CN112094901A

CN112094901A - Application of long-chain non-coding RNA in diagnosis and treatment of bone diseases

Info

Publication number: CN112094901A
Application number: CN202011053399.1A
Authority: CN
Inventors: 杨承刚; 向常娟
Original assignee: Gu'an Bojian Biotechnology Co ltd
Current assignee: Gu'an Bojian Biotechnology Co ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2020-12-18

Abstract

The invention discloses application of long-chain non-coding RNA in diagnosis and treatment of bone diseases. The number of the long non-coding RNA is ENSG 00000265478. The invention also discloses a new drug target for treating osteoarthritis, thereby providing a theoretical basis for developing a drug for treating osteoarthritis.

Description

Application of long-chain non-coding RNA in diagnosis and treatment of bone diseases

Technical Field

The invention belongs to the field of biomedicine, and relates to application of long-chain non-coding RNA in diagnosis and treatment of bone diseases.

Background

Osteoarthritis (OA), also known as osteoarthropathy, is the earliest known as proliferative arthritis or hypertrophic arthritis, and is frequently seen in middle-aged and elderly people, and the incidence of the osteoarthritis is higher and higher as the aging process of the population in the whole society is increased. Osteoarthritis is better caused by heavier and more active joints, the knee joint is one of the most common incidence sites of osteoarthritis, according to relevant data statistics, the incidence rate of osteoarthritis is second to heart disease in the population over 50 years old worldwide, and the world health organization determines the global health topic of 2000 + 2010 as 'bone and joint ten years'. Osteoarthritis is not only a medical problem but also a social problem, and is increasingly attracting attention.

Synovial tissue is the lining structure located inside the joint cavity, and various intra-articular diseases affect the synovium. Synovial cells are important tissue structures for maintaining the normal function of joints, and are also the main lesion sites in various joint diseases. Osteoarthritis (OA) is characterized by degenerative changes in the articular cartilage, the pathological changes affecting the various components of the joint, but in no way limited to cartilage, including subchondral bone, synovium, meniscus and ligaments. Pathological changes of the components affect each other and interact with each other to accelerate joint degeneration. With the intensive research on OA in recent years, it was found that the synovium is involved in the entire pathological process of OA. In the early stage of OA, the proliferation and fibrosis of the synovium, secretion of inflammatory mediators and cartilage degrading enzymes are important factors in the pathogenesis of OA, and the synovium is always involved in its pathological process as OA further develops. Synovial fibroblasts are the most active component cells of synovial tissues, can produce a large amount of inflammatory cytokines and immune response mediators in an autocrine or paracrine manner, accelerate the degradation of cartilage matrix, and are the main factors causing various clinical symptoms. In OA joint synovium, synovial membrane lower layer fibroblasts proliferate, especially around blood vessel fibroplasia, thereby causing the decrease of synovial membrane vascular permeability, hindering the normal exchange of synovial fluid and blood plasma, and making the intra-articular synovial fluid component unable to obtain oxygen and nutrient substances in blood. The cartilage cells in the joints lack nutrition and have atrophy functions, the capacity of synthesizing matrixes is reduced, the buffering capacity on force is continuously reduced, and the microenvironment for survival of the cartilage cells is further deteriorated. Therefore, inhibition of synovial cell proliferation is expected to be a new strategy for the treatment of osteoarthritis.

Disclosure of Invention

The invention aims to provide a long-chain non-coding RNA marker for diagnosing and treating osteoarthritis. The QPCR experiment proves that the expression level of the long-chain non-coding RNA in the blood of the osteoarthritis patient is obviously higher than that in the blood of a healthy control, and in addition, in vitro cells prove that the inhibition of the expression of the long-chain non-coding RNA can inhibit the proliferation of synovial cells, so the long-chain non-coding RNA can be used as a molecular marker for diagnosing and treating osteoarthritis.

In order to test the purpose, the invention adopts the following technical scheme:

the invention provides an application of a reagent for detecting long-chain non-coding RNA expression in preparing osteoarthritis diagnosis products; the ID of the long non-coding RNA is ENSG00000265478, and a specific sequence can be inquired at an Ensembl website.

Further, the reagent comprises a PCR amplification primer used for detecting the expression quantity of the long-chain non-coding RNA by using SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a composite probe.

In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.

The invention provides a product for osteoarthritis diagnosis, which comprises a reagent for detecting the expression level of the long-chain non-coding RNA.

Further, the reagent comprises SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a PCR amplification primer used for detecting the expression quantity of the long-chain non-coding RNA by using a composite probe.

