JP7080516B2 - Application of LncRNA-32598 in the production of drugs that suppress the hypertrophic differentiation of chondrocytes or in gene delivery systems - Google Patents

Description

The present invention belongs to the technical field of tissue engineering and relates to the application of LncRNA-32598 in the production of a drug that suppresses the hypertrophy and differentiation of chondrocytes or in a gene delivery system.

The cartilage tissue of the human body is composed of chondrocytes and extracellular matrix. Among them, chondrocytes, which make up only 1% of the total tissue volume, cause cartilage formation during growth and maintain the metabolic balance of extracellular matrix in the tissue. Nevertheless, collagen fibrils are substrates and structural frameworks of tissues that provide the tensile strength of cartilage. Articular cartilage is a type of hyaline cartilage, the main component of its collagen fibrils is type II collagen protein, and the secondary components are type IX collagen protein and type XI collagen protein. The collagen fiber network encapsulates proteoglycans and sugar proteins, thus giving the cartilage tissue mechanical elasticity. Chondrocytes require oxygen for metabolism, but articular cartilage has no blood vessels, so nutrients and other cytokines can only be obtained from articular synovial fluid and subchondral bone. Joint movement and load can help the diffusion of nutrients. Osteoarthritis and trauma are the two most common causes of cartilage damage. Osteoarthritis is a degenerative disease of articular cartilage. In the early stages of the disease, chondrocytes proliferate and repair surface cartilage defects, but over time, the rate of cartilage substrate synthesis gradually slows below the rate of degradation, the cartilage surface begins to fibrosis, and then. The inside of the cartilage is damaged. When inflammation breaks the highest point, blood vessels grow into cartilage, causing the formation of fibrous cartilage and ultimately the loss of cartilage function. Traumatic cartilage injuries fall into three categories: microinjuries, cartilage fractures, and osteochondral fractures. Microinjury refers to the destruction of chondrocytes and extracellular matrix so as not to affect the articular surface, as in the early stages of osteoarthritis. Cartilage fracture refers to the destruction of the articular surface so that it does not invade the subchondral bone. Increased matrix anabolic activity is observed about 2 weeks after injury and eventually returns to normal levels. Osteochondral fracture refers to the injury breaking through the highest point and reaching the subchondral bone. In this case, the vasculature of the subchondral bone may activate the inflammatory response of the tissue. In addition, vascular mesenchymal stem cells deposit in the fibrin network and eventually form fibrocartilage. This has a serious effect on cartilage function. The ability to repair avascular articular cartilage is limited, and it can be seen that how to restore the function of articular cartilage after injury is a major issue in the medical field.

Tissue engineering is an ever-developing interdisciplinary field and is expected to provide new solutions for cartilage defect repair. Cell scaffolds, biomaterials and seed cells are three important elements of tissue engineering, but the selection of seed cells is the focus of tissue engineering to make up cartilage. Mesenchymal stem cells (MSCs) are easily isolated, rapidly expand outside the body, and are derived from various tissues such as bone marrow, fat, synovial membrane, bone membrane, umbilical vein, and placenta, and bone formation, cartilage formation, and lipids. It is currently the most potential seed cell in cartilage tissue engineering due to its potential for multi-directional differentiation such as formation. However, during the construction of chondrocytes by histological engineering, it is difficult for chondrocytes derived from MSCs to maintain the metabolic balance of extracellular substrates, so some mature chondrocytes begin to express the chondrocyte hypertrophy marker gene, which is the same. Destroys chondrocyte homeostasis. Changes in the cell microenvironment cause tissue engineering regenerated cartilage cell spontaneous death and matrix degradation, with serious implications for the repair and functional reconstruction of articular cartilage defects. Osteocyte hypertrophy is also the cause of fibrocartilage development during tissue engineering cartilage repair. Therefore, suppressing the progression of MSCs hypertrophy after cartilage differentiation and maintaining the metabolic balance of chondrocyte substrates is a top priority for improving the application of tissue engineering techniques to cartilage repair.

Sox9 and Runx2 are two important regulators in the process of chondrocyte differentiation and hypertrophy. Various signaling pathways regulate the progression of chondrocyte proliferation or hypertrophy by altering the relative transcriptional activity of Sox9 and Runx2. In the Wnt pathway, the LEF / TCF / β-catenin complex may promote Runx2 expression. The TGF-β signaling pathway induces Sox9 expression via phosphorylated Smad2 / 3 and promotes cartilage differentiation, whereas the BMP signaling pathway promotes chondrocyte hypertrophy via phosphorylated Smad1 / 5/8. do. PTHrP can suppress the progression of chondrocyte hypertrophy, and the Ih signaling pathway increases the transcriptional activity of Runx2, thereby actively regulating the progression of chondrocyte hypertrophy. The balance between these signaling pathways is key to normal chondrocyte differentiation and cartilage growth. Over the last decade, significant progress has been made in the study of signaling pathways and transcriptional regulatory networks in the process of chondrocyte differentiation and hypertrophy. A complete understanding of the molecular mechanism is crucial for the development of new drugs to prevent and treat a variety of bone diseases. At the same time, avoiding cell hypertrophy during cartilage repair is an important research topic, but understanding important pathways and molecular regulatory networks during cartilage growth is the basis for solving the above problems.

