CN104083761A - Application of microRNA-101 inhibitor in the preparation of drugs for preventing or treating osteoarthritis - Google Patents

Abstract

The invention discloses an application of a microRNA-101 (micro-ribonucleic acid-101) inhibitor in preparing medicaments for preventing or treating osteoarthritis, and belongs to the field of biomedicine. In an osteoarthritis animal model, the articular cartilage injury is exacerbated when a microRNA-101 analog is injected into the knuckle articular cavity, and the expression levels of extracellular matrix II type collagen and proteoglycan causing cartilage specificity are reduced; while the cartilage injury of the analog can be remarkably improved by injecting the microRNA-101 inhibitor into the knuckle articular cavity, the cartilage injury process of the osteoarthritis animal model can be retarded, and a role of protecting cartilage can be played, so that degradation of cartilage tissues can be retarded by inhibiting expression of the microRNA-101, namely through the microRNA-101 inhibitor, and the effect of stabilizing the cartilage cell phenotype can be achieved, so that articular cartilage injury can be controlled, and cartilage repairing can be promoted. Therefore, the microRNA-101 inhibitor can be used for preparing medicaments for controlling osteoarticular injury and promoting cartilage repairing.

Description

Application of microRNA-101 inhibitor in the preparation of drugs for preventing or treating osteoarthritis

technical field

The invention relates to the field of biomedicine, in particular to the application of a microRNA-101 inhibitor in the preparation of drugs for preventing or treating osteoarthritis.

Background technique

microRNA (miRNA for short) is a type of endogenous non-coding single-stranded RNA with a length of 21-25 nucleotides, which is highly conserved, time-sequenced and tissue-specific in evolution, and exists in almost all multicellular organisms middle. So far, miRBase, a database that specifically collects miRNA sequences, covers more than 8,000 miRNAs including plants, animals, and viruses, among which more than 800 human miRNAs exist in the miRBase database. It is speculated that miRNA regulates one-third of human genes and participates in many physiological and pathological processes of the body. miRNA mainly acts on the 3'untranslated region of the target gene to directly cause the degradation of the target gene mRNA or inhibit the translation of the target gene mRNA.

miRNA can participate in the regulation of cell function and plays an important role in the regulation of human diseases. At present, miRNAs related to cartilage damage in osteoarthritis have been preliminarily studied. Miyaki reported that the expression of miRNA-140 decreased in human chondrocytes stimulated by interleukin (hereinafter referred to as IL-1β), and overexpression of miRNA-140 could inhibit the upregulation of the expression of chondrocyte extracellular matrix degrading enzymes caused by IL-1β , the reduction of proteoglycan expression of chondrocyte extracellular matrix gene caused by IL-1β (see Miyaki S et al., MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses.Arthritis and rheumatism2009,60(9):2723- 2730). In IL-1β-stimulated chondrocytes, miRNA-34a was highly expressed, and inhibiting the expression of miRNA-34a could counteract the decrease in the expression of Col2a1 protein and inducible nitric oxide synthase Nos2 (iNOS) induced by IL-1β. Cartilage destruction can be prevented by inhibiting miRNA-34a (see Abouheif MM et al., Silencing microRNA-34a inhibits chondrocyte apoptosis in a ratosteoarthritis model in vitro. Rheumatology (Oxford) 2010, 49(11): 2054-2060). In contrast, suppression of miRNA-27b expression in chondrocytes by IL-1β resulted in increased expression of the microRNA-27b direct target gene MMP13 (another enzyme involved in the degradation of cartilage ECM), leading to degradation of the cartilage ECM (see Akhtar et al. N et al., MicroRNA-27b regulates the expression of matrixmetalloproteinase13 in human osteoarthritis chondrocytes.Arthritis and rheumatism2010,62(5):1361-1371). Studies have also shown that miRNA-675 can promote the expression of cartilage-specific extracellular matrix gene type II collagen (Dudek KA et al., Type II collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes. J Biol Chem2010, 285( 32):24381-24387).

It can be seen that in the treatment of osteoarthritis cartilage damage, it is of great significance to seek a new miRNA for the treatment of osteoarthritis cartilage damage.

Contents of the invention

In order to solve the problems in the prior art, an embodiment of the present invention provides an application of a microRNA-101 inhibitor in the preparation of a drug for preventing or treating osteoarthritis. Described technical scheme is as follows:

In the first aspect, the embodiment of the present invention provides an application of a microRNA-101 inhibitor in the preparation of a drug for preventing or treating osteoarthritis.

