CN108823300A

CN108823300A - Application of the circRNA on the product of preparation diagnosis osteoarthritis

Info

Publication number: CN108823300A
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: 2018-11-16
Anticipated expiration: 2038-06-20
Also published as: CN108823300B

Abstract

The invention discloses provide hsa_circ_0045714 and/or hsa_circ_0005567 as molecular marked compound preparation diagnosis or treatment osteoarthritis product on application, and further confirm hsa_circ_0045714 and hsa_circ_0005567 in osteoarthritic tissue expression up-regulation.The present invention further discloses the diagnostic kits that hsa_circ_0045714 and/or hsa_circ_0005567 are used to prepare detection osteoarthritis, and the kit includes the primer and specification of specific amplification osteoarthritis correlation circRNA.It can not only quickly and effectively accomplish early detection using hsa_circ_0045714 and/or hsa_circ_0005567 detection osteoarthritis, and provide therapy target and important evidence for clinical applications such as gene therapy, drug therapies.

Description

Application of the circRNA on the product of preparation diagnosis osteoarthritis

Technical field

The present invention relates to biomedicine fields, and in particular to hsa_circ_0045714 and/or hsa_circ_0005567 Application on the product of preparation diagnosis or treatment osteoarthritis.

Background technique

Osteoarthritis (osteoarthritis, OA) is the chronic disease that articular cartilage is gradually degenerated at any time, the pass Section cartilage is covered on bone end, forms the articular surface in joint.It is reported that there are many factors, and patient to be made to be susceptible to suffer from osteoarthritis, including lose Pass neurological susceptibility, obesity, accident or athletic injury, operation, drug and weight body demand force.Osteoarthritis is considered soft from joint What the damage of bone started.Most common two kinds are sports-related injury and long-term " Reusability " joint damage to the damage in joint Wound.The joint most frequently influenced by osteoarthritis is knee, hip and hand.In most cases, due to knee and the necessary load-bearing function of hip Can, the osteoarthritis in these joints more easily leads to deformity than Hand osteoarthritis.With the development of cartilage degradation, in joint and close Secondary variation occurs in its hetero-organization (including bone, muscle, ligament, meniscus and synovial membrane) around saving.Cartilaginous tissue primary declines Exhausting with the net effect of its hetero-organization secondary damage is that pain, swelling, weakness and diseased joints forfeiture function occur for patient.These diseases Shape is often developed to the degree seriously affected, is such as made patient disability or is influenced quality of life.

Articular cartilage is mainly made of cartilage cell, II Collagen Type VI, proteoglycans and water.Articular cartilage does not have blood or nerve Distribution, cartilage cell is only cell type in the tissue.The appearance of cartilage cell's responsibility system at cartilage matrix II Collagen Type VI And proteoglycans.Thereafter the matrix has the physicochemical characteristics for allowing to be made with water matrix to be saturated again.This structure-function relationship Net effect be that articular cartilage has special wear resistance characteristics, and it is mobile that almost friction free occurs between articular cartilage face. When being not suffering from osteoarthritis, articular cartilage often provides lifelong painless load-bearing and unlimited under the even physical qualification of high demand The joint of system is mobile.

As all living tissues, articular cartilage constantly undergoes renewal process, wherein removal (catabolic activity) " " cell and matrix components simultaneously generate (anabolic activity) " newly " cell and molecule always.It is organized relative to majority, articular cartilage Anabolism or catabolism turnover rate are lower.The long-term maintenance of mature cartilage structure integrality is synthesized and is degraded dependent on matrix Between balance appropriate.Cartilage cell is by response in chemistry and mechanical irritation maintenance matrix equilibrium in its environment.It is soft It is necessary to cartilage dynamic equilibrium that osteocyte, which irritates suitable and effective response to these,.By anabolic activity it is insufficient or Catabolic activity surplus destroy dynamic equilibrium can cause cartilage degradation and osteoarthritis (Adams etc., 1995, Nature377Suppl:3-174).Majority, which is damaged, can start the synthesis of enhancing with the tissue of catabolic activity raising It is metabolized response, tissue is allowed to restore.Unfortunately, articular cartilage response is damaged or is consumed in cartilage matrix and raises it and synthesize generation The ability for thanking activity and raising synthetic proteins glycan and II Collagen Type VI is very limited.