With the development of genomics, new techniques such as deep sequencing have shown that most DNA nucleotide sequences are transcribed into RNA. However, RNA is classified into coding RNA and non-coding RNA (ncRNA) because only 1-2% of the human genome encodes proteins. In the past, ncRNAs were considered "evolutionary debris," but there is increasing evidence that ncRNAs have important regulatory effects on various biological behaviors of cells. MicroRNAs (microRNAs) with a size of about 20 nt are also a type of ncRNA, and their main action is to bind to the target mRNA and induce its degradation or suppress translation, thereby expressing the gene. Is to adjust negatively. Our previous study found that miR-145-5p can target Sox9 and regulate chondrocyte differentiation and hypertrophy at post-transcriptional levels. NcRNAs longer than 200 nt compared to microRNAs are called long non-coding RNAs (lncRNAs). Its biological function is more complex because LncRNA has a high transcription rate in the cell and can be folded into complex high-level structures. LncRNAs play a role similar to transcription factors and can bind to target genes and regulate their expression. For example, lncRNA RP11-714G18.1 directly binds to the nearby gene LRP2BP, increasing the expression of LRP2BP and ultimately suppressing the migration of endothelial cells, thereby affecting the hardening process of atherosclerosis. be able to. In addition, lncRNA acts to protect the target mRNA from inhibition by binding to and separating specific miRNAs as competing endogenous RNAs (ceRNAs). For example, lncRNA Gm10768 competitively binds to miR-214 as a molecular sponge, reducing inhibition of activated transcription factor 4 (ATF4) and affecting the hepatic gluconeogenic process. The above results suggest that lncRNA regulates the expression of target genes at the transcriptional and post-transcriptional levels by various mechanisms and affects various pathophysiological processes. However, the regulation of lncRNA on chondrocyte biological behavior, especially chondrocyte hypertrophy and differentiation, is currently unknown. Furthermore, the application of lncRNA to the study of chondrocyte hypertrophy differentiation has not yet been reported.

Currently, there are many ways to control chondrocyte hypertrophy. At the protein level, parathyroid hormone-related peptides and inhibitors of matrix metalloproteinases can reduce the degradation of extracellular substrates and slow the progression of cell hypertrophy. At the genetic level, transcription factors such as Sox9 and Nkx3.2 due to overexpression of mesenchymal stem cells may reduce the expression of hypertrophic proteins. Addition of chondroitin sulfate to the medium promotes the synthesis of extracellular matrix of chondrocytes and significantly reduces the expression of type X collagen. In addition, a hypoxic environment and biomechanical stimuli cause a regulatory effect on chondrocyte hypertrophy. A wide variety of plant extracts from broadband sources have the advantages of high efficiency, safety and low side effects, and are gradually gaining attention for research and application in the fields of biology and medicine. Since plant extracts from different sources have different active ingredients, they can regulate cell growth and proliferation via different signaling pathways. In addition, some plant extracts also have effects such as complement activation, anti-inflammatory, delayed aging, and inhibition of tumor growth. Many scholars now report the effect of plant extracts on osteoblast and osteoclast differentiation, for example, curcumin on mesenchymal stem cell-derived bone by inducing mild endoplasmic reticulum stress. It has been reported to promote differentiation. Furthermore, cordisepine has been reported to suppress the formation of osteoclasts by removing reactive oxygen species to prevent bone loss. Therefore, looking for plant extracts that suppress chondrocyte hypertrophy can provide new strategies for reducing the appearance of fibrocartilage in tissue engineering. It has not been reported that lncRNA is applied to the study of chondrocyte hypertrophy differentiation as a marker for drug screening.

In view of these, it is an object of the present invention that when repairing defects using histologically engineered chondrocytes, seed cells often undergo hypertrophy, which makes it difficult to maintain cell phenotype and is prone to fibrosis. By screening lncRNA-LncRNA-32598, which is associated with the progression of chondrocyte hypertrophy derived from mesenchymal stem cells, the effect and molecular mechanism of the problem are studied, and the chondrocyte derived from stem cells is used. After confirming that the progress of cell hypertrophy can be effectively suppressed, screening of a plant extract that suppresses the progress of chondrocyte hypertrophy using LncRNA-32598 as a marker is performed. The present invention provides the application of long non-coding RNA LncRNA-32598 in the production of drugs that suppress the hypertrophy and differentiation of chondrocytes or in gene delivery systems. The present invention provides the following technical solutions.

The application of LncRNA-32598 in the production of drugs that suppress the hypertrophy and differentiation of chondrocytes or in the gene delivery system is that the base sequence of LncRNA-32598 is SEQ ID NO. Indicated by 1.

Furthermore, the LncRNA-32598 can promote the expression of the Sox9 gene.

Furthermore, the LncRNA-32598 can competitively bind to miR-145a-5p and miR-509-5p.

Furthermore, the LncRNA-32598 can suppress the hypertrophic differentiation of chondrocytes.