Specifically, the nucleotide sequence of the microRNA-101 is 5'-UACAGUACUGUGAUAACUGAA-3'.

Specifically, preferably, the microRNA-101 is further modified, and the modified microRNA-101 maintains the activity of the microRNA-101.

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

Specifically, preferably, the nucleotides in the microRNA-101 are further partially substituted or increased or decreased, and the microRNA-101 after the nucleotides are further partially substituted or increased or decreased maintains the activity of the microRNA-101.

In a second aspect, an embodiment of the present invention provides a pharmaceutical composition for preventing or treating osteoarthritis, the pharmaceutical composition comprising an effective amount of an inhibitor of microRNA-101.

Specifically, preferably, the pharmaceutical composition further includes other drugs compatible with the microRNA-101 inhibitor, as well as pharmaceutically acceptable carriers and/or adjuvants.

Specifically, preferably, the dosage form of the pharmaceutical composition is selected from a solution, a suspension, an emulsion, a controlled-release preparation or a sustained-release preparation.

Specifically, preferably, the administration mode of the pharmaceutical composition is selected from injection administration or oral administration.

The beneficial effects brought by the technical solution provided by the embodiments of the present invention are:

The embodiment of the present invention provides the application of a microRNA-101 inhibitor in the preparation of drugs for the prevention or treatment of osteoarthritis. In the rat osteoarthritis animal model induced by iodoacetic acid, microRNA was injected into the knee joint cavity of rats -101 mimics can aggravate cartilage damage in rats with osteoarthritis, and cause a decrease in the expression levels of cartilage-specific extracellular matrix genes type II collagen and proteoglycan. Injection of microRNA-101 inhibitors into rat knee joints can significantly improve their cartilage damage, delay the process of cartilage damage in animal models of osteoarthritis, and play a protective role in cartilage. Therefore, by inhibiting the expression of microRNA-101, that is, the inhibitor of microRNA-101 can slow down the degradation of cartilage tissue and stabilize the phenotype of chondrocytes, thereby controlling the destruction of bone joints and promoting cartilage repair. The above microRNA-101 inhibitor can stabilize the chondrocyte phenotype and prevent the degradation of the cartilage extracellular matrix by regulating the key transcription factor Sox9 gene in chondrocytes related to chondrocyte differentiation and phenotype stability. This indicates that inhibitors of microRNA-101 can be used to prepare drugs that control bone and joint destruction and promote cartilage repair.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.

Figure 1a is a schematic diagram of the expression level of microRNA-101 in the iodoacetic acid-induced osteoarthritis cartilage damage model provided in Example 1 of the present invention;

Figure 1b is a schematic diagram of the expression level of Sox9mRNA in the iodoacetic acid-induced osteoarthritis cartilage damage model provided in Example 1 of the present invention;

Figure 1c is a schematic diagram of the expression level of Sox9 protein in the iodoacetic acid-induced osteoarthritis cartilage damage model provided in Example 1 of the present invention;

Figure 2a is a schematic diagram of confocal microscope observation results of frozen sections of rat knee articular cartilage in each animal model group provided in Example 1 of the present invention;

Figure 2b is a schematic diagram of the expression levels of miR-101a in each animal model group provided in Example 1 of the present invention;

Figure 2c is a schematic diagram of the expression levels of Sox9mRNA in each animal model group provided in Example 1 of the present invention;

Figure 2d is a schematic diagram of the expression levels of Sox9 protein in each animal model group provided in Example 1 of the present invention;

Figure 3a is a schematic diagram of the observation results of cartilage tissue in each animal model group provided in Example 1 of the present invention;

Figure 3b is a schematic diagram of the histological scoring results of each animal model group provided in Example 1 of the present invention after 7 days;

Figure 3c is a schematic diagram of the histological score results of each animal model group provided in Example 1 of the present invention after 14 days;

4 is a schematic diagram of HE staining results of cartilage tissues of each animal model group provided in Example 1 of the present invention (magnification 10 times, scale bar=400 microns);

Fig. 5 is a schematic diagram of proteoglycan staining results of cartilage tissues of each animal model group provided in Example 1 of the present invention (magnification 10 times, scale bar = 400 microns);

Fig. 6 is a schematic diagram of the immunohistochemical staining results of type II collagen in each animal model group provided in Example 1 of the present invention (magnification 10 times, scale bar = 400 μm).

