Application of histone modification enzyme genes KDM3A and G9A in regulating cartilage formation and cartilage repair of dental pulp mesenchymal stem cells

Abstract

The present invention belongs to the field of gene functions and applications, and relates to the application of histone modification enzyme genes KDM3A and G9A in regulating cartilage formation and cartilage repair of dental pulp mesenchymal stem cells. The KDM3A and G9A genes bidirectionally regulate the cartilage differentiation of dental pulp mesenchymal stem cells. The present invention provides a potential molecular therapeutic target for improving the application efficacy of dental pulp mesenchymal stem cells in cartilage tissue engineering, and has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of gene functions and applications, and in particular, relates to the application of histone modification enzyme genes KDM3A and G9A in regulating cartilage formation and cartilage repair of dental pulp mesenchymal stem cells. Background Art

[0002] Cartilage defects caused by trauma or degenerative lesions are closely related to disability, usually leading to joint pain, strangulation, and functional impairment or disorder. Unfortunately, due to its inherent characteristics of being avascular and nerveless, articular cartilage has extremely low self-repair ability, and its regeneration has always been the most challenging clinical problem in the field of orthopedics. Existing clinical treatment methods (autologous chondrocyte transplantation, autologous / allogeneic cartilage transplantation, and joint replacement, etc.) all have obvious defects, including insufficient chondrocyte sources, production of fibrous cartilage that cannot perform normal functions, immune rejection, and limited prosthesis life. Recently, tissue engineering technology based on mesenchymal stem cells (MSCs) is expected to achieve in situ repair of cartilage defects with both function and appearance through a one-step method, and has therefore become a hot topic in biomedical research.

[0003] Dental pulp mesenchymal stem cells (DPSCs) are mesenchymal stem cells isolated from the pulp of lost deciduous teeth or extracted permanent teeth (such as wisdom teeth). This method of obtaining the stem cells has the least morbidity and damage to the donor site. Unlike the most widely studied invasive and painful method of obtaining bone marrow mesenchymal stem cells, the DPSCs method is low-cost and easy to obtain, which makes it have a broader clinical application prospect. At the same time, DPSCs have been shown to have multidirectional differentiation potential, including osteogenic, adipogenic and chondrogenic differentiation capabilities. It is reported that human DPSCs can generate cartilage-like tissue in nude mice or repair cartilage defects in different animal models. Therefore, DPSCs have the potential to become seed cells for cartilage tissue engineering. Unfortunately, the epigenetic mechanism regulating DPSCs chondrogenesis is still unknown. Therefore, it is quite necessary to discover key regulatory factors to obtain higher chondrogenesis efficiency of DPSCs.

[0004] KDM3A (lysine demethylase 3A) is a member of the histone demethylase family. KDM3A catalyzes the demethylation of mono- and di-methylated histone H3 lysine 9 (H3K9me1 / me2), thereby mediating transcriptional activation. The methyltransferase G9A, which forms a heterodimer with its highly homologous analog GLP, catalyzes H3K9me1 and H3K9me2, acting opposite to KDM3A. In addition to histone H3, several non-histone proteins have been found to be substrates of KDM3A and G9A. For example, KDM3A was found to mediate lysine demethylation of PGC-1α, an important regulator of mitochondrial biogenesis that responds to oxygen availability. Accumulating evidence suggests that KDM3A and G9A play key regulatory roles in diverse biological events, such as metabolism, cancer progression, spermatogenesis, stem cell function, and sex determination. In bone marrow mesenchymal stem cells (BMSCs), KDM3A and G9A were found to play an important role in osteogenesis through their classical histone demethylation or methylation activities. KDM3A has also been shown to regulate the senescence of mesenchymal stromal cells and bone aging through lectin-mediated heterochromatin reorganization, but the involvement of KDM3A and G9A in the chondrogenic differentiation of mesenchymal stem cells has not been reported. Summary of the invention

[0005] The present invention verifies the association between histone modification enzyme genes KDM3A and G9A and the regulation of cartilage differentiation of dental pulp mesenchymal stem cells, and provides relevant applications based on this.

[0006] The first aspect of the present invention provides the use of histone modification enzyme genes KDM3A and G9A in regulating cartilage differentiation and cartilage repair of dental pulp mesenchymal stem cells. The KDM3A and G9A genes bidirectionally regulate the cartilage differentiation of dental pulp mesenchymal stem cells.

