CN113265459B - Application of miRNA-601 as a molecular marker in the diagnosis and treatment of osteoarthritis - Google Patents

Abstract

The invention discloses application of miR-601 as a molecular marker in diagnosis and treatment of osteoarthritis, and belongs to the field of biological medicines. The invention provides a molecular marker miR-601 related to osteoarthritis, and the nucleotide sequence is 5'-UGGUCUAGGAUUGUUGGAGGAG-3' (SEQ ID No. 1). The marker is expressed and reduced in osteoarthritis patients, and the purpose of diagnosing OA can be achieved by carrying out specific detection on miR-601; and the detection of proliferation capability and apoptosis capability of the OA chondrocyte shows that the miR-601 can improve the proliferation capability of the OA chondrocyte and reduce the OA chondrocyte apoptosis. Therefore, by detecting miR-601, the early detection of osteoarthritis can be rapidly and effectively achieved, and an important basis is provided for miR-601 targeted treatment.

Description

Application of miRNA-601 as a molecular marker in the diagnosis and treatment of osteoarthritis

technical field

The invention relates to the field of biomedicine, in particular to the application of miR-601 as a molecular marker in the diagnosis and treatment of osteoarthritis.

Background technique

Osteoarthritis (OA) is a common degenerative joint disease in the elderly, which brings a huge burden to the patient's family and society (Nelson AE. Osteoarthritis year in review2017: clinical. Osteoarthritis Cartilage. 2018, 26(3):319-325.). The main pathological features of osteoarthritis include articular cartilage erosion, synovitis and subchondral bone degeneration, but the exact mechanism of its occurrence is unclear (Hunter DJ, Bierma-Zeinstra S. Lancet. 2019, 393(10182):1745- 1759.). Existing studies have shown that multiple pathological factors such as overload, trauma, imbalance of the inflammatory system, and damage to the anti-inflammatory pathway are jointly involved in the occurrence and development of osteoarthritis, resulting in cell senescence, decreased cell density, abnormal secretion activity, extracellular Matrix (ECM) degradation, impaired articular cartilage development, etc. (Robinson WH, Lepus CM, Wang Q et al. Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 2016, 12(10):580- 92.).

Conservative treatment is currently the main treatment for OA, mainly limited to control pain. Multiple drugs such as NSAIDs, cyclooxygenase-2 (COX-2) inhibitors, glucosamine, steroids, and hyaluronic acid (Philp AM, Davis ET, Jones SW. Developing anti-inflammatory therapeutics for patients withosteoarthritis. Rheumatology (Oxford). 2017,56(6):869-881. ) has been clinically used to delay the progression of OA, but these drugs are limited to pain control and cannot reverse osteoarthritis, and long-term use is Has significant side effects (Cutolo M, Berenbaum F, Hochberg M, et al. Commentary on recent therapeutic guidelines for osteoarthritis. Semin Arthritis Rheum. 2015, 44(6):611-617.). Joint replacement is the main method for the treatment of advanced OA. Although its effect is remarkable, it is expensive and has a certain period of use (Zhang W, Ouyang H, Dass CR, et al. Current research onpharmacologic and regenerative therapies for osteoarthritis. Bone Res. 2016 , 4:15040. ).

MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded RNA molecules with a total length of about 19-23 nucleotides, which mainly regulate gene expression levels by degrading target mRNAs or interfering with translation, and then participate in various biological processes. . A large number of studies have confirmed that miRNAs play an important role in the occurrence and development of tumors, inflammation, diabetes and other diseases. Recently, miRNA was found to be involved in the occurrence and development of osteoarthritis (Tavallaee G, Rockel JS, Lively S, et al. MicroRNAs in Synovial Pathology Associated With Osteoarthritis. Front Med (Lausanne). 2020, 7:376.), therefore, it was found that osteoarthritis Specific miRNA is expected to provide a new treatment or detection method for the diagnosis and treatment of OA.

Although more and more miRNAs have been discovered as biomarkers and therapeutic targets for human diseases, to find miRNAs specifically expressed in OA and involved in chondrocyte proliferation and apoptosis, to clarify their mechanism of action, and to develop strategies based on miRNAs The diagnosis and treatment of OA have important significance and application prospects.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides the application of miR-601 as a molecular marker in the diagnosis and treatment of osteoarthritis, including the preparation of a kit for diagnosing OA and a drug for treating OA. Using miR-601 to detect osteoarthritis can not only quickly and effectively achieve early detection, but also provide an important basis for miR-601-targeted therapy for OA.

In order to achieve the above objects, the present invention provides the following technical solutions.

The present invention provides the application of miR-601 as a molecular marker in the diagnosis and treatment of osteoarthritis, characterized in that the nucleotide sequence of the miR-601 is 5'-UGGUCUAGGAUUGUUGGAGGAG-3' (SEQ ID No. 1) .