Existing osteoarthritis treatment method include movement, drug, rest and joint care, operation, pain relief techniques, Replacement therapy and body weight control.Treating common drug in osteoarthritis includes nonsteroidal anti-inflammatory such as aspirin, brufen Deng；It is directly used in the topical pain-relieving creams, rubber and spray etc. of skin.It can be performed the operation so that bone surface reconstruction, bone Reset and replacement joint.Although oneself be used to treat disease for various medicinal treatments, they are nothings to long-term control and prevention Effect.Further, since osteoarthritis hidden occur and develop slowly, therefore osteoarthritis often disease develop advanced stage It is just identified, rather than potential treatment may more effective early in disease development.Therefore prevention, change or treatment osteoarthritis Further development in lysis depends critically upon the early diagnosis marker of identification disease, so that early stage be allowed to interfere.

Summary of the invention

The purpose of the present invention is to provide a kind of application of new marker on the product for diagnosing or treating osteoarthritis.

To achieve the above object, the present invention provides hsa_circ_0045714 and/or hsa_circ_0005567 conducts Application of the molecular marked compound on the product of preparation diagnosis or treatment osteoarthritis.

Further, the hsa_circ_0045714 and hsa_circ_0005567 is in osteoarthritis biological sample Expression up-regulation.

Still further, the marker includes such as SEQ ID NO:1 and SEQ ID NO:Nucleotide sequence shown in 2.

Preferably, the product be being capable of quantitative detection hsa_circ_0045714 and/or hsa_circ_0005567 table Up to the product of amount.

More preferably, the product is osteoarthritis diagnostic kit.

Further, the kit include the relevant hsa_circ_0045714 of specific amplification osteoarthritis and/or The primer and specification of hsa_circ_0005567.

Still further, the primer sequence of the hsa_circ_0045714 such as SEQ ID NO:3 and SEQ ID NO:4 institutes Show；The primer sequence of the hsa_circ_0005567 such as SEQ ID NO:5 and SEQ ID NO:Shown in 6.

Further, the primer is suitable for the inspection of SYBR Green, TaqMan probe and/or molecular hybridization probe It surveys.

Also further, the kit further includes 10 × Buffer, dNTP, Mg²⁺, Taq enzyme and fluorescent dye.

Beneficial effects of the present invention are as follows：

Present invention firstly discloses hsa_circ_0045714 and hsa_circ_0005567 are related to osteoarthritis, go forward side by side One step confirms that hsa_circ_0045714 and hsa_circ_0005567 express up-regulation in osteoarthritic tissue, more than utilization CircRNA detects osteoarthritis, can not only quickly and effectively accomplish early detection, is also gene therapy, drug therapy etc. is faced Bed application provides therapy target and important evidence.

Detailed description of the invention

Fig. 1 hsa_circ_0045714 and hsa_circ_0005567 and its possible target gene are in osteoarthritic tissue sample Expression in this.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means.With reference to the accompanying drawings and examples to the present invention It is described in detail.

Test method without specific conditions in embodiment, usually conventional method in that art, such as according to normal conditions Such as Sambrook et al., molecular cloning, the condition in laboratory manual (third edition) (Science Press, 2002), or according to Condition proposed by reagent manufacturing firm.