Further, the aforementioned products include, but are not limited to, chips, kits, test strips, or high throughput sequencing platforms; the high-throughput sequencing platform is a special tool for diagnosing osteoarthritis, and with the development of a high-throughput sequencing technology, the construction of an RNA expression profile of a person becomes very convenient work. By comparing the RNA expression profiles of patients with disease and normal populations, it is easy to identify which RNA abnormalities are associated with disease. Therefore, the application of the long-chain non-coding RNA to the high-throughput sequencing to obtain the correlation between the expression abnormality of the long-chain non-coding RNA and osteoarthritis also belongs to the protection scope of the invention.

The kit comprises a reagent for detecting the expression quantity of the long non-coding RNA, the reagent comprises nucleic acid combined with the long non-coding RNA or a DNA sequence thereof, and the nucleic acid comprises SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a PCR amplification primer used when a composite probe is used for detecting the expression quantity of the long non-coding RNA.

The chip comprises a reagent for detecting the expression quantity of the long non-coding RNA, the reagent comprises nucleic acid combined with the long non-coding RNA or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression quantity of the long non-coding RNA.

The test paper comprises a reagent for detecting the expression quantity of the long non-coding RNA, the reagent comprises nucleic acid combined with the long non-coding RNA or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression quantity of the long non-coding RNA.

The present invention provides a pharmaceutical composition for the treatment of osteoarthritis comprising an agent that inhibits the long non-coding RNA described above.

Further, the agent is not limited as long as it can inhibit the expression level of the long non-coding RNA or the functional activity of the long non-coding RNA.

In a specific embodiment of the present invention, the agent for inhibiting the long non-coding RNA is an agent for inhibiting the expression of the long non-coding RNA. The agent for inhibiting the expression of the long non-coding RNA includes an interfering RNA of the long non-coding RNA. The interfering RNA includes siRNA and shRNA.

The pharmaceutical composition of the present invention may be administered alone or together with other drugs as a medicine. The other drug that can be administered together with the pharmaceutical composition of the present invention is not limited as long as it does not impair the effect of the therapeutic or prophylactic pharmaceutical composition of the present invention.

The pharmaceutical composition of the invention can be prepared into various dosage forms according to requirements. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The route of administration of the pharmaceutical composition of the present invention is not limited as long as it can exert the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, cutaneous, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.

The dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic or prophylactic pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.

The invention also provides the application of the long-chain non-coding RNA in preparing a medicament for treating osteoarthritis.

The invention also provides application of the reagent for inhibiting the expression of the long-chain non-coding RNA in preparing a medicament for treating osteoarthritis.

The present invention also provides a method for diagnosing osteoarthritis, the method comprising the steps of:

(1) obtaining a sample from a subject;

(2) detecting the expression level of the long-chain non-coding RNA in a sample of the subject;

(3) correlating the expression level of the long non-coding RNA with the disease of the subject.

(4) If the expression level of the long non-coding RNA is significantly increased as compared to a normal control, the subject is judged to have osteoarthritis, or the risk of the subject being judged to have osteoarthritis is high.

The present invention also provides a method for treating osteoarthritis, which comprises inhibiting the expression level of the long-chain non-coding RNA or inhibiting the regulatory activity of the long-chain non-coding RNA.

In the context of the present invention, "diagnosing osteoarthritis" includes determining whether a subject has suffered from osteoarthritis, determining whether a subject is at risk of suffering from osteoarthritis.

As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:

(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;

(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or

(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".

The invention has the advantages and beneficial effects that:

the invention discovers the molecular marker for diagnosing osteoarthritis, and the molecular marker can be used for judging the early stage of osteoarthritis occurrence, thereby improving the cure rate of patients.

The invention discovers a new drug target for treating osteoarthritis, thereby providing a theoretical basis for developing drugs for treating osteoarthritis.

Drawings

FIG. 1 shows a statistical graph of the differential expression of long non-coding RNAs using QPCR, where A: ENSG 00000265478; b: LLNLR-268E 12.1; c: CTC-338M 12.5;

FIG. 2 shows the results of CCK8 assay for the effect of long non-coding RNA expression on cell proliferation.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 differential expression of non-coding RNAs in osteoarthritis patients

1. Case collection

57 patients with osteoarthritis were collected, 28 men and 29 women with average age of about 61 years, and diseases such as rheumatoid arthritis, rheumatic arthritis and suppurative arthritis were excluded. As a control, 40 additional physical healthy persons were collected.

Osteoarthritis diagnosis is performed according to the diagnosis standard in "osteoarthritis diagnosis and treatment guidelines" issued by the Chinese medical society.