Further, the nucleotide sequence of the miR-601 is further partially replaced or increased or decreased to maintain the activity of the miR-601.

The present invention also provides an application of miR-601 as a molecular marker in a kit for diagnosing and treating osteoarthritis, characterized in that the kit comprises primers and probes for specific detection of miR-601;

Preferably, the miR-601-specific primers have the following sequences:

5'-TGGTCTAGGATTGTTGGA-3' (SEQ ID No. 2)

5'-CAGTGCGTGTCGTGGAGT-3' (SEQ ID No. 3).

The present invention also provides an application of miR-601 as a molecular marker in the preparation of a drug for targeted treatment of osteoarthritis, characterized in that the drug comprises miR-601, which promotes chondrocyte proliferation and inhibits chondrocyte proliferation. apoptotic activity.

Further, an application of miR-601 as a molecular marker in the preparation of a drug for the targeted treatment of osteoarthritis, characterized in that, the miR-601 is further modified, and the modified miR-601 maintains the Activity of miR-601.

Preferably, the modification is selected from at least one of ribose modification, base modification, and phosphate backbone modification.

Further, the application of the miR-601 as a molecular marker in the preparation of a drug for the targeted treatment of osteoarthritis is characterized in that the drug can be prepared into various dosage forms as required, including but not Limited to solutions, tablets, granules, patches, capsules, ointments, aerosols or suppositories.

Further, the application of the miR-601 as a molecular marker in the preparation of a drug for the targeted treatment of osteoarthritis is characterized in that the administration route of the drug is not limited, including but not limited to joints Intravenous, oral, intravenous, intramuscular, subcutaneous, sublingual, nasal spray, rectal infusion, oral spray, topical or systemic transdermal administration.

Compared with the prior art, the beneficial effects of the present invention are as follows.

The invention discloses a molecular marker miR-601 related to osteoarthritis, and further confirms that the expression of miR-601 is down-regulated in patients with osteoarthritis, and the purpose of diagnosing OA can be achieved by specific detection of miR-601 And through the detection of OA chondrocyte proliferation ability and apoptosis ability, miR-601 can improve the OA chondrocyte proliferation ability and reduce OA chondrocyte apoptosis. Therefore, the detection of miR-601 can not only quickly and effectively achieve the early detection of osteoarthritis, but also provide an important basis for miR-601 targeted therapy.

Description of drawings

Figure 1 Expression of miR-601 in patients with osteoarthritis.

Figure 2 ROC curve to evaluate the diagnostic value of miR-601 for OA.

Figure 3 Validation of miR-601 mimic transfection effect.

Figure 4 The effect of miR-601 on the proliferation of OA chondrocytes.

Figure 5 The effect of miR-601 on apoptosis of OA chondrocytes.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental method of unreceipted specific conditions in the embodiment is usually a routine method in the area, such as according to routine conditions such as people such as Sambrook, molecular cloning, the condition described in the experimental manual (New York: Cold Spring Harbor Laboratory Press, 1989) , or as recommended by the manufacturer. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The term "osteoarthritis" as used herein refers to a specific form of arthritis, particularly a chronic disease in which the articular cartilage over time gradually degenerates, the articular cartilage covering the ends of the bones that re-form the articular surfaces.

The miR-601 of the present invention is a known miRNA before the present invention, which is derived from the published human genome sequence and located on human chromosome 9, and the specific position is chr 9: 123402525-123402603.

In the embodiment of the present invention, miR-601 refers not only to an endogenous, non-coding microRNA containing a 5'-UGGUCUAGGAUGUUGGAGGAG-3' sequence, but also to a molecule that can exercise the core sequence of miR-601 functions, and can also refer to Micromolecules synthesized in vitro with 5'-UGGUCUAGGAAUUGUUGGAGGAG-3' sequence can also be called mimics of miR-601. The in vitro synthesis methods of the above-mentioned micromolecules are well known in the art.

Example 1 Abnormal expression of miR-601 in patients with osteoarthritis.

1. Sample collection.

Thirty-one patients with OA who underwent knee replacement surgery were included as the observation group, and 31 patients with limb mutilation and amputation were included as the control group. Diseases such as rheumatoid arthritis, rheumatoid arthritis, and septic arthritis were excluded, and the blood samples of the patients were collected. Samples and cartilage tissue samples were used for follow-up testing, and there were no significant differences in age and gender between the OA group and the control group.

2. The total RNA in the sample was extracted by Trizol method.

Step 1. Take out the cartilage tissue and put it into a pre-cooled mortar for grinding. After the tissue sample is in powder form, fresh whole blood is lysed to red blood cells, and the leukocyte pellet is collected.

Step 2. Add pre-cooled TRizol to the cartilage powder and leukocyte pellet, respectively, and store at room temperature for 5 minutes.