[0007] Specifically, the present study found that the expression of KDM3A (lysine demethylase 3A) gene was upregulated during the chondrogenic differentiation of dental pulp mesenchymal stem cells and was positively correlated with the expression of cartilage markers, while the expression of G9A (lysine methyltransferase) gene was negatively correlated with the expression of cartilage markers. Inhibiting G9A can promote the chondrogenic differentiation of dental pulp mesenchymal stem cells.

[0008] Therefore, the second aspect of the present invention provides the use of a promoter of the histone modifying enzyme gene KDM3A and / or an inhibitor of G9A in screening and / or preparing a promoter for the chondrogenic differentiation of dental pulp mesenchymal stem cells.

[0009] The present invention has found that inhibiting KDM3A can inhibit the chondrogenic differentiation of dental pulp mesenchymal stem cells and weaken their in situ repair ability at the cartilage defect of rat knee joint, while overexpressing KDM3A can promote the chondrogenic differentiation of dental pulp mesenchymal stem cells. Therefore, any substance that can promote KDM3A can promote the upregulation of KDM3A, thereby promoting the chondrogenic differentiation of dental pulp mesenchymal stem cells. The promoter can be a specific small molecule promoter of KDM3A.

[0010] The present invention has found that inhibiting G9A can promote the chondrogenic differentiation of dental pulp mesenchymal stem cells and improve their in situ repair ability at the cartilage defect of rat knee joint. Therefore, any substance that can inhibit G9A can also promote the upregulation of KDM3A, thereby promoting the chondrogenic differentiation of dental pulp mesenchymal stem cells. The inhibitor can be a specific small molecule inhibitor of G9A.

[0011] Specifically, the inhibitor is a specific small molecule inhibitor of G9A, BIX-01294. Further studies have shown that the specific small molecule inhibitor BIX-01294 has the effect of promoting cartilage formation of dental pulp mesenchymal stem cells and can be used as a drug to promote cartilage formation.

[0012] The third aspect of the present invention provides the use of a promoter of the histone modification enzyme gene KDM3A and / or an inhibitor of G9A in the preparation of a cartilage repair drug.

[0013] The promoter and inhibitor are as described above and will not be described in detail here.

[0014] In the present invention, the cartilage repair drug is a cartilage in situ repair drug.

[0015] The present invention provides a potential molecular therapeutic target for improving the application efficacy of dental pulp mesenchymal stem cells in cartilage tissue engineering, and has good practical application value.

[0016] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0018] Figure 1 : This is a graph showing the expression level of KDM3A in the chondrogenic process of DPSCs cells in the embodiment of the present invention. Among them, Figure A shows the increase in the expression level of KDM3A at 0, 3, 7, and 14 days of chondrogenic differentiation of dental pulp mesenchymal stem cells by RT-qPCR; Figure B shows the increase in the expression level of KDM3A at 0, 3, and 7 days of chondrogenic differentiation of DPSCs cells by western blot analysis.

[0019] Figure 2 This is a series of graphs showing the effect of KDM3A on the chondrogenic differentiation ability of DPSCs in the examples of the present invention.

[0020] Among them, A is RT-qPCR showing that after DPSCs were transfected with lentivirus shKDM3A to knock out KDM3A, the expression levels of related cartilage markers were significantly downregulated; B is western blot analysis showing that after knocking out KDM3A, the protein level of cartilage marker ACAN was significantly downregulated; C is RT-qPCR showing that after DPSCs were overexpressed with lentivirus transfection KDM3A, the expression levels of related cartilage markers were significantly upregulated; D is western blot analysis showing that after overexpressing KDM3A, the protein levels of cartilage markers Col2A1 and ACAN were significantly upregulated (*P<0.05; **P<0.01; ***P<0.001).

[0021] Figure 3 It is the effect of KDM3A or G9A on the key transcription factor SOX9 in cartilage formation in the embodiment of the present invention and the effect of G9A on the cartilage-forming ability of DPSCs. Among them, A is a western blot analysis showing that after knocking out KDM3A, the expression level of SOX9 decreased significantly; B is a western blot analysis showing that after knocking down G9A using siRNA, the expression level of SOX9 increased significantly; C is a western blot analysis showing that after knocking down KDM3A using siRNA, the expression level of SOX9 decreased, and after knocking down KDM3A and G9A simultaneously using siRNA, the expression level of SOX9 increased significantly; D is a histological section staining of chondrocytes showing that after knocking down KDM3A and G9A at the same time, the cartilage matrix content generated by DPSCs increased significantly compared with knocking down G9A alone.