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the implementation manner of the present invention will be further described in detail below in conjunction with the accompanying drawings.

Osteoarthritis (OA), also known as osteoarthritis and degenerative joint disease, is the degeneration of articular cartilage caused by trauma, joint deformity and other reasons. Clinically, it can produce joint pain, limited mobility, joint deformity and even dysfunction. Because OA is not easy to cure, it seriously hinders work and affects the quality of life. It has become the second most common cause of loss of labor after the age of 50, second only to heart disease. The clinical treatment of OA is mainly aimed at relieving pain, relieving symptoms, and delaying disease. For example, drug treatment: use drugs to relieve pain symptoms (such as analgesics, non-steroidal anti-inflammatory drugs, etc.), hormones, and chondroprotective agents (such as intra-articular injection of sodium hyaluronate, oral glucosamine, etc.). Surgical treatment: use arthroscopic joint irrigation, drilling, microfracture, osteochondral transplantation, chondrocyte or mesenchymal stem cell transplantation. For those with severe joint deformity, osteotomy orthopedic surgery is required; for those with joint destruction and severe dysfunction, artificial joint replacement is required. The above-mentioned treatment methods can only alleviate the symptoms of pathological changes that have occurred in the bones and joints, but cannot reverse or stop the symptoms of pathological changes.

Studies have found that the occurrence and development of OA include several important pathological changes: articular cartilage degeneration, subchondral bone sclerosis, synovial inflammation and osteophyte formation. Among them, articular cartilage degeneration is the first occurrence in osteoarthritis, which cannot be reversed by current treatment methods, and is also the most important pathological change. Articular cartilage is composed of chondrocytes and extracellular matrix (ECM). Chondrocytes are the only cell type in articular cartilage that synthesize and break down extracellular matrix. The extracellular matrix of cartilage is mainly composed of type II collagen (Collagen type II, COL2A1) and proteoglycan (Aggrecan). Chondrocytes and the extracellular matrix work together to maintain the structure of articular cartilage and the homeostasis of its extracellular environment. When OA occurs, this homeostasis is disrupted, manifested as degradation of the extracellular matrix of cartilage, degeneration of chondrocytes, etc. In the process of OA, the degeneration of chondrocytes and the degradation of extracellular matrix are mainly divided into two stages: the biosynthesis stage and the degradation stage. During the biosynthetic phase, chondrocytes attempt to repair the damaged extracellular matrix of cartilage. However, due to the low level of gene expression for synthesizing extracellular matrix, sufficient extracellular matrix cannot be efficiently synthesized. In the degradation stage, under the action of inflammatory factors, chondrocytes produce extracellular matrix-degrading enzymes that will digest the extracellular matrix and cause extracellular matrix degradation. At the same time, inflammatory factors can also cause a further decline in the expression of genes that synthesize extracellular matrix, and the synthesis of extracellular matrix is even more insufficient, leading to a vicious cycle and causing rapid destruction of cartilage. Therefore, insufficient synthesis of cartilage extracellular matrix and degradation of cartilage extracellular matrix are the main pathological changes in OA cartilage injury.

According to the research on the pathological process of OA, in order to slow down the symptoms of osteoarthritis, and effectively reverse or stop osteoarthritis, the present invention further studies the genes, gene regulatory networks, epigenetics that play an important role in the pathological process of OA. The change and related miRNA provide an application of a microRNA-101 inhibitor in the preparation of a drug for preventing or treating osteoarthritis.

The study of the present invention found that in the rat model of cartilage destruction induced by iodoacetic acid, the level of microRNA-101 (ie, microRNA-101a) in rat cartilage tissue was significantly increased. Injection of microRNA-101 mimics into rat knee joints can cause aggravation of cartilage damage in rats with osteoarthritis. Injection of microRNA-101 inhibitors into rat knee joints can significantly improve their cartilage damage and play a protective role in cartilage. Therefore, by inhibiting the expression of microRNA-101, that is, the inhibitor of microRNA-101 can slow down the degradation of cartilage tissue and stabilize the phenotype of chondrocytes, thereby controlling the destruction of bone joints and promoting cartilage repair. The above microRNA-101 inhibitor can stabilize the chondrocyte phenotype and prevent the degradation of the cartilage extracellular matrix by regulating the key transcription factor Sox9 gene in chondrocytes related to chondrocyte differentiation and phenotype stability. It can be seen that the inhibitor of microRNA-101 can be used to prepare drugs for preventing or treating osteoarthritis.