2. Blood Total RNA extraction

1. Extraction of Total RNA from Whole blood

1.1 homogenization treatment: subject lml blood was collected using an EDTA-containing anticoagulation vacuum tube, following a 1: 3M1 TRIPURE LS Reagent (Bioteke) was added and vortexed with a vortex mixer for 15-20s to help lyse the cells in the blood. And opening the high-speed low-temperature centrifuge for precooling.

1.2 incubation at 15-30 ℃ for 5min to completely break down the nucleoprotein body.

1.3 add 0.6m1 chloroform, cover the sample tube tightly, shake vigorously for 15s and incubate at room temperature for 3 min.

1.4 centrifugation was carried out for 10min at 12000rpm at 4 ℃ in a high speed cryocentrifuge, after which the sample was divided into three layers, whereupon the RNA was dissolved in the upper aqueous phase and the supernatant was transferred to a new centrifuge tube.

1.5 adding 75% ethanol with the same volume, mixing by gently inverting, and pouring the precipitate and liquid together into an adsorption column RA sleeved with a collecting tube.

1.6 repeated centrifugation at 10000rpm at 4 ℃ for 45s, and waste liquid is discarded until all precipitates and solution pass through the column.

1.7 Add 500. mu.l deproteinised RE (Bioteke), centrifuge at 12000rpm for 45s and discard the waste.

1.8 Add 700. mu.l of Wash RW (Bioteke), centrifuge at 12000rpm for 60s and discard the waste.

1.9 centrifuging at 4 deg.C and 12,000rpm for 2min to remove the rinsing liquid as much as possible to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction.

1.10 taking out the adsorption column RA, putting into a centrifuge tube of RNase free, adding 80. mu.l of RNase free water (hot bath at 65-70 ℃ in advance), standing at room temperature for 2min, and centrifuging at 12000rpm for 1 min.

4、QPCR

Reagent: the reverse transcription kit (DDR037A) was purchased from Bao bioengineering (Dalian) Co., Ltd. SYBR Premix Ex Taq for fluorescent Real-time (Real-time) quantitative PCR (polymerase chain reaction)^TM(Tli RNaseH Plus) kit was manufactured by Takara, Japan.

4.1 reverse transcription

Taking the extracted total RNA (1 mu g) as a template, adding the following reaction system:

Buffer 4μL，

RT Enzyme Mix 1. mu.L, Oligo dT Primer (50. mu.M) 1. mu.L, Random 6mers (100. mu.M) 1. mu.L, as RNase-free ddH₂O make up the reaction volume to 20. mu.L. The mixture was incubated at 37 ℃ for 15min and 85 ℃ for 5s to obtain cDNA. The cDNA can be used for IncRNA Real-time PCR detection.

4.2QPCR

According to Takara, Japan

Premix Ex Taq^TM(Tli RNaseH Plus) kit instructions.

The result is calculated by a relative quantitative method and a formula 2-delta ct. The experiment was repeated 3 times.

Designing a primer: primers were designed by the Primer design tool of NCBI (Primer BLAST) based on the long non-coding RNA transcript sequences as follows:

ENSG00000265478：

an upstream primer: 5'-TATAAGCAGCATCTACTAC-3' (SEQ ID NO. 1); a downstream primer: 5'-GATACGAACTTGAACTCT-3' (SEQ ID NO. 2);

LLNLR-268E12.1：

an upstream primer: 5'-TAGGAAGCCACAGAAGTAT-3' (SEQ ID NO. 3);

a downstream primer: 5'-CGAAGCGGAATCTACATT-3' (SEQ ID NO. 4);

CTC-338M12.5：

an upstream primer: 5'-AACTGGAGGATCACTTGA-3' (SEQ ID NO. 5);

an upstream primer: 5'-GCTTCTGGAACTTTATTTACTC-3' (SEQ ID NO. 6).

Primers were designed based on the GAPDH (internal reference gene) sequence, the upstream primer: 5'-AGAAGGCTGGGGCTCATTTG-3' (SEQ ID NO. 7); a downstream primer: 5'-AGGGGCCATCCACAGTCTTC-3' (SEQ ID NO. 8).

5. Results

The results are shown in figure 1, where long non-coding RNA: enug 00000265478, LLNLR-268E12.1, CTC-338M12.5 expression was significantly elevated with statistical significance (. P < 0.05).

Example 2 Effect of Long non-coding RNA on synovial cell proliferation

1. Cell culture

Cell: human osteoarthritic synovial cells (cat 408OA-05A) were purchased from Cell applications.

Culture medium: synovial Cell growth medium (cat. No.: 415-500) was purchased from Cell applications.