Step 3. Add chloroform, shake the centrifuge tube vigorously, mix well, and place at room temperature for 10 minutes.

Step 4. Centrifuge at 12000rpm for 15 minutes at 4°C, suck the upper aqueous phase into another new centrifuge tube, add an equal volume of -20°C pre-cooled isopropanol, invert and mix well, and place on ice for 10 minutes.

Step 5. After high-speed centrifugation at 12,000 rpm for 15 minutes at 4°C, the supernatant was carefully discarded, and pre-cooled 75% ethanol was added to wash the precipitate.

Step 6. Centrifuge at 12,000 rpm for 5 minutes at 4°C, discard the ethanol liquid, place at room temperature for 5 minutes to fully dry the precipitate, and add RNase-free water to dissolve the precipitate.

Step 7. Use NanoDrop2000 to measure the RNA purity and concentration. The RNA quality is determined by the ratio of OD260/OD280. The ratio of 1.8-2.2 is regarded as qualified.

3. Real-time quantitative PCR to detect the expression of miR-601.

Reagents: Reverse transcription kit One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Code No.D350A) and real-time quantitative PCR reagent SYBR PrimeScipt miRNA RT-PCR Kit (Takara, Code No.RR716) were purchased from Bao Bioengineering ( Dalian) Co., Ltd., produced by Japan Takara Company.

Reverse transcription: One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, CodeNo.D350A) was used for reverse transcription. A 20 μL reaction system was used, and 1 μg of total RNA was taken from each sample as a template. The experimental operation was carried out according to the product instructions. The reaction program was 37 °C for 60 min; 37 °C for 5 sec. The reaction system is shown in Table 1. The obtained cDNA was stored in a -20°C refrigerator for later use.

Table 1 Reverse transcription reaction system.

Real-Time PCR: SYBR PrimeScipt miRNA RT-PCR Kit (Takara, Code No. RR716) was used for amplification. The experimental operation was carried out according to the product instructions. The reaction system is shown in Table 2. The expression detection of miR-601 set up 3 parallel tube reactions each time. The forward primer sequence for amplifying miR-601 is shown in SEQ ID NO.2, and the downstream primer is shown in SEQ ID NO.3. Using U6 as an internal reference, the upstream primer is shown in SEQ ID NO.4, and the downstream primer is shown in SEQ ID NO.5. Amplification program: 95°C for 30s; 40x (95°C for 5s, 60°C for 34s). The relative quantitative analysis of the data was carried out by the 2- ^ΔΔCt method using ABI7500 fluorescence quantitative PCR instrument.

Table 2 Real-Time PCR reaction system of miR-601.

4. Statistical methods.

All data are expressed in the form of mean ± standard deviation (mean ± SD). SPSS 17.0 software was used for statistical analysis. Differences between the two groups were compared by t test. All results were plotted using GraphPad Software software. , set P<0.05 as statistically significant. ROC curve analysis was performed on the variable miR-601 to determine its diagnostic efficacy, sensitivity and specificity for OA.

5. result.

As shown in Figure 1, compared with the control group, the expression of miR-601 in the blood and tissue of patients with osteoarthritis was significantly down-regulated by 65% and 55%, respectively, and the difference was statistically significant (P<0.05). .

As shown in Figure 2, miR-601 can be used as a biomarker for the diagnosis of osteoarthritis, and its area under the curve is 0.880 (blood sample) and 0.806 (tissue sample), which can effectively distinguish osteoarthritis patients from normal controls .

Example 2 Effects of miR-601 analogs on chondrocytes.

1. Acquisition and culture of primary human chondrocytes.

The cartilage tissue on the surface of the tibia and femur of the knee joint of patients with OA was scraped with a blade, washed three times with PBS containing 1% double antibody, and cut into ^1mm3 pieces with sterile ophthalmic scissors, and placed in a 10cm petri dish , add 10 mL of 0.25% trypsin, digest in a 37°C, 5% CO ₂ incubator for 30 min, add DMEM medium containing 10% FBS to stop trypsin digestion, and wash twice with PBS. Add 10 ml of 0.2% type II collagenase, and place it in a 37°C, 5% CO ₂ incubator to digest for 4 h. The cell suspension was filtered with a 200-mesh cell sieve, and the filtrate was transferred into a centrifuge tube and centrifuged at 1000 r/min for 10 min, and the supernatant was discarded. Mix well by pipetting with DMEM medium containing 10% FBS, and put it into a 25cm culture flask for culture.

2. transfection.