It can be understood that the inhibitor of microRNA-101 has the meanings known in the art. Design and preparation methods of inhibitors of microRNA-101 are also well known in the art. For example, those skilled in the art design antisense RNA based on microRNA-101, or contact microRNA-101 with specific substances, detect the expression of microRNA-101, and select candidate substances that specifically inhibit the expression of microRNA-101, so as to obtain Inhibitors of microRNA-101. It can be understood that the inhibitor of microRNA-101 can be any substance that can specifically inhibit the regulation of microRNA-101 on the target gene, it can also be a substance that specifically inhibits the expression of microRNA-101 in cells, and it can also be a substance combined with microRNA-101. 101 has a substance that specifically interacts, such as a substance that specifically binds to microRNA-101, or a substance that specifically inhibits the interaction between microRNA-101 and DICER. Specifically, the inhibitor of microRNA-101 can be a small molecule nucleic acid, which can effectively inhibit the expression of microRNA-101.

Specifically, the nucleotide sequence of microRNA-101 is 5'-UACAGUACUGUGAUAACUGAA-3'.

Since the microRNA-101 nucleotide sequence is very conserved among various species (such as mice, rats, pigs, etc.), in the embodiments of the present invention, microRNA-101 is selected from human microRNA-101, and its sequence in miRbase The number is: MI0000103, the nucleotide sequence is 5'-UACAGUACUGUGAUAACUGAA-3', which has the specific biological activity of aggravating the symptoms of osteoarthritis.

In the embodiment of the present invention, microRNA-101 not only refers to an endogenous, non-coding microRNA containing a 5'-UACAGUACUGUGAUAACUGAA-3' sequence, but also refers to a microRNA molecule capable of performing the core sequence of the function of microRNA-101, or refers to A microRNA molecule synthesized in vitro with a sequence of 5'-UACAGUACUGUGAUAACUGAA-3' (also called a mimetic of microRNA-101). Wherein the in vitro synthesis method of the above-mentioned microRNA molecule is well known in the art. Therefore, the inhibitor of microRNA-101 may be an inhibitor of microRNA-101 obtained from the aforementioned microRNA-101.

Specifically, preferably, the microRNA-101 is further modified, and the modified microRNA-101 maintains the activity of the microRNA-101.

In the embodiment of the present invention, in order to maintain the stability of microRNA-101 during in vitro operation, it can be further modified, but the modified microRNA-101 maintains the activity of the microRNA-101. It can be understood that the inhibitor of microRNA-101 can be the inhibitor obtained according to the above-mentioned modified microRNA-101.

Among them, "microRNA-101 activity" refers to microRNA-101 that can cause the expression levels of cartilage-specific extracellular matrix gene type II collagen (Col2A1) and proteoglycan (Aggrecan) to decrease, and cause specific biological factors that aggravate cartilage damage. active. Specifically, the above modification is selected from at least one of ribose modification, base modification, and phosphate backbone modification.

Specifically, preferably, the nucleotides in the microRNA-101 are further partially substituted or increased or decreased, and the microRNA-101 after the nucleotides are further partially substituted or increased or decreased maintains the activity of the microRNA-101.

Among them, "the activity of microRNA-101" also refers to the specificity of microRNA-101 that can cause the expression levels of cartilage-specific extracellular matrix genes type II collagen (Col2A1) and proteoglycan (Aggrecan) to decrease, and cause the aggravation of cartilage damage. biological activity.

In the second aspect, the embodiment of the present invention provides a pharmaceutical composition for preventing or treating osteoarthritis, the pharmaceutical composition comprising an effective amount of an inhibitor of microRNA-101.

The pharmaceutical composition containing an effective amount of an inhibitor of microRNA-101 also has the specific effect of an inhibitor of microRNA-101, that is, stabilizes the phenotype of chondrocytes and prevents the degradation of the extracellular matrix of cartilage.

Wherein, "effective amount" refers to the amount that produces functions or activities on human or animal body and can be accepted by human or animal.

When the microRNA-101 inhibitor is a small molecular nucleic acid, it can effectively inhibit the expression of microRNA-101. Further, the nucleotides in the microRNA-101 inhibitor are further partially substituted or increased or decreased, and the microRNA-101 inhibitor after the nucleotides are further partially substituted or increased or decreased maintains the activity of inhibiting the expression of microRNA-101.

Specifically, preferably, the pharmaceutical composition further includes other drugs compatible with the microRNA-101 inhibitor, as well as pharmaceutically acceptable carriers and/or adjuvants.