Furthermore, the LncRNA-32598 can suppress the degeneration of the cartilage substrate.

In addition, the LncRNA-32598 can be used as a marker for effective screening of plant extracts that suppress the progression of chondrocyte hypertrophy.

In addition, the gene delivery system is a lentivirus packaging system, an adenovirus packaging system, a reverse transcription packaging system or a naked plasmid.

In addition, the chondrocytes include normal phenotypic chondrocytes and chondrocytes derived from mesenchymal stem cells.

Further, the mesenchymal stem cells are bone marrow mesenchymal stem cells, peripheral blood mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord mesenchymal stem cells.

Further, the mesenchymal stem cells require 10 to 14 days of induced culture time for cartilage differentiation, and 10 to 14 days for induced culture from hypertrophy to hypertrophy of the chondrocytes.

The specific application steps are as follows.
1. Highly expressed lncRNA-LncRNA-32598 is identified via the lncRNA chip at the stage of chondrocyte hypertrophy and differentiation.

After treating the mesenchymal stem cells with the cartilage-inducing medium for 14 days, the medium is replaced with the hypertrophy-inducing medium and treated for another 14 days. The total period is 28 days. Samples are collected at three time points: day 0 (non-induced group), day 14 (cartilage group), and day 28 (hypertrophy group). After screening a large number of candidate lncRNAs that are negatively correlated with miR-145a-5p expression and have significant changes, bioinformatics analysis is used to compare candidate lncRNAs to miR-145a-5p and lncRNAs. The highest score associated with -32598 and miR-145a-5p is found. By qPCR validation, LncRNA-32598 expression is elevated during cartilage differentiation and decreased during cartilage hypertrophy.

2. LncRNA-32598 has an action of regulating hypertrophic differentiation of chondrocytes.

To study the regulatory effect of lncRNA-32598 on chondrocyte hypertrophy, the lncRNA overexpresses and interferes with lentivirus-infected chondrocyte progenitor cells (from mesenchymal stem cells that have induced chondrocyte differentiation for 14 days). .. After treating cartilage progenitor cells with hypertrophy-inducing medium for 14 days, at the nucleic acid level, the expression of hypertrophy markers Runx2 and Col10a1 that interfered with the LncRNA-32598 cell group was higher than that of the control group, and Runx2 and Runx2 overexpressing the LncRNA-32598 group. The expression of Col10a1 is lower than that of the control group. WB results also show interference with the expression of LncRNA-32598, which can increase the expression of Runx2 and Col10a1. In addition, Alcian blue staining represents the metabolism of the extracellular matrix, and the result is that the degradation and metabolism of the extracellular matrix that interfered with the LncRNA-32598 group was strong, and the degradation and metabolism of the extracellular matrix that overexpressed the LncRNA-32598 group. It shows that it is weak. The above data show that LncRNA-32598 can suppress chondrocyte hypertrophy and suppress cartilage substrate degradation.

3. LncRNA-32598 targets and regulates miR-145a-5p and miR-509-5p to regulate Sox9 expression.

lncRNA-32598 can be regulated by directly targeting miR-145a-5p and miR-509-5p with a dual luciferase reporter gene system. The two mechanisms are different. Here, 1) when lncRNA-32598 is overexpressed, the expression of the hypertrophy markers Runx2, Col10a1 and Mmp13 is reduced as compared with the control group, and due to the overexpression of miR-145a-5p, Runx2, Col10a1 and Mmp13 are overexpressed. Expression is increased. Furthermore, overexpression of miR-145a-5p can antagonize the increased inhibitory effect on the expression of Runx2, Col10a1 and Mmp13 by the expression of lncRNA-32598. At the protein level, overexpression of lncRNA-32598 causes an increase in Sox9 expression, overexpression of miR-145a-5p causes a decrease in Sox9 expression, and overexpression of miR-145a-5p is due to expression of lncRNA-32598. It antagonizes the increase in promoting action on the expression of Sox9. The effect of Runx2 on expression is the exact opposite of that of Sox9. The results of alcian blue staining showed that overexpression of lncRNA-32598 suppressed extracellular matrix degradation, overexpression of miR-145a-5p promoted extracellular matrix degradation, and overexpression of miR-145a-5p. Has been shown to antagonize the increased inhibitory effect on extracellular matrix degradation by the expression of lncRNA-32598. 2) Id1 is a downstream molecule of the BMP signaling pathway and can regulate Sox9 expression at the transcriptional level. The dual luciferase reporter gene system confirms a direct target relationship between miR-509-5p and Id1, and co-expression experiments show that Id1 expresses Sox9 caused by overexpression of miR-509-5p. Shows that it can antagonize the decline. In the overexpression experiment of lncRNA-32598, it is found that lncRNA-32598 competitively binds to miR-509-5p to regulate the expression of Sox9 and suppress the hypertrophy of chondrocytes.

4. LncRNA-32598 can be screened as a biomarker for plant extracts that suppress chondrocyte hypertrophy.

It was found that cordisepine, curcumin, xanthotoxin, dihydroartemisinin and anthocyanin promote the expression of LncRNA-32598 and suppress the expression of chondrocyte hypertrophy marker genes at the nucleic acid and protein levels. Correspondingly, resveratrol suppresses the expression of LncRNA-32598 and promotes the progression of chondrocyte hypertrophy. Therefore, LncRNA-32598 can be screened as a biomarker for plant extracts that suppress chondrocyte hypertrophy.