The present inventor carries out high-throughput transcript profile to OA patient's cartilaginous tissue sample and normal articular cartilage sample Sequencing, by bioinformatics method carry out genescreen, pick out the apparent hsa_circ_0045714 of differential expression and Hsa_circ_0005567, there is no hsa_circ_0045714 and hsa_circ_0005567 and osteoarthritis in existing research Relevant report, further, inventor have carried out molecular biology method verifying, it was confirmed that hsa_circ_0045714 and hsa_ Circ_0005567 expresses up-regulation in osteoarthritic tissue.

Hsa_circ_0045714 and hsa_circ_0005567 of the invention is before making the present invention known CircRNA can be retrieved (http by circBase://circbase.org), essential information is as follows：

Circbase ID：Hsa_circ_0045714 is located at No. 17 chromosome of the mankind, chr17:73808192- 73809959；Circbase ID：Hsa_circ_0005567 is located at No. 1 chromosome of the mankind, chr1:51868106- 51874004, derive from human genome.

The particular form of terms used herein " osteoarthritis " articulations digitorum manus inflammation, especially articular cartilage are gradually moved back at any time The chronic disease of change, the articular cartilage covering re-form on the bone end of articular surface.

Terms used herein " expression up-regulation " refer to the sequence corresponding to expressed gene, wherein the measurement card of sequence amount It is bright, it is determined in the individual for having identified morbid state different with osteoarthritis by stages from from normal individual or from by osteoarthritis Same gene in isolated biological sample is compared, and the gene is from suffering from osteoarthritis or determined by osteoarthritis by stages Osteoarthritis identified the expression in the biological sample separated in the individual of morbid state increase.According to the present invention, " expression up-regulation " refer to intensity for hybridization measurement by the method for the invention at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or higher expression increases, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more It is high or be higher than 1 times, up to 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or higher.

Terms used herein " expression " refer to through the measurement of known to those skilled in the art and method described herein Given nucleic acid or protein can measure.When being related to the corresponding circRNA of molecular marked compound of the present invention, expression can lead to Hybridization or more is crossed to measure, such as includes green, TaqMan and quantitative real-time RT-PCR using SYBR.

1 high-flux sequence of embodiment screens difference expression gene

1, it samples

Take acquisition 19 (average ages 64.5 of cartilaginous tissue sample in BJ Union Hospital's orthopaedics osteoarthritis surgery Year, the range of age 58-75 years old, 2 males, 17 women), all samples are confirmed through pathological examination, are diagnosed as knee joint bone Arthritis；Normal articular cartilage is trauma surgery patient articular cartilage tissue from BJ Union Hospital's orthopaedics.The group of acquisition - 80 DEG C of low temperature refrigerators of number postposition are woven in save.Clinical sample used in this research know to patient and informs and pass through Ethics Committee of the court passes through.

2, Total RNAs extraction is carried out to tissue samples

UsingReagent (Invitrogen, Carlsbad, CA, USA) carries out sample rna extraction, experiment Operation is carried out by product description, and concrete operations are as follows：

It freezes to be put into the mortar being pre-chilled tissue after liquid nitrogen, taking-up after collection sample and be ground, sample to be organized This is at after powdered：

1. Trizol is added, room temperature preservation 5 minutes；

2. chlorination imitates 0.2mL, with forced oscillation centrifuge tube, mix well, places -10 minutes 5 minutes at room temperature；

3. drawing upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15 minutes into another new centrifuge tube pipe, pay attention to The protein substance that be not drawn between two layers of water phase.New pipe is moved into, the pre- cold isopropanol of isometric -20 DEG C is added, it is sufficiently reverse It mixes, is placed in 10 minutes on ice；

4. 12000rpm high speed from 15 minutes after carefully discard supernatant, in 1mL/mL Trizol ratio be added 75% DEPC ethanol washing precipitating (4 DEG C of preservations), washing precipitate, oscillation mixes, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；

5. discarding ethanol liquid, 5 minutes are placed at room temperature sufficiently to dry precipitating, it is heavy that the processed water dissolution of DEPC is added It forms sediment；

6. usingSpectrophotometer (IMPLEN, CA, USA) measures RNA purity and use RNA Assay Kit in 2.0Flurometer detection kit (Life Technologies, CA, USA) concentration, it freezes in -80 DEG C.Pass throughThe extraction feelings of spectrophotometer detection RNA sample Condition, the sample requirement of RNA-seq sequencing：OD₂₆₀/OD₂₈₀For 1.8-2.2.