When the microRNA-101 inhibitor is used to prevent or treat osteoarthritis, it can be used alone or in combination with other compatible drugs and pharmaceutically acceptable carriers and/or adjuvants. Among them, "pharmaceutically acceptable carrier and/or adjuvant" refers to a carrier that is suitable for human animals without excessive adverse side effects (such as toxicity, irritation and allergy), that is, a carrier with a reasonable benefit/risk ratio and / or excipients.

Specifically, the carrier can be nanoparticles, liposomes, cholesterol, chitosan, viruses and the like.

Specifically, preferably, the dosage form of the pharmaceutical composition is selected from solution, suspension, dry powder, emulsion, controlled release or sustained release preparation. The administration mode of the pharmaceutical composition is selected from injection administration (for example, intravenous injection, joint cavity local injection or intramuscular injection, etc.) or oral administration.

The present invention will be further illustrated by specific examples below.

Those who do not indicate the conditions in the following specific examples are all carried out according to the conventional conditions or the conditions suggested by the manufacturer. All reagents and instruments used without manufacturer’s indication are conventional products that can be purchased from the market.

Among them, the mimetic of microRNA-101 was synthesized from Shanghai Jikai Company, product number: GCD950817.

The inhibitor of microRNA-101 was synthesized from Shanghai Jikai Company, product number: GCD950818.

Example 1

1) Establishment of animal model of iodoacetic acid-induced osteoarthritis

The SD rat was fixed in the assistant's hand in the supine position, and the surgeon touched the rat's joint space and patellar ligament with his left hand. Then use a 26-gauge needle to pass through the patellar ligament of the rat, and feel a sense of hollowness, which proves that the needle has entered the joint cavity, and inject iodoacetic acid (2 mg of iodoacetic acid dissolved in 50 μl of PBS (phosphate buffered saline)) with a micro-injector. middle). Normal diet, let the rats move freely in the cage.

2) Animal grouping

Thirty-two (64 knees in total) SD rats with a body weight of 150 g were divided into 4 groups on average, including: the first group (only injected with iodoacetic acid without miRNA intervention); the second group (injected with iodoacetic acid, and treated with control miRNA Intervention; the third group (injection of iodoacetic acid, the mimetic intervention of microRNA-101, that is, the overexpression of microRNA-101 group); the fourth group (injection of iodoacetic acid, the intervention of the inhibitor of microRNA-101, that is, the inhibition of 101 expression group). The two knee joints of the same rat were respectively made into different treatment groups.

3) Experimental animals are treated with miRNA intervention

Three days after the injection of iodoacetic acid, SD rats began to receive miRNA intervention every three days until the experiment was terminated.

4) Animals were killed and materials were collected

On the 7th and 14th day after injection of iodoacetic acid, the SD rats were sacrificed by overdose anesthesia, and the knee joints were removed and put into fixative solution after trimming.

5) Histological evaluation

After the SD rats were sacrificed, the front end of the femur (8 samples in each group) was cut off, then fixed in 4% paraformaldehyde (pH 7.4) for 48 hours at 4°C and then the samples were subjected to rapid decalcification. Decalcification in solution for 2 days. Decalcified specimens were trimmed, dehydrated with graded ethanol, and embedded in paraffin. Cut into 5 μm thick sagittal sections and perform hematoxylin-eosin staining (HE staining for short) and toluidine blue staining, and type II collagen antibody for immunohistochemical analysis. Histological scoring was performed using the modified Mankin scoring method. (See Mankin, H.J., Dorfman, H., Lippiello, L. & Zarins, A. Biochemical and metabolic abnormalities intracellular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. The Journal of int bone and jo American volume 53, 523-537(1971).)

6) Immunofluorescence analysis of microRNA-101 expression in rat knee articular cartilage

Rat knee articular cartilage (after day 1 injection) was rinsed in PBS and embedded in OCT compound. Cryosections were cut into 8 μm thick frozen sections on glass slides using a cryostat. Then, the sections were stained with Hoechst33342 staining solution for 5 minutes. After washing with PBS three times, the slides were observed under a confocal microscope.