The beneficial effects of the present invention are as follows.

In the process of constructing chondrocytes by histological engineering, it is difficult for chondrocytes derived from MSCs to maintain the metabolic balance of extracellular substrates, so some mature chondrocytes begin to express marker genes for chondrocyte hypertrophy. It destroys chondrocyte homeostasis. Changes in the cell microenvironment cause tissue engineering regenerated cartilage cell spontaneous death and matrix degradation, with serious implications for the repair and functional reconstruction of articular cartilage defects. Osteocyte hypertrophy is also the cause of fibrocartilage development during tissue engineering cartilage repair. Therefore, suppressing the progression of MSCs hypertrophy after cartilage differentiation and maintaining the metabolic balance of chondrocyte substrates is an important problem to be solved for improving the application of tissue engineering technology to cartilage repair. .. The present invention mainly achieves the following beneficial technical effects.

1) During repair, some tissue-engineered cartilage exhibits fibrous changes, and the fibrocartilage lacks the biomechanical properties necessary to withstand compression stress, but is predominantly in the formation of fibrocartilage. The reason is the hypertrophy and differentiation of chondrocytes. Hypertrophic chondrocytes secrete X-type collagen and matrix metalloproteinase 13, which is a degradation of extracellular matrix, and have a serious effect on the effect of cartilage repair. In addition, chondrocyte hypertrophy is also one of the important pathological features of osteoarthritis, as the extracellular matrix degrades, the hyaline cartilage becomes fibrotic, which usually deteriorates over time. The differentiation of chondrocytes derived from mesenchymal stem cells and the subsequent enlargement of chondrocytes are important processes that influence the repair of cartilage by tissue engineering and the development and development of osteoarthritis. By using the method of the present invention, LncRNA-32598 can bind directly to miR-145a-5p and miR-509-5p, and LncRNA-32598 can express Sox9 by competitively binding to the target site together with microRNA. By promoting and increasing the expression of LncRNA-32598, the degradation of cartilage matrix can be reduced, thereby suppressing the hypertrophic differentiation of mesenchymal stem cell-derived chondrocytes.

2) Since LncRNA-32598 plays an important role in maintaining the chondrocyte phenotype and the metabolic balance of the matrix, it is possible to find a strategy to prevent the appearance of fibrous cartilage in tissue engineering more quickly as a marker. can. The present invention screens plant extracts that suppress chondrocyte hypertrophy using LncRNA-32598 as a biomarker. It was found that cordisepine, curcumin, xanthotoxin, dihydroartemisinin and anthocyanin promote the expression of LncRNA-32598 and suppress the expression of chondrocyte hypertrophy marker genes at the nucleic acid and protein levels. Correspondingly, resveratrol suppresses the expression of LncRNA-32598 and promotes the progression of chondrocyte hypertrophy. Therefore, LncRNA-32598 can be screened as a biomarker for plant extracts that suppress chondrocyte hypertrophy. Plant extracts screened based on increased expression of LncRNA-32598 can suppress the progression of chondrocyte hypertrophy by a variety of molecular mechanisms. It helps to quickly and conveniently discover new ways to maintain cartilage phenotype and stability, and can be used to discover and develop new drugs for the treatment of osteoarthritis. It can also be used to find good strategies for solving clinical application problems of cartilage by tissue engineering.

In order to further clarify the object, technical solution and beneficial effect of the present invention, the present invention will be described with reference to the following drawings.
FIG. 1 shows the results of changes over time in LncRNA-32598 by qPCR verification. Expression of LncRNA-32598 increases during cartilage differentiation and decreases during cartilage hypertrophy. FIG. 2 shows the results of LncRNA-32598 suppressing chondrocyte hypertrophy in LncRNA-32598 interference and overexpression experiments. FIG. 3 shows the results of competitive binding of LncRNA-32598 to miR-145a-5p. FIG. 4 shows the possibility that LncRNA-32598 competitively directly binds to miR-509-5p. FIG. 5 shows the results of MiR-509-5p targeting Id1 and regulating Sox9 expression. FIG. 6 shows the results of regulating chondrocyte hypertrophy by competitively binding LncRNA-32598 to miR-145a-5p. FIG. 7 shows the results of regulatory binding of chondrocyte hypertrophy by competitive binding of LncRNA-32598 to miR-509-5p. FIG. 8 shows the results of plant extracts affecting chondrocyte expression of lncRNA-32598.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. If no specific conditions are described in a preferred embodiment, the experimental method is usually performed according to conventional conditions or according to conditions recommended by the reagent manufacturer.