RNA quality judging standard：The OD of RNA sample₂₆₀/OD₂₈₀Value is between 1.7-2.2；Total serum IgE electrophorogram has clearly 28S, 18S band；70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.

RNA integrity assessment uses 2100 system of 6000 detection kit of RNA Nano and biological analyser (Agilent Technologies,CA,USA).Normal group and each sample of osteoarthritis group are diluted to same concentration, by normal group Sample and osteoarthritis group sample prepare building and sequencing for library respectively.

3, sequencing preparation and high-flux sequence

Each RNA sample inputs 3 μ g.First with Epicentre Ribo-zero^TMRRNA Removal kit The total serum IgE that (Epicentre, USA) extracts upper step removes rRNA, then removes rRNA free residual object with ethanol precipitation. It recycles Ultra^TMDirectional RNA Library Prep kit (NEB, USA) is according to explanation Book prepares sequencing library, and index coding is added in the sequence of attributes of each sample.By sequencing library the first of NEBNext Bivalent cation is utilized in chain synthesis reaction buffer (First Strand Synthesis Reaction Buffer, 5 ×) Cracked under high temperature.The first chain is synthesized using random primer (random hexamer primer) and M-MuLV reverse transcriptase cDNA.The second chain cDNA then is carried out with DNA polymerase i and RNase H to synthesize, and in reaction buffer, replaces dNTPS with dUTP In dTTP.CDNA segment is converted into flat end with polymerase and exonuclease again.By DNA fragmentation 3 ' hold phosphorylation it Afterwards, it is connect with the NEBNext Adaptor with hairpin structure in case hybridization.It is about 150~200bp's to select length CDNA segment purifies library fragments with ApHealthXP system (Beckman Coulter, Beverly, USA).Then Size selection is carried out with the USER Enzyme (NEB, USA) of 3 μ L, cDNA is connected at 37 DEG C and is carried out 15 minutes, then at 95 DEG C It carries out 5 minutes.PCR is carried out using Phusion high-fidelity DNA polymerase.According to specification in cBot Cluster Volume is indexed using TruSeq PE Cluster Kit v3-cBot-HS kit (Illumia) in Generation system The cluster of code sample, then high-flux sequence is carried out by 4000 platform of Illumina Hiseq, Sequenced Reads is obtained, Later with reference to related species reference sequences or genome, analysis of biological information is carried out.

4, detection data is analyzed

4.1 Reads mass controls

The data of the Fastq form obtained in upper step by Script Programming inside initial data, remove belt lacing first Reads, the reads comprising poly N and low-quality reads obtain clean data.Meanwhile calculating clean data's Q20, Q30 and G/C content.All subsequent analysis are all based on the clean data of high quality.

4.2 Reads, which are compared, refers to gene

Genome and gene group model comment file are directly downloaded from genome website.Pass through with reference to gene group index Bowtie2v2.2.8 is constructed, and the clean reads of both-end by HISAT2v2.0.4.HISAT2 and refers to genome Match.

The identification of 4.4 circRNA

Use find_circ (Sebastian Memczak et al., 2013) identification circRNA.The base of find_circ Present principles are never to compare to the both ends of the reads of reference sequences the anchor sequence for respectively extracting 20-nt, by every a pair Anchor sequence compares reference sequences again, if the end 5' of anchor sequence is compared to reference sequences (starting and termination site It is denoted as A3, A4 respectively), while the end 3' of the anchor sequence compares to the upstream in this site and (originates and remember respectively with termination site For A1, A2), and there are splice site (GT-AG) between A2 to the A3 of reference sequences, then using this read as candidate circRNA.Finally the candidate circRNA using read count more than or equal to 2 is as the circRNA of identification.