[0022] Figure 4The in vivo experiments in the examples of the present invention prove the effects of KDM3A and G9A on the cartilage repair ability of dental pulp mesenchymal stem cells. Among them, A is a representative macroscopic image of each group after 8 weeks of cartilage defect repair in rats. After knocking out KDM3A, the cartilage repair ability of dental pulp mesenchymal stem cells decreased, the surface of the repair area was irregular, and the height of the repair area was significantly lower than that of normal cartilage tissue. When KDM3A and G9A were knocked down at the same time, the repair ability of DPSCs was improved compared with knocking down KDM3A alone, and the repair area was well integrated with normal cartilage and the height was comparable. B is the hematoxylin-eosin (H&E) and toluidine blue staining of rat knee cartilage defects 8 weeks after repair, showing that the repair area was better integrated with normal cartilage tissue and had more cartilage matrix after simultaneous knockdown of KDM3A and G9A than after simple knockdown of KDM3A; C is the immunohistochemical staining of SOX9 and type II collagen expression levels after 8 weeks of repair of rat knee cartilage defects, showing that the expression levels of SOX9 and type II collagen in the repair area were higher after simultaneous knockdown of KDM3A and G9A than after simple knockdown of KDM3A.

[0023] Figure 5 It is a series of diagrams of the small molecule inhibitor BIX-01294 promoting chondrogenic differentiation of DPSCs in the embodiment of the present invention. Among them, A is a CCK8 experiment showing that the small molecule inhibitor BIX-01294 is used at a concentration higher than 1μM, showing toxicity to DPSCs; B is a western blot analysis showing that the expression of SOX9 increases with the addition of BIX-01294; C is RT-qPCR showing that the expression level of related cartilage markers in DPSCs is significantly upregulated after the addition of the inhibitor BIX-01294; D is a histological section staining of chondrocytes of DPSCs, showing that the content of proteoglycan and type II collagen increases after the addition of the inhibitor BIX-01294 (*P<0.05; **P<0.01; ***P<0.001). DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0025] If no specific techniques or conditions are specified in the examples, they are all carried out according to the commonly used technical means in the art or the methods in the product instructions. If the manufacturers of the reagents or instruments used in the experiments are not specified, they are all conventional products that can be purchased commercially.

[0026] The specific operation steps of RT-qPCR, western blot and histological staining experiments in the present invention are as follows:

[0027] 1. RT-qPCR

[0028] The total RNA of DPSCs was extracted with Trizol (Invitrogen, USA), and the RNA in the cells was extracted using a kit according to the instructions and stored in a -80°C refrigerator. In this example, 1 μg of RNA was reversely synthesized into cDNA using a reverse transcription kit from Quanshijin Company. The specific system is shown in Table 1:

[0029] Table 1 Reverse transcription system

[0030]

[0031] After extracting total RNA, the amount can be adjusted according to the concentration of the extracted RNA, with a total mass of about 1 μg as a reference, and the volume of RNase-free water can be changed accordingly. The reaction was carried out on a PCR instrument: incubated at 42°C for 15 minutes, and then the obtained cDNA was stored at -80°C.

[0032] qPCR was performed using the Quanshijin SuperMix kit, and the relevant primer sequences are shown in Table 2:

[0033] Table 2: Related primer sequences in this example

[0034]

[0035] The program was: pre-denaturation at 94°C for 30 s, and the cycle program was 94°C for 5 s, 60°C for 30 s, for 40-45 cycles.

[0036] 2. Western blot

[0037] The cells were washed three times with PBS. After absorbing all the PBS, an appropriate amount of lysis solution mixed with RIPA and Cocktail was added, and the mixture was placed on a 4°C shaker for 30 minutes. Then the cells and RIPA suspension were transferred to a 1.5mL EP tube and shaken on a shaker for 1 minute. Then, the cells were centrifuged at 4°C at 12000r / min for 20 minutes. After centrifugation, cell debris was precipitated, and the supernatant (the supernatant is the total protein to be extracted) was collected and stored at -80°C.