Example 1: Bone composition by histological engineering based on the intrachondral bone formation system of LncRNA-32598 1. Cultivation of bone marrow MSCs and establishment of a model for inducing hyperdifferentiation of chondrocytes derived from MSC Human bone marrow MSC with lymphocyte isolation solution After culturing and centrifuging, the soft-film mononuclear cells are washed twice with DMEM (centrifugal at 1000 r / min for 5 minutes), and the supernatant is discarded. Cells were resuspended in DMEM-L complete medium containing 15% fetal bovine serum, then inoculated into culture flasks at a cell density of 1 × ¹⁰⁶ / mL and recorded as primary at 37 ° C., 5% CO ₂ . Incubate in a constant temperature incubator. The liquid is changed every 3 days, and after the liquid is changed, cell proliferation is observed with an inverted microscope. As cells approach fusion, they are digested with 0.25% trypsin and passaged at a ratio of 1: 2 to 1: 3. The basic medium formulation is as follows: 89% DMEM-F12 medium, 10% FBS, 1% dual antibody (100 mg / ml streptomycin / 100 U / ml penicillin). When about 80% of the cultured bone marrow MSCs are fused, the MSCs cells are digested and centrifuged, and the cells are resuspended and counted in the cartilage induction medium. Next, the cells ( ^2.5 × 105) were dropped into a 15 ml centrifuge tube and centrifuged at 3500 rpm for 15 minutes to aggregate the cells, and then the centrifuge tube was carefully inserted into the centrifuge tube rack at 37 ° C. Incubate in a constant temperature incubator. In addition, cells are uniformly inoculated into cell culture plates (6 well plates, 8 × 10 ⁴ / well) for monolayer culture. The time for inducing differentiation of mesenchymal stem cell-derived cartilage is 14 days, and the induction medium is changed every 3 days. After 14 days of induction of differentiation of mesenchymal stem cell-derived cartilage, the medium is replaced with a hypertrophy-inducing medium, and culture is continued for 14 days. The total period was 28 days. The induction medium needs to be changed every 3 days. The formulation of the cartilage induction medium is as follows: DMEM high glucose medium, 5% FBS, 1% dual antibody (100 mg / ml streptomycin / 100 U / ml penicillin), 1% ITS mixture, 40 μg / ml proline, 100 nM. Dexamethasone, 50 μg / ml ascorbic acid, 10 ng / ml TGF-β3. The formulation of the hypertrophy induction medium is as follows: DMEM high glucose medium, 5% FBS, 1% dual antibody (100 mg / ml streptomycin / 100 U / ml penicillin), 1% ITS mixture, 40 μg / ml proline, 1 nM dexamethasone, 50 μg / ml ascorbic acid, 100 ng / LT3.

2. 2. LncRNA-32598 is reduced during the differentiation stage of cartilage hypertrophy.
After treating MSCs with cartilage-inducing medium for 14 days, the medium was replaced with hypertrophy-inducing medium and treated for another 14 days. The total period was 28 days. Samples are collected at three time points: day 0 (non-induced group), day 14 (cartilage group), and day 28 (hypertrophy group). Expression of LncRNA-32598 in chondrocyte differentiation and hypertrophy was detected by qPCR. The results are shown in FIG. The results show that the expression of LncRNA-32598 (corresponding to NCBI sequence NR_045420.2) is increased at the stage of cartilage differentiation and decreased at the stage of cartilage hypertrophy. This is consistent with the expression profiling results of the lncRNA chip detected by the Arraystar Mouse lncRNA Microarray V3.0. Using bioinformatics analysis and comparison, we find that LncRNA-32598 and miR-145a-5p have the highest scores.

The gene sequence of LncRNA-32598 is as follows.
５'－ＡＴＣＴＣＴＧＣＣＣＣＴＣＡＧＡＧＴＣＣＴＧＧＧＧＡＧＧＡＴＧＧＡＴＧＧＡＡＣＡＧＧＡＧＧＡＧＣＡＴＣＡＴＧＡＧＡＡＧＧＣＴＴＣＣＴＧＴＡＡＡＣＴＡＣＧＧＡＧＧＧＴＧＡＧＧＧＧＡＣＧＧＡＧＣＧＴＧＡＧＧＧＡＡＣＧＴＣＧＴＧＡＧＡＴＧＧＴＧＣＴＣＧＴＧＧＡＡＧＣＣＡＧＣＡＧＡＧＧＧＴＧＴＡＧＧＧＴＣＴＣＣＴＧＧＡＧＣＣＡＣＡＴＣＴＣＴＧＧＡＧＴＴＡＣＡＴＧＡＡＧＴＴＧＴＧＧＡＣＣＴＣＴＣＡＡＣＡＴＧＧＧＴＴＣＴＧＧＧＡＡＣＴＧＡＡＣＣＣＡＧＧＴＣＣＴＣＴＧＧＡＡＧＧＡＣＡＴＣＧＡＧＴＧＣＴＣＴＴＡＡＣＣＡＣＴＧＡＧＣＣＡＴＣＴＣＴＧＣＡＧＣＣＴＡＡＣＣＣＣ
ＡＴＣＣＣＴＴＧＧＡＧＧＣＴＧＡＡＧＣＡＡＡＧＣＡＡＧＴＴＣＡＡＴＧＡＣＣＧＴＧＣＧＡＣＣＴＧＧＣＴＣＴＴＧＴＴＴＧＧＧＣＣＴＡＡＧＧＡＡＡＡＧＣＣＧＡＣＴＧＴＡＡＧＣＣＧＧＧＡＡＧＣＡＴＣＴＣＴＧＧＣＡＡＧＡＡＣＡＡＧＧＡＡＧＧＡＴＴＡＡＧＣＡＣＧＴＣＴＧＴＧＧＴＴＧＴＧＣＧＣＡＧＧＧＡＴＣＡＣＡＡＧＧＴＧＣＴＣＣＴＧＣＡＧＴＧＧＧＡＡＧＡＧＣＣＡＣＣＡＣＴＡＧＧＧＧＣＧＣＴＣＣＡＡＧＴＣＴＴＧＴＡＴＴＴＴＣＴＡＣＣＴＴＧＡＡＡＡＣＴＣＧＴＴＴＣＣＣＴＧＴＧＧＣＴＧＣＣＣＴＣＣＡＡＡＴＣＣＡＡＣＧＧＡＧＡＴＧＡＡＴＧＧＣＴＴＴＴＡＧＴＴＴＡＣＣＴＡＧＣＴＧＡＴＴＴＡＡＴＡＡＡＣＴＴＴＡＡＡＡＡＴＡＡＡＴＧＴＡＣＧＡＡＴＧＡＣＴＴＴＣＡＡＡＡＡ－３' （ＳＥＱ ＩＤ ＮＯ．１）