7) RNA extraction and Real-time PCR analysis

Total RNA was extracted from MIA rat knee articular cartilage using TRIzol reagent. Isolated RNA was reverse-transcribed, and real-time PCR was performed using the ABI Step One Plus QPCR Analyzer (ABI, Foster City, CA, USA) with the SYBR Selection Master Mix (ABI). The conditions of Real-time PCR were as follows: 50°C for 2 minutes, 95°C for 2 minutes, followed by 95°C for 15 seconds, 40 cycles of 60°C for 30 seconds. Melting curves were added to ascertain the absence of non-specific amplification. Primers for Real-timePCR are as follows:

Sox9 (FW), 5'-AGGAAGCTGGCAGACCAGTA-3' and (RV), 5'-ACGAAGGGTCTCTTCTCGCT-3';

18S RNA FW, 5'-GTAACCCGTTGAACCCCATT-3', and RV, 5'-CCATCCAATCGGTAGTAGCG-3'.

For the reverse transcription and expression analysis of microRNA-101, the Bulge-loop ^TM miRNA kit was used to operate according to the instructions (purchased from Guangzhou Ruibo Biotech). Expression of Sox9 and miR-101 was determined using the 2-ΔΔCT method.

8) Protein separation and immunoblotting

Protein was extracted with lysis buffer (50 mM Tris-hydrochloric acid, pH 7.4, 150 mM sodium chloride, 1% NP-40 and 0.1% sodium dodecyl sulfate), and the concentration was measured using BCA protein assay kit (Pierce company) , using bovine serum albumin as a standard. Proteins were run on SDS-PAGE gels (10%) for 1.5 hours and then electrotransferred to nitrocellulose membranes at 4°C for 2 hours. The primary antibody (type II collagen) was diluted 1:4000 overnight at 4°C; the horseradish peroxidase-labeled secondary antibody was diluted 1:1000 and incubated at room temperature for 1 hour. Protein expression levels were detected by chemiluminescence. β-actin (Sigma, 1:10000 dilution) was used as an internal reference.

Statistical Analysis

The significance of differences between groups was calculated using analysis of variance (ANOVA) analysis. The results of the same group were evaluated using the Student-t test. A P value of less than 0.05 was considered statistically significant. All data are presented as mean ± standard deviation.

Research result

Expression of microRNA-101 and Sox9 genes in the knee articular cartilage of the first group of rats

In order to examine whether microRNA-101 is involved in the process of cartilage degeneration in the first group of rats, the rat knee articular cartilage was harvested one day after iodoacetic acid injection for further analysis. As shown in Figure 1a, compared with normal rats (i.e. rats not treated with iodoacetic acid), the Real-time PCR results for the first group showed that the expression level of microRNA-101 was significantly increased after injection of iodoacetic acid. However, the RNA and protein levels of Sox9 were significantly decreased (see Figure 1b and Figure 1c). It can be seen that Sox9 is a target of microRNA-101, and Sox9 is regulated by microRNA-101 during the degradation of cartilage extracellular matrix. In summary, microRNA-101 is involved in the cartilage degeneration process in MIA rats, and its function is realized by regulating the expression of Sox9.

Effects of microRNA-101 mimics and microRNA-101 inhibitor expression vectors on articular cartilage

In order to test whether adenovirus-mediated delivery of microRNA-101 can penetrate into cartilage and play a role, first, mice injected with miRNA-101 mimics (group 3) and inhibitors of microRNA-101 (group 4) Frozen sections of mouse knee articular cartilage were analyzed and observed by immunofluorescence under a confocal microscope. The nuclei of the chondrocytes to be detected are fluorescently labeled (see Figure 2a), and the green fluorescent protein marker is provided in the carrier of the mimetic of microRNA-101 and the carrier of the inhibitor of microRNA-101, whether the expression of green fluorescent protein It can be indicated whether miRNA-101 mimetics and microRNA-101 inhibitors are expressed.

Confocal microscopy results showed that GFP could be expressed in cartilage, menisci, and even synovium (see Figure 2a). This suggests that carriers of microRNA-101, and its mimics or inhibitors, are able to penetrate cartilage. Secondly, in order to detect whether the expression vector can play a role in cartilage, the RNA of rat knee articular cartilage tissue was detected by Real-time PCR to detect the expression level of microRNA-101 in the cartilage tissue injected with the overexpression vector. The results showed that the injection of microRNA In the samples of mimics of -101 (third group) and in samples of microRNA-101 inhibitors (fourth group), the expression level of microRNA-101 was significantly increased relative to the second group (see Figure 2b). In samples injected with microRNA-101 mimics (the third group), the mRNA expression level of Sox9 decreased (see Figure 2c and 2d); The expression level of mRNA was significantly increased (see Figures 2c and 2d). It can be seen that adenovirus-mediated microRNA-101 mimics and microRNA-101 inhibitors can penetrate into cartilage and play a role in regulating the expression of its target Sox9.