The miR-601 mimic was purchased from Guangzhou Ribo Biotechnology Co., Ltd., the forward sequence is shown in SEQ ID NO.6, and the reverse sequence is shown in SEQ ID NO.7. The chondrocytes in the logarithmic growth phase were passaged to 12-well culture plates. When the cell confluence density reached about 30%, the miR-601 mimic was transfected into the chondrocytes using Lipofectamine 2000 produced by Invitrogen. Negative control and blank control were set simultaneously. For specific operations, refer to the instructions. After 48 hours of transfection, follow-up experimental studies were carried out.

3. The transfection effect of miR-601 mimic was detected by Real-time PCR.

The total RNA of chondrocytes was extracted by Trizol method, and the expression of miR-601 in chondrocytes was detected by reverse transcription and real-time quantitative PCR according to the method in Example 1.

4. The effect of miR-601 on the proliferation activity of chondrocytes was detected by CCK-8 assay.

Chondrocytes 48h after transfection were taken and seeded in 96-well plates at 5000 cells/well, with 5 duplicate wells in each group; after cells adhered, CCK-8 reagent was added, 100μL/well, and incubated at 37°C for 1h in the dark , and detect the absorbance value (OD) with a microplate reader at a wavelength of 450 nm.

5. Apoptosis was detected by flow cytometry.

Apoptosis was detected by Annexin V-FITC/PI Apoptosis Detection Kit (Shanghai Yisheng Biotechnology Co., Ltd.) and flow cytometry. For specific steps, refer to the kit instructions. Chondrocytes were collected 48h after transfection, resuspended with 100μL of 1×Binding buffer solution, added 5μL of AnnexinV-FITC and 5μL of PI, mixed well, incubated in the dark for 15min, added with 400μL of 1×Binding buffer, and analyzed by flow cytometer. Apoptosis detection.

6. Statistical methods.

All data are expressed in the form of mean ± standard deviation (mean ± SD). SPSS 17.0 software was used for statistical analysis. One-way ANOVA was used for comparison between multiple groups. SNK (Student-Newman-Keuls) test was used for comparison. All results were plotted using GraphPad Software, and statistical significance was set at P < 0.05.

7. Results.

The results are shown in Figure 3, the expression of miR-601 in chondrocytes in the negative control group was not significantly different from that in the blank control group, and the expression of miR-601 in chondrocytes in the transfection miR-601 mimic group was significantly higher than that in the negative control group ( P<0.05).

The results are shown in Figure 4. Compared with the blank control group, the proliferation of cells in the negative control group did not change significantly, while the proliferation of chondrocytes transfected with miR-601 mimics was significantly improved compared with the negative control group (P<0.05).

The results are shown in Figure 5. Compared with the blank control group, the apoptosis of the negative control group did not change significantly, while the chondrocyte apoptosis level transfected with miR-601 mimic was significantly lower than that of the negative control group (P<0.05).

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

SEQUENCE LISTING

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Claims (8)

1. The application of miR-601 as a molecular marker in preparation of a reagent for diagnosing and treating osteoarthritis is characterized in that the nucleotide sequence of miR-601 is 5'-UGGUCUAGGAUUGUUGGAGGAG-3' (SEQ ID No. 1).
2. 2. The application of miR-601 as a molecular marker in preparation of a kit for diagnosing osteoarthritis is characterized in that the kit comprises primers and probes for specifically detecting miR-601.
3. 3. The application of miR-601 as a molecular marker in the preparation of a kit for diagnosing osteoarthritis according to claim 2, wherein the miR-601 specific primers have the following sequences:

   5' -TGGTCTAGGATTGTTGGA -3'（SEQ ID No.2）

   5' -CAGTGCGTGTCGTGGAGT- 3'（SEQ ID No.3）。
4. 4. the application of miR-601 in preparation of a medicine for targeted treatment of osteoarthritis is characterized in that the medicine comprises miR-601, the miR-601 has the activities of promoting chondrocyte proliferation and inhibiting chondrocyte apoptosis, and the nucleotide sequence of miR-601 is shown in SEQ ID No. 1.
5. 5. The use of miR-601 in the preparation of a medicament for the targeted treatment of osteoarthritis according to claim 4, wherein the miR-601 is further modified, and the modified miR-601 maintains the activity of miR-601.
6. 6. The use of claim 5, wherein the modification is selected from at least one of a ribose modification, a base modification, and a phosphate backbone modification.
7. 7. The use of miR-601 in the preparation of a medicament for the targeted treatment of osteoarthritis according to claim 4, wherein the medicament can be prepared into various dosage forms as required, including but not limited to solution, tablet, granule, patch, capsule, paste, aerosol or suppository.
8. 8. The use of miR-601 in the preparation of a medicament for the targeted treatment of osteoarthritis according to claim 4, wherein the route of administration of the medicament is not limited, and includes but is not limited to joint cavity, oral, intravenous, intramuscular, subcutaneous, sublingual, nasal spray, rectal infusion, oral spray, topical or systemic transdermal administration of the skin.

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