Example 2: LncRNA-32598 that suppresses chondrocyte hypertrophy and cartilage substrate degradation

To detect the regulatory effect of LncRNA-32598 on chondrocyte hypertrophy, lenchvirus-infected chondrocyte progenitor cells are overexpressed and interfered with by LncRNA (MSCs cells induced and differentiated in cartilage for 14 days). It was confirmed by qPCR that a cartilage progenitor cell line overexpressed and disturbed by LncRNA-32598 was successfully established (Fig. 2A). At the nucleic acid level, the expression of the hypertrophy markers Runx2 and Col10a1 that interfered with the LncRNA-32598 cell group was higher than that of the control group, and the expression of Runx2 and Col10a1 that overexpressed the LncRNA-32598 group was lower than that of the control group (FIGS. 2B, C). ). The WB results show that the expression of Runx2 and Col10a1 that interfered with the LncRNA-32598 group was increased, and the expression of Runx2 and Col10a1 that overexpressed the LncRNA-32598 group was decreased (FIG. 2D). Alcian blue staining represents the metabolism of the extracellular matrix, and the result is that the decomposition metabolism of the extracellular matrix that interfered with the LncRNA-32598 group is strong, and the decomposition metabolism of the extracellular matrix that overexpresses the LncRNA-32598 group is weak. Is shown (Fig. 2E). In addition, immunochemical staining of Col10a1 showed that the expression of Col10a1 that interfered with the LncRNA-32598 group was higher than that of the control group, and the expression of Col10a1 that overexpressed the LncRNA-32598 group was lower than that of the control group (). E).

The above data indicate that lncRNA-32598 suppresses chondrocyte hypertrophy and cartilage substrate degradation.

Example 3: LncRNA-32598 regulates Sox9 expression by competitively binding to miR-145a-5p and miR-509 and suppressing chondrocyte hypertrophy.

1. LncRNA-32598 targets and regulates miR-145a-5p and miR-509-5pm.

The cells were disturbed by LncRNA-32598 to form overexpressed cells, and the expression level of miR-145a-5p was detected by qPCR, and the results are shown in FIG. It was shown that the expression of miR-145a-5p in the disturbing group was higher than that in the control group, and the expression of miR-145a-5p in the overexpressing group was lower than that in the control group (FIG. 3A). The direct target relationship between lncRNA-32598 and miR-145a-5p is further validated by the dual luciferase reporter gene system. The wild-type Lnc3'-UTR plasmid and the mutant Lnc3'-UTR are transfected into HEK293 cells and a precursor or control of microRNA-145 is added, respectively. The results showed that the fluorescence intensity detected in cell samples cotransfected with microRNA precursors and wild-type Lnc3'-UTR plasmids was significantly reduced, and microRNA-145 precursors and mutant Lnc3'-UTR plasmids were obtained. It is shown that the fluorescence intensity detected in the co-transfected cell sample is not different from that of the control group (Fig. 3B).

The cells were disturbed by LncRNA-32598 to form overexpressed cells, and the expression level of miR-509-5p was detected by qPCR, and the results are shown in FIG. It was shown that the expression of miR-509-5p in the disturbing group was higher than that in the control group, and the expression of miR-509-5p in the overexpressing group was lower than that in the control group (FIG. 4A). The wild-type Lnc3'-UTR plasmid and the mutant Lnc3'-UTR are transfected into HEK293 cells and the precursor or control of miR-509-5p is added, respectively. The results showed a significant reduction in fluorescence intensity detected in cell samples cotransfected with microRNA precursors and wild-type Lnc3'-UTR plasmids, with miR-509-5p precursors and mutant Lnc3'-UTR. It is shown that the fluorescence intensity detected in the cell sample cotransfected with the plasmid is not different from that of the control group (Fig. 3B).