The expression of 4.5 circRNA is analyzed

Carry out the statistics of expression quantity to known in each sample and new circRNA, and with TPM (Zhou et al., 2010) into Row expression quantity normalized, obtains the readcount of sample.

4.6 circRNA Differential expression analysis results

The input data of circRNA differential expression is readcount data obtained in the analysis of circRNA expression. For there is the duplicate sample of biology, analyze we using based on negative binomial distribution DESeq2 (Michael et al., 2014) it is analyzed；Sample duplicate for abiology is first standardized readcount data using TMM, Variance analysis is carried out with DEGseq (Wang et al., 2010) later.

The further analysis of 4.7 differential expression circRNA

In order to better understand the function of difference expression gene, we have carried out Gene to difference expression gene Ontology (GO) analysis, signal path (KEGG) analysis and circRNA miRNA bindingsite assay (for animal and Plant, respectively using the binding site of the miRNA of the circRNA after the shearing of miRanda and psRobot software prediction), and to difference Different expressing gene carries out functional annotation, in view of above data analysis as a result, we have screened differential expression in conjunction with document Hsa_circ_0045714 and hsa_circ_0005567 is used for the research of the application, cartilage of the above circRNA in OA patient Up-regulation is expressed in tissue samples.

Cartilaginous tissue hsa_circ_0045714 and the hsa_circ_0005567 table of embodiment 2RT-PCR verifying OA patient Up to situation

1, material

17 osteoarthritic cartilage tissues samples are derived from BJ Union Hospital's osteoarthritis surgery (average age 66 years old), It is grouped and is numbered.All sample standard deviations confirm that control group is meniscus injury and ligamentaum cruciatum by pathological examination Damage carries out the patient of arthrocsopic surgery treatment.- 80 DEG C of low temperature refrigerators of all sample number postpositions save.

2, expression analysis method

2.1 pairs of tissue samples carry out Total RNAs extraction, with the extracting method of embodiment 1.

2.2 reverse transcriptions synthesize cDNA

UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) CDNA reverse transcription is carried out, experimental implementation is carried out by product description, and concrete operations are as follows：

Using Reverse Transcriptase kit, reverse transcription synthesis cDNA is carried out to l μ g total serum IgE with RT Buffer.Using 25 μ L Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid.- 20 DEG C are stored in after the cDNA sample of acquisition is diluted 10 times Refrigerator is spare.

2.3Real-Time PCR

2.3.1 instrument and analysis method

With 7500 type fluorescence quantitative PCR instrument of ABI, using 2^-ΔΔCtMethod carries out data relative quantitative assay.

2.3.2 design of primers

Using online primer-design software, gene order is referring to Circbase ID：Hsa_circ_0045714 and hsa_ Circ_0005567, interior participation in the election GAPDH are synthesized by invitrogen company after design of primers.Specific primer sequence is as follows：

1 primer sequence of table

Operating process is as follows：

2 Real Time reaction system of table

Component	Additional amount
		2×mix	10μL
Upstream primer (10 μM)	0.5μL
		Downstream primer (10 μM)	0.5μL
Template	2μL
		Sterile purified water is added	To 25 μ L

Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) is respectively to mesh Gene primer and reference gene primer expanded.Experimental implementation is carried out by product description.Amplification program is：95℃ 5min, (95 DEG C of 15sec, 60 DEG C of 45sec, 72 DEG C of 35sec) × 40 circulations.Simultaneously in 60-95 DEG C of progress solubility curve analysis. After reaction, the PCR product of 5 μ l is taken to carry out 2% agarose electrophoresis, with quick plastic recovery kit (invitrogen company) The segment that size is 252bp and 147bp is subjected to gel extraction and is sequenced, as a result carries out homology analysis with blast software.