The culture method comprises the following steps: adding 10% fetal calf serum and double antibodies into the synovial cell growth culture medium to prepare a complete culture medium; taking out the frozen cells from the liquid nitrogen, placing the cells in a constant-temperature water bath box at 37 ℃, and shaking for several minutes until the cells are completely dissolved; sucking out the cell suspension, transferring the cell suspension into a centrifugal tube, centrifuging the cell suspension at 1000rpm for 5min, and removing the supernatant; adding complete culture medium, resuspending cells, counting cells, inoculating in culture flask, 37 deg.C, 5% CO₂Culturing in a cell culture box; the liquid is changed after 24 hours, and then the liquid is changed every 2 to 3 days.

2. Gene expression interference

siRNA and general negative control RNA aiming at long-chain non-coding RNA are synthesized from Shanghai Jima pharmaceutical technology GmbH, and the sequences are as follows:

siRNA against ENSG 00000265478:

sense strand: 5'-AGAGAAAUCCUAUAUAUCCCAtt-3' (SEQ ID NO. 9); antisense strand: 5'-GGAUAUAUAGGAUUUCUCUUAtt-3' (SEQ ID NO. 10);

siRNA against LLNLR-268E 12.1:

sense strand: 5'-UCAUUCAGCGCAUGGUUACUGtt-3' (SEQ ID NO. 11);

antisense strand: 5'-GUAACCAUGCGCUGAAUGAAUtt-3' (SEQ ID NO. 12);

siRNA against CTC-338M 12.5:

sense strand: 5'-UAAUUUUCUUUUAAGAGACUGtt-3' (SEQ ID NO. 13);

antisense strand: 5'-GUCUCUUAAAAGAAAAUUAAAtt-3' (SEQ ID NO. 14).

Human osteoarthritic synovial cells cultured in 6cm plates at logarithmic growth phase were transfected (see Liposome 2000 transfection instructions for specific procedures). After transfection, cells were cultured for 48h and tested for gene interference by QPCR, and the results are shown in table 1, where siRNA was effective in inhibiting expression of long non-coding RNA compared to negative control, and the difference was statistically significant (P × 0.05).

4. CCK-8 assay for cell proliferative Activity

1) Taking cells, digesting the cells by a conventional method, preparing a cell suspension, and counting the cells.

2) According to 1 x 10⁴The cell suspension was diluted at a concentration of one ml and seeded in 96-well plates at 200. mu.l per well, with 3 duplicate wells per set.

3) Cell transfection was performed as described previously.

4) At 72 hours post-cell transfection, 10. mu.l of CCK-8 solution was added to each well, the plates were gently tapped to aid mixing, and incubated in an incubator for 4 hours. After being taken out, the absorbance value (OD value) of the sample with the wavelength of 450nm is detected on a microplate reader.

5. Statistical analysis

All data were statistically calculated and analyzed using SPSS19.0 software, the measured data were expressed as means ± standard deviation, the difference between the two groups was analyzed by two-way variance, and if P <0.05, it was considered to be statistically significant.

6. Results

The proliferation activity of synovial cells was detected by CCK-8 method, and the result is shown in FIG. 2, the proliferation rate of the interfering group cells was slower than that of the negative control group cells, and the difference was statistically significant (P x <0.05) by statistical test analysis. The results show that the inhibition of the expression of the long-chain non-coding RNA can inhibit the proliferation of osteoarthritis synovial cells, thereby effectively inhibiting osteoarthritis.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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Claims

1. The application of the reagent for detecting the expression of the long-chain non-coding RNA in the preparation of osteoarthritis diagnosis products; the ID of the long non-coding RNA is ENSG 00000265478.

2. The use of claim 1, wherein the reagent comprises PCR amplification primers used for detecting the expression level of the long non-coding RNA by using SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, or composite probes.

3. The use according to claim 4, wherein the primer sequences are shown as SEQ ID No.1 and SEQ ID No. 2.

4. A product for use in the diagnosis of osteoarthritis comprising an agent that detects the expression of the long non-coding RNA of claim 1.

5. The product of claim 4, wherein the reagent comprises SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, or PCR amplification primers used in the detection of the expression level of the long non-coding RNA.

6. The product of claim 5, wherein the primer sequences are shown as SEQ ID No.1 and SEQ ID No. 2.

7. A pharmaceutical composition for treating osteoarthritis comprising an agent that inhibits the long-chain non-coding RNA of claim 1.

8. The pharmaceutical composition of claim 7, wherein the agent comprises an agent that inhibits the expression level of a long-chain non-coding RNA, or an agent that inhibits the functional activity of the long-chain non-coding RNA.

9. The pharmaceutical composition of claim 8, wherein the agent that inhibits the expression level of a long non-coding RNA comprises an interfering RNA to a long non-coding RNA.

10. Use of the long non-coding RNA of claim 1 in the manufacture of a medicament for the treatment of osteoarthritis.