefore, the diagnosis kit can clearly distinguish osteoarthritis patients and provide diagnosis clues for clinic.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Application of circRNA in preparation of product for diagnosing osteoarthritis

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Claims

Use of hsa _ circ _0045714 and hsa _ circ _0005567 as molecular markers in the manufacture of a kit for the diagnosis of osteoarthritis, said hsa _ circ _0045714 and hsa _ circ _0005567 both being up-regulated in osteoarthritic tissue; the hsa _ circ _0045714 acts on the UNK gene, the UNK gene expression is down-regulated when the hsa _ circ _0045714 expression is up-regulated, the hsa _ circ _0005567 acts on the HEATR5B gene, and the HEATR5B gene expression is up-regulated when the hsa _ circ _0005567 expression is up-regulated;

the hsa _ circ _0045714 marker is a nucleic acid sequence shown as SEQ ID NO. 1; the hsa _ circ _0005567 marker is a nucleotide sequence shown in SEQ ID NO. 2;

the kit comprises a primer sequence for specifically amplifying a nucleic acid sequence shown by SEQ ID NO.1, wherein the primer sequence is shown as SEQ ID NO.3 and SEQ ID NO. 4; the kit comprises primer sequences for specifically amplifying the nucleotide sequence shown by SEQ ID NO.2, which are shown as SEQ ID NO.5 and SEQ ID NO. 6.
2. The use of claim 1, wherein the kit further comprises 10 x Buffer, dNTP, Mg²⁺Taq enzyme and fluorescent dye.