3, experimental result

Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and baseline is put down without the phenomenon that raises up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR：2^-ΔΔCt× 100%, compare hsa_circ_0045714 and hsa_circ_0005567 in osteoarthritic tissue and normal tissue Expression, and the circRNA- of hsa_circ_0045714 and hsa_circ_0005567 are predicted by CircNET MiRNA-mRNA acts on network, and the expression of corresponding target gene mRNA, prediction hsa_circ_0005567 are acted on HEATR5B gene, hsa_circ_0045714 act on UNK gene.Result above demonstrates high-throughput transcript profile expression data Confluence analysis hsa_circ_0045714 and hsa_circ_0005567 expressed in Human Osteoarthritis up-regulation as a result, point It Shang Tiao not be about 128 times and 512 times of normal tissue, log2Fold_change=log2 (normal tissue sample/osteoarthritis Tissue samples), concrete outcome is shown in Fig. 1；After RT-PCR product is recovered, it is sequenced on the full-automatic sequenator of ABI3730, it should The nucleotide sequence of the segment of 252bp and 147bp is as shown in SEQ ID NO.1 and SEQ ID NO.2.With Vector NTI 10 software of advance (Invitrogen company) is whole by the sequence and hsa_circ_0045714 and hsa_circ_0005567 A circRNA sequence is compared, and comparison result is shown, such as SEQ ID NO:Nucleotides sequence shown in 1 is classified as hsa_circ_ A part of sequence of 0045714 gene, coincidence rate 100%, SEQ ID NO:Nucleotides sequence shown in 2 is classified as hsa_circ_ A part of sequence of 0005567 gene, coincidence rate 100%.

3 osteoarthritis diagnostic kit of embodiment

Based on the primer sets that embodiment 2 obtains, the kit of the present invention for osteoarthritis diagnosis is assembled, it is described Kit includes that the primer sets of specific amplified hsa_circ_0045714 and hsa_circ_0005567 are as shown in table 1, specially：

1, kit includes specific amplification hsa_circ_0045714：SEQ ID NO.3 and SEQ ID NO.4；

2, kit includes specific amplification hsa_circ_0005567：SEQ ID NO.5 and SEQ ID NO.6；

3, kit includes 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.

With the primer pair such as SEQ ID NO of specific amplified house-keeping gene (GAPDH):7 and SEQ ID NO:Shown in 8；Also wrap SYBR Green polymerase chain reaction system is included, such as PCR buffer, SYBR Green fluorescent dye, dNTPs.The PCR is slow The ingredient of fliud flushing is 25mM KCl, 2.5mM MgCl₂, 200mM (NH₄)₂SO₄；It further include bone articular cartilage normal tissue cDNA： As negative control and the detection common quantitative PCR detection of sample cDNA, each reaction system use is identical as detection sample cDNA Amount.

By the optimization to primer concentration and annealing temperature, determine that optimal reaction system is as shown in table 5：

5 PCR reaction system of table

Component	Additional amount
		SYBR Green polymerase chain reaction system	12.5μL
Upstream primer (10 μM)	0.5μL
		Downstream primer (10 μM)	0.5μL
Template cDNA	2.0μL
		Sterile purified water is added	To 25 μ L

Optimum reaction condition is：

95 DEG C of initial denaturation 5min, (95 DEG C of denaturation 15sec, 60 DEG C of annealing 45sec, 72 DEG C of extension 35sec) × 40 circulations, 72 DEG C of extension 15min.