[0038] Solebow's 10% SDS-PAGE gel was used for electrophoresis. The required loading amount of 40 μg was calculated based on the sample concentration. The gel was first run at 90 V for 20 min. After the sample ran to the separation gel, a voltage of 120 V was used to run until the bromophenol blue was 1 cm away from the bottom of the gel.

[0039] A PVDF membrane of appropriate width was used and activated with anhydrous ethanol. The electrotransfer was performed with a current of 200 mA and the electrotransfer time was selected according to the molecular weight of the target protein.

[0040] After electrotransfer, the PVDF membrane was blocked with 5% skim milk for 2 h.

[0041] After the blocked PVDF membrane is cleaned with TBST, it is placed in the prepared primary antibody solution and shaken at 4°C overnight. Wash with TBST three times, 10 minutes each time. Dilute the secondary antibody with TBST according to the proportion in the instructions, and incubate on a shaker at room temperature for 1 hour. Wash with TBST three times, 10 minutes each time, prepare the developer according to the instructions, and place the band on the gel imaging system for development.

[0042] 3. Histological Staining

[0043] For chondrocyte culture and histological staining, 3 × 10 5 DPSCs were placed in a 15 mL conical centrifuge tube and centrifuged at 250 g for 5 min to form a cell sphere culture. Then, the DPSC microspheres were cultured in chondrogenic differentiation medium for 14 days. The culture medium was changed every 2 days. After 14 days of induction, the cells were fixed with 4% paraformaldehyde at room temperature for 24 h, then embedded in paraffin, and the cell spheres were cut into 5 μm frozen sections. The samples were stained with alcian blue, safranin-O, toluidine blue, and sirius red according to the manufacturer's standard procedures.

[0044] For histological staining of rat knee joints, the knee joints were fixed with 4% paraformaldehyde for 3 days, decalcified with EDTA decalcification solution for 1 month, dehydrated with gradient alcohol, embedded into paraffin blocks, and sections were cut with 5 μm thickness and stained with hematoxylin-eosin and toluidine blue.

[0045] For immunohistochemical staining, after the slides were dewaxed and washed, endogenous peroxidase was removed with hydrogen peroxide. After rinsing with PBS, the samples were blocked. The sections were incubated with primary antibodies at 4°C overnight, and finally the secondary antibodies were added for incubation at room temperature for 1 h. After blocking, the sections were observed under a microscope and photographed. Semi-quantitative expression levels were calculated by Image J.

[0046] Example 1: Expression level of KDM3A during chondrogenic differentiation of DPSCs.

[0047] Logarithmic phase cells were plated in well plates. When the cell density reached 80-90%, the culture medium was replaced with chondrogenic differentiation induction medium. RNA was collected for RT-qPCR after 0, 3, 7, and 14 days of induction. Figure 1 The results showed that after chondrogenic induction, the expression of KDM3A increased at 3, 7, and 14 days, with the highest expression at 7 days. Total protein was collected after 0, 3, and 7 days of induction for western blot ( Figure 1B), the results showed that the expression level of KDM3A protein increased significantly during chondrogenic differentiation.

[0048] Example 2: Effects of KDM3A on relevant cartilage markers.

[0049] shKDM3A was designed according to the coding sequence of KDM3A in NCBI, and shNC was a negative control that did not target any gene, and then synthesized at Genetron. When the cell density reached 30%-50%, transfection was performed. Fresh culture medium was replaced after 12 hours. The transfected cells were collected for RT-qPCR experiments and western blot. Figure 2 A shows that after knocking out KDM3A, the expression levels of cartilage markers (ACAN, COMP, and Col2A1) were significantly decreased; Figure 2 B shows that after knocking out KDM3A, the protein levels of cartilage markers were significantly decreased.

[0050] Overexpression of KDM3A was designed based on the coding sequence of KDM3A in NCBI, and the vector was a negative control that did not target any gene, and then synthesized at Genema Gene. When the cell density reached 30%-50%, transfection was performed. Fresh culture medium was replaced after 12 hours. The transfected cells were collected for RT-qPCR experiments and western blot. Figure 2 C shows that after overexpression of KDM3A, the expression levels of cartilage markers (ACAN, COMP, and Col2A1) were significantly increased; Figure 2 D shows that after overexpression of KDM3A, the protein levels of cartilage markers were significantly increased.

[0051] Example 3: Effects of KDM3A or G9A on SOX9, a key transcription factor in chondrogenesis, and the effect of G9A on the chondrogenic ability of DPSCs.