2. MiR-509-5p targets Id1 and regulates Sox9 expression.

Experiments have shown that miR-509-5p suppresses Sox9 expression, but as shown in FIG. 5, bioinformatics analysis shows that miR-509-5p directly targets Sox9 expression. It cannot be regulated, suggesting that it directly targets and regulates the expression of Id1.

FIG. 5A is a bioinformatics analysis suggesting that miR-509-5p directly targets and regulates Id1 expression.

FIG. 5B is the result of WB, when the expression of miR-509-5p is disturbed, the expression of Id1 is increased as compared with the control group, and when the expression of miR-509-5p is overexpressed, the expression of Id1 is controlled. It is shown to decrease compared to the group. This suggests that miR-509-5p can regulate the expression of Id1.

FIG. 5C shows the fluorescence intensity detected in cell samples cotransfected with the precursor of miR-509-5p and the mutant Lnc3'-UTR plasmid, showing a significant decrease in fluorescence intensity. The fluorescence intensity detected in the cell sample cotransfected with the precursor of miR-509-5p and the mutant Lnc3'-UTR plasmid was not different from that of the control group, and miR-509-5p directly targeted Id1. Shows the possibility of adjusting.

Id1 is a downstream molecule of the BMP signaling pathway and can regulate Sox9 expression at the transcriptional level. To validate the speculation, cells were treated with medium containing various components for 7 days. The experiments were grouped as follows: Anti-miR NC group, Anti miR-509-5p group, Pre-miR NC group and PremiR-509-5p group.

To further verify the regulatory relationship of miR-509-5p to Sox9 by Id1, it constitutes Id1 overexpression and a control lentivirus, infects cells and is described above in a medium containing a miR-509-5p precursor or control. Cell lines were treated for 7 days. The experiments were grouped as follows: control group, miR-509-5p group, Id1 group and co-expression group. The WB results shown in FIG. 5D show that when miR-509-5p is overexpressed alone, the expression of Id1 and Sox9 is reduced compared to the control group, which is consistent with the previous results. ing. When Id1 is overexpressed alone, the expression of Id1 and Sox9 is increased as compared with the control group, indicating that the virus overexpressing Id1 is effective. Furthermore, the results of the co-expression group shown in FIG. 5D show that Id1 can antagonize the decrease in Sox9 expression caused by the overexpression of miR-509-5p. The above data show that miR-509-5p directly targets Id1 and regulates Sox9 expression.

3. LncRNA-32598 regulates Sox9 expression via miR-145a-5p and miR-509 to suppress chondrocyte hypertrophy differentiation.

To further examine the extranuclear mechanism of LncRNA-32598 that regulates chondrocyte hypertrophy, overexpression of LncRNA-32598 and control chondrocyte precursor cells are composed to induce hypertrophy of precursors or controls containing miR-145a-5p. Treated with medium for 14 days. The experiments were grouped as follows. Control group, LncRNA-32598 group, miR-145a-5p group and co-expression group. The results are shown in FIG.

6A, 6B and 6C show that when qPCR detects overexpression of lncRNA-32598, the expression of hypertrophy markers Runx2, Col10a1 and Mmp13 is reduced compared to the control group, miR-145a. Overexpression of -5p can cause elevated expression of Runx2, Col10a1 and Mmp13.

FIG. 6D shows that at the protein level, overexpression of lncRNA-32598 causes increased expression of Sox9, and overexpression of miR-145a-5p causes a decrease in Sox9 expression, the effect on Runx2 expression. It's the exact opposite of that of Sox9.

In FIG. 6E, the results of alcian blue staining and immunochemical staining showed that overexpression of lncRNA-32598 suppressed extracellular matrix degradation, and overexpression of miR-145a-5p promoted extracellular matrix degradation. , MiR-145a-5p is shown to antagonize the increased inhibitory effect on extracellular matrix degradation by the expression of lncRNA-32598. The results of immunochemical staining show that overexpression of LncRNA-32598 suppresses Col10a1 synthesis and overexpression of miR-145a-5p antagonizes the increased inhibitory effect on Col10a1 synthesis by expression of LncRNA-32598.

In summary, LncRNA-32598 regulates Sox9 expression by competitively binding to miR-145a-5p, suppressing chondrocyte hypertrophy and cartilage substrate degradation.

Overexpressing and control cartilage precursor cells of normally constructed LncRNA-32598 were treated with hypertrophy-inducing medium containing miR-509-5p precursor or control for 14 days. The experiments were grouped as follows. Control group, LncRNA-32598 group, miR-509-5p group and co-expression group. The detection method and index are the same as miR-145a-5p.

7A, 7B and 7C show that when qPCR detects overexpression of lncRNA-32598, the expression of hypertrophy markers Runx2, Col10a1 and Mmp13 is reduced compared to the control group, miR-145a-5p. Overexpression of can cause an increase in Runx2, Col10a1 and Mmp13 expression.

FIG. 7D shows that at the protein level, overexpression of lncRNA-32598 causes increased expression of Sox9, and overexpression of miR-145a-5p causes a decrease in Sox9 expression, the effect on Runx2 expression. It's the exact opposite of that of Sox9.