Observation of trochlear cartilage in each group

There was no death or infection in the rats after the intra-articular injection and during the treatment. Rats in the above four groups were collected 7 days and 14 days after the injection of iodoacetic acid. Observations showed that at 7 days, in the above four groups of rats, the cartilage destruction of the trochlear part of the knee joint was obvious, and there was a slight exposure of the subchondral bone. However, there was little difference in the degree of cartilage degeneration among the four groups (see Figure 3a). At 14 days, the cartilage degeneration at the trochlear part of the knee joint was different among the four groups. Specifically, the cartilage degeneration in the first and second groups was similar, with a slight exposure of the subchondral bone (see Figure 3a) ; while the cartilage degeneration of the trochlear surface in the third group was relatively obvious, and the subchondral bone was exposed (see Figure 3a); in the fourth group, the cartilage degeneration was very slight, and the degree of cartilage degeneration was smaller than that of the first and second groups. The cartilaginous surface was relatively smooth without exposure of subchondral bone (see Figure 3a).

Histological score of trochlear cartilage in each group

The knee joint score of each group is mainly evaluated according to the modified Mankin scoring system, and the degree of cartilage degeneration is mainly evaluated from three aspects: cartilage structure (including irregular cartilage surface, cracks to radial layer, pannus, surface disappearance, Minor tissue destruction, fissures to subchondral bone, tissue destruction, etc.), chondrocyte abnormalities (including chondrocyte hypertrophy, hypertrophy, clustering), matrix staining (including reduced surface staining, reduced intracellular matrix staining, and only extracellular matrix staining , no staining). A low score indicates mild cartilage degeneration, while a high score indicates severe cartilage degeneration.

From the perspective of tissue scores, there was no significant difference in the scores of each group at 7 days. However, compared with the other groups, the score of the fourth group was low, and there was a statistical difference between the histological scores of the other groups, indicating that the cartilage damage of the fourth group was small (see Figure 3b). At 14 days, the overall histological scores of each group were significantly different, mainly in that the scores of the fourth group were statistically significant compared with the first and second groups; the scores of the third group were compared with the first and second groups. The comparison between the two groups was also statistically significant (see Figure 3c). It can be seen that the low score of the third group indicates less cartilage degeneration; the higher score of the fourth group indicates more cartilage degeneration, indicating that the mimic of microRNA-101 aggravates osteoarthritis, and the inhibitor of microRNA-101 improves osteoarthritis. inflammation.

HE staining results of knee trochlear cartilage tissue of animals in each group

As shown in Figure 4, from the perspective of HE staining, specific cartilage degeneration in animal models includes: unevenness and cracks on the surface of cartilage, thinning of the calcified layer of cartilage, and unclear tide lines. At 7 days, there was no significant difference between the overall HE staining of each group. At 14 days, the thickness of cartilage in each group was thinner compared with that at 7 days, but the degree of thinning in the fourth group was relatively mild. The connection between articular cartilage and subchondral bone in groups 1, 2, and 3 was not tight, and there were cracks on the cartilage surface reaching deep into the subchondral bone, while the cartilage surface in group 4 was not smooth and had no cracks. It can be seen that after the injection of microRNA-101 mimics into the joint cavity, the cartilage damage was aggravated, the structure was disordered, and the cartilage layer became thinner; while after the injection of microRNA-101 inhibitors, the cartilage structure was relatively normal.

The results of toluidine blue staining and type II collagen immunohistochemical staining of extracellular matrix of knee trochlear cartilage in animals in each group

Type II collagen and proteoglycans are the main components of the extracellular matrix of cartilage tissue, and they are also specific markers of cartilage. When the cartilage tissue degenerates, the extracellular matrix of the cartilage tissue gradually degrades, and the amount of type II collagen and proteoglycan gradually decreases; Reflect changes in the amount of type II collagen. Therefore, type II collagen and proteoglycans are also the main detection indicators of cartilage degeneration.

As shown in Figure 5 and Figure 6, the results of toluidine blue staining and type II collagen immunohistochemical staining showed that there was no significant difference in the expression of proteoglycan and type II collagen among the groups.