4 osteoarthritis diagnostic kit detection effect of embodiment

The a small amount of cartilaginous tissue cell for taking the medical patient of 30 osteoarthritis to be detected, comes from BJ Union Hospital's orthopaedics. All clinical samples of this research know to patient and inform and pass through through this Hospital Ethical Committee.Use conventional side Method (or using specific kit) extracts RNA from cartilaginous tissue cell, using seminal plasma fructose detection kit, according to optimum response body System reacts with condition progress PCR, uses in kit normal articular cartilage cDNA as in Real-Time PCR quantitative detection CDNA is compareed, detects the hsa_circ_0045714 and/or hsa_circ_0005567 of tissue samples with respect to normal articular cartilage Expression quantity variation, analysis detection are examined as a result, comparing between sample and control using t, P<0.05 is significant difference, is judged to detection sample This positive.

1, detection effect of the hsa_circ_0045714 as the kit of molecular marked compound

Testing result shows in 30 patients to be detected, have the hsa_circ_0045714 of 24 patients in Bones and joints Scorching chondrocytes expressed level is 40-100 times in normal tissue, and in addition 6 expression quantity do not find differences.Through clinical further Detection, it is as a result almost the same with kit test result prepared by the present invention.Infer accordingly, this diagnostic kit can specify area Human Osteoarthritis is separated, and provides diagnostic clue as clinic.

2, detection effect of the hsa_circ_0005567 as the kit of molecular marked compound

Testing result shows in 30 patients to be detected, have the hsa_circ_0005567 of 22 patients in Bones and joints Scorching chondrocytes expressed level is 150-300 times in normal tissue, and in addition 8 expression quantity do not find differences.Through clinic into one Step detection, it is as a result almost the same with kit test result prepared by the present invention.Infer accordingly, this diagnostic kit can define Human Osteoarthritis is distinguished, and provides diagnostic clue as clinic.

3, detection effect of the hsa_circ_0045714 and hsa_circ_0005567 as the kit of molecular marked compound

Testing result is shown, in 30 patients to be detected, there is the hsa_circ_0005567 and hsa_ of 26 patients Circ_0005567 is at 50-250 times that osteoarthritis chondrocytes expression is in normal tissue, and in addition 4 expression quantity are not It finds differences.It is as a result almost the same with kit test result prepared by the present invention through clinical further detection.Infer accordingly, This diagnostic kit can clearly distinguish Human Osteoarthritis, and provide diagnostic clue as clinic.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

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Claims

1.hsa_circ_0045714 and/or hsa_circ_0005567 is closed as molecular marked compound in preparation diagnosis or treatment bone Save the application on scorching product.

2. application as described in claim 1, which is characterized in that the hsa_circ_0045714 and hsa_circ_0005567 The expression up-regulation in osteoarthritis biological sample.

3. application as claimed in claim 2, which is characterized in that the hsa_circ_0045714 acts on UNK gene, described UNK down regulation of gene expression described in timing, the hsa_circ_0005567 are acted in hsa_circ_0045714 expression HEATR5B gene, HEATR5B gene expression described in timing is also raised in the hsa_circ_0005567 expression.

4. application as described in claim 1, which is characterized in that the marker includes such as SEQ ID NO:1 and SEQ ID NO:Nucleotide sequence shown in 2.

5. application as described in claim 1, which is characterized in that the product is osteoarthritis diagnostic kit.

6. application as claimed in claim 5, which is characterized in that the kit includes that specific amplification osteoarthritis is relevant The primer and specification of hsa_circ_0045714 and/or hsa_circ_0005567.

7. application as claimed in claim 5, which is characterized in that the primer sequence of the hsa_circ_0045714 such as SEQ ID NO:3 and SEQ ID NO:Shown in 4.

8. application as claimed in claim 5, which is characterized in that the primer sequence of the hsa_circ_0005567 such as SEQ ID NO:5 and SEQ ID NO:Shown in 6.

9. application as claimed in claims 6 or 7, which is characterized in that the primer is suitable for SYBR Green, TaqMan probe And/or the detection of molecular hybridization probe.

10. application as claimed in claim 5, which is characterized in that the kit further includes 10 × Buffer, dNTP, Mg²⁺、 Taq enzyme and fluorescent dye.