In FIG. 7E, the results of alcian blue staining and immunochemical staining show that overexpression of miR-509-5p antagonizes the increased inhibitory effect on extracellular matrix degradation by the expression of lncRNA-32598.

Therefore, in addition to miR-145a-5p, LncRNA-32598 regulates the expression of Sox9 by competitively binding to miR-509-5p to suppress chondrocyte hypertrophy and cartilage substrate degradation.

Example 4: LncRNA-32598 can be screened as a biomarker for plant extracts that suppress chondrocyte hypertrophy differentiation.

The establishment of an in vitro induction model of chondrocyte hypertrophy differentiation is the same as above. Alcian blue or toluidine blue stains evaluate the metabolism of extracellular matrix. The qPCR method detects the expression of genes associated with chondrocyte hypertrophy and differentiation and LncRNA-32598. In the experiment, it is necessary to detect the primer sequence of the LncRNA-32598 gene as shown in SEQ ID NO: 2 and SEQ ID NO: 3. With GAPDH expression as an internal control, Delta Ct was used to analyze the relative expression of the gene. The primer sequence of the LncRNA-32598 gene is as follows.

Downstream: 5'-GGGGAGCCGTCTCATAGT-3'(SEQ ID NO. 2);
Upstream: 5'-GGAGGTAAGCGGTCTTGG-3'(SEQ ID NO.3).

Safe concentrations of selected anti-inflammatory plant extracts were determined using Cell Counting Kit-8, and ATDC5 cells 14 days after cartilage induction were treated with various plant extracts for 72 hours to collect RNA. The expression of LncRNA-32598 in cells was detected by qPCR. The results shown in FIG. 8 show that Cordycepin, Curcumin, Xanthotoxin (XAT), Dihydroartemisinin (DHA) and Cyanidin (Cyanidin) are nucleic acids-25 compared to the control group. Although promoting expression, resveratrol suppresses the expression of lncRNA-32598, indicating that it suppresses the expression of chondrocyte hypertrophy marker genes at the nucleic acid and protein levels. In addition, resveratrol can suppress the expression of LncRNA-32598 and promote the hypertrophic differentiation of chondrocytes. The above results indicate that the present invention screens plant extracts that suppress chondrocyte hypertrophy using LncRNA-32598 as a biomarker.

Finally, it should be explained that the preferred embodiments described above are used only to illustrate the technical solutions of the invention and are not limited thereto. Although the present invention has been described in detail through the preferred embodiments described above, one of ordinary skill in the art may make various changes in form and detail without departing from the scope defined by the claims of the invention. You should understand what you can do.

Claims (10)

The use of LncRNA-32598 in the production of a drug that suppresses hypertrophic differentiation of chondrocytes, wherein the base sequence of LncRNA-32598 is SEQ ID NO. Use of LncRNA-32598 in the manufacture of a drug that suppresses the hypertrophic differentiation of chondrocytes, as shown in 1. The use of LncRNA-32598 in the production of a drug that suppresses hypertrophy and differentiation of chondrocytes according to claim 1, wherein the LncRNA-32598 can promote the expression of the Sox9 gene. The LncRNA-32598 in the production of a drug that suppresses hypertrophy and differentiation of chondrocytes according to claim 1, wherein the LncRNA-32598 can competitively bind to miR-145a-5p and miR-509-5p. Use of. The use of LncRNA-32598 in the production of a drug that suppresses the hypertrophy and differentiation of chondrocytes according to claim 1, wherein the LncRNA-32598 can suppress the hypertrophy and differentiation of chondrocytes. The use of LncRNA-32598 in the production of a drug that suppresses hypertrophy and differentiation of chondrocytes according to claim 1, wherein the LncRNA-32598 can suppress degeneration of a cartilage substrate. The chondrocyte hypertrophy differentiation according to claim 1, wherein the LncRNA-32598 can be used as a marker for effective screening of a plant extract that suppresses the progression of chondrocyte hypertrophy. Use of LncRNA-32598 in the manufacture of drugs to be used . The LncRNA-in the production of a drug that suppresses hyperdifferentiation of chondrocytes according to claim 1, wherein the chondrocytes include chondrocytes of normal phenotype and chondrocytes derived from mesenchymal stem cells. Use of 32598. The seventh aspect of claim 7 , wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells, peripheral blood mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells, or umbilical cord mesenchymal stem cells. Use of LncRNA-32598 in the production of drugs that suppress the hypertrophy and differentiation of cartilage cells. The mesenchymal stem cells require 10 to 14 days of induced culture time for chondrocyte differentiation, and the induced culture time for chondrocyte hypertrophy to hypertrophy differentiation is 10 to 14 days. Item 7. Use of LncRNA-32598 in the production of a drug that suppresses the hyperdifferentiation of chondrocytes according to Item 7. It is an agent for suppressing hypertrophy of chondrocytes containing LncRNA-32598, and the base sequence of the LncRNA-32598 is SEQ ID NO. A chondrocyte hypertrophy differentiation inhibitor, which is represented by 1.

Images