At 14 days, there were differences in the results of toluidine blue staining and type II collagen immunohistochemical staining among the groups, as shown in the following: compared with the other three groups, the toluidine blue staining and type II collagen immunohistochemical staining in the fourth group The deep staining indicates that the extracellular matrix of the cartilage tissue in the fourth group has less degeneration (see Figure 5 and Figure 6). At the same time, there was no significant difference in toluidine blue staining and type II collagen immunohistochemical staining in the other three groups.

In summary, after injecting microRNA-101 mimics into the joint cavity, the extracellular matrix of cartilage: Proteoglycan staining becomes lighter, indicating that the extracellular matrix of cartilage is degraded; Compared with other groups, the staining was darker, indicating that the injection of microRNA-101 inhibitors could prevent the degradation of cartilage extracellular matrix.

After intra-articular injection of microRNA-101 mimics, another extracellular matrix of cartilage: the staining of type II collagen becomes lighter, indicating that the extracellular matrix of cartilage is degraded; after injection of inhibitors of microRNA-101, type II collagen of cartilage tissue is Histochemical staining was darker than other groups, indicating that the injection of microRNA-101 inhibitors could prevent the degradation of cartilage extracellular matrix.

Example 2

Preparation of inhibitor drugs for microRNA-101:

1) The design of the inhibitor of microRNA-101: according to the sequence of microRNA-101, the antisense oligonucleotide of microRNA-101 is designed according to the principle of complementarity, and the antisense oligonucleotide sequence of microRNA-101 is: 5'ATGTCATGACACTATTGACTT3' .

2) Modification of microRNA-101 inhibitors using locked nucleic acid technology: 5'ATGTCATGACACTATTGACTT3' in the antisense oligonucleotide sequence of microRNA-101, 1, 2, 3, 4 in the 1-21st position from the 5' end , 5, 6, 7 or more nucleotides are modified by locked nucleotides (LNA). The locked nucleotides are selected from β-D-oxygen-locked nucleotides, (beta-D-sulfur-locked nucleotides, β-D-amino-locked nucleotides and/or α-L- Oxy-locked nucleotides for substitution). The locked nucleotides are prepared according to the following method (see international patents: WO99/14226, WO00/56746, WO00/56748, WO00/66604, etc.). Thus, the modified microRNA-101 inhibitor drug is prepared. The stability of the prepared microRNA-101 inhibitor drug is improved, and it can specifically bind to the microRNA molecule that inhibits the endogenous maturation of cells

IL-1β and TNF-α acting on human cartilage tissue can cause chondrocyte degeneration, extracellular matrix degradation, and cartilage damage. Adding an effective amount of the prepared microRNA-101 inhibitor drug to the culture medium of human chondrocytes, and then stimulating IL-1β and TNF-α, it was found that the microRNA-101 inhibitor drug could reverse the expression of IL-1β and TNF-α. TNF-α-induced degeneration of human chondrocytes and degradation of extracellular matrix can protect chondrocytes from cartilage damage caused by inflammatory factors. It can be seen that the microRNA-101 inhibitor drug provided in the embodiment of the present invention has the effect of protecting articular chondrocytes and facilitating cartilage repair.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (9)

1. Application of inhibitors of microRNA-101 in the preparation of drugs for the prevention or treatment of osteoarthritis. 2. The application according to claim 1, wherein the nucleotide sequence of the microRNA-101 is 5'-UACAGUACUGUGAUAACUGAA-3'. 3. The application according to claim 2, wherein the microRNA-101 is further modified, and the modified microRNA-101 maintains the activity of the microRNA-101. 4. The application according to claim 3, wherein the modification is at least one selected from ribose modification, base modification, and phosphate backbone modification. 5. The application according to claim 2, wherein the nucleotides in the microRNA-101 are further partially replaced or increased or decreased, and the nucleotides are further retained by the partially substituted or increased or decreased microRNA-101. Describe the activity of microRNA-101. 6. A pharmaceutical composition for preventing or treating osteoarthritis, said pharmaceutical composition comprising an effective amount of an inhibitor of microRNA-101. 7. The pharmaceutical composition according to claim 6, characterized in that, the pharmaceutical composition further comprises other drugs compatible with the microRNA-101 inhibitor and pharmaceutically acceptable carriers and/or adjuvants. 8. The pharmaceutical composition according to claim 7, wherein the dosage form of the pharmaceutical composition is selected from a solution, a suspension, a dry powder, an emulsion, a controlled release agent or a sustained release preparation. 9. The pharmaceutical composition according to claim 7, characterized in that, the administration mode of the pharmaceutical composition is selected from injection administration or oral administration.