[0052] Figure 3 A western blot experiment showed that the expression level of SOX9 decreased significantly after knocking out KDM3A.

[0053] siKDM3A and siG9A were designed according to the coding sequence of KDM3A in NCBI, and siNC was a negative control that did not target any gene, and then synthesized at Genentech. After the cell density reached 50-60%, the cells were transfected using the RNAiMAX kit from Thermo Fisher Scientific, and total cell protein was collected 48 hours after transfection for western blot. Figure 3 B shows that after knocking down G9A, the expression level of SOX9 was significantly upregulated. Figure 3 C shows that after knocking down KDM3A and G9A simultaneously, the expression level of SOX9 was restored compared with that of knocking down KDM3A alone.

[0054] After cells were transfected with siKDM3A or transfected with siKDM3A and siG9A simultaneously, chondrocytes were cultured and induced into chondrogenic cartilage for 14 days before paraffin embedding and staining. Figure 3 D shows that knockdown of KDM3A significantly reduced the content of proteoglycan and fiber in spheroid cartilage matrix, while knockdown of both KDM3A and G9A increased the content of proteoglycan and fiber.

[0055] Example 4: In vivo experiments demonstrated the effects of KDM3A and G9A on the repair capacity of dental pulp stem cartilage.

[0056] A 2 mm drill was used to create a full-thickness articular cartilage defect of 2 mm in diameter and 1 mm in depth in the middle of the femoral trochlea of ​​5-6 week-old SD rats. After removing the cartilage and bone fragments, the border around the drill was trimmed with a surgical knife and rinsed to remove the fragments. SD rats with cartilage defects were randomly divided into 4 groups according to the type of shRNA and siRNA transfected: (1) control group (control); (2) shNC; (3) shKDM3A; (4) shKDM3A+siG9A (n=6 in each group). Then, 2×10 6 A mixture of 100 cells / ml DPSCs and matrix gel was transplanted into the cartilage defect. A control group was implanted with matrix gel without cells. The animals were killed 8 weeks after surgery to evaluate the cartilage repair. Figure 4 The macroscopic photograph A shows that after knocking out KDM3A, the cartilage repair ability of DPSCs decreased, while after knocking down KDM3A and G9A at the same time, the cartilage repair ability of cells recovered. Figure 4 B shows that the cartilage repair area in the shKDM3A group was poorly integrated with the normal cartilage tissue, and toluidine blue staining showed that the proteoglycan content was significantly decreased. However, in the shKDM3A+siG9A group, the cartilage repair area was well connected with the normal tissue, and toluidine blue staining showed that the proteoglycan content was also significantly increased. Figure 4 C immunohistochemical staining showed that the expression levels of SOX9 and type II collagen in the shKDM3A group were significantly decreased, while those in the shKDM3A+siG9A group were significantly increased.

[0057] Example 5: Effect of BIX-01294 on the chondrogenic differentiation ability of DPSCs.

[0058] Count the cells in good condition and plate them in a 96-well plate. After the cells adhere to the wall, change the medium and add 100 μl of culture medium treated with different concentrations of BIX-01294 (0, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, 1.5 μM, 2 μM) to each well; add 100 μL of prepared CCK8 solution (prepared according to the instruction manual 10:1) on days 1, 3, 6, and 8 respectively, place the 96-well plate in an incubator for 2 hours, and use an ELISA reader to detect the absorbance of each sample at 450 nm. Figure 5 A shows that when the concentration of BIX-01294 is higher than 1 μM, it becomes toxic to DPSCs.

[0059] The cells in good condition were counted and plated in 6-well plates. After the cells adhered to the wall, the medium was changed, and 2 mL of culture medium treated with different concentrations of BIX-01294 (0, 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM) was added to each well. After 48 hours, the cells were collected to extract total protein for western blot experiments. Figure 5 B shows that with the addition of BIX-01294, the expression of SOX9 increased significantly. Combined with the CCK-8 experiment, it was determined that 1 μM BIX-01294 was used for subsequent experiments.

[0060] Count the cells in good condition and plate them in 6-well plates. After the cells adhere to the wall, change the medium and add 2 mL of medium treated with 1 μM BIX-01294 to each well. After 48 hours, collect the cells and extract RNA for RT-qPCR experiments. Figure 5 C shows that the expression of cartilage markers increased significantly after the addition of BIX-01294.

[0061] After the cells were treated with 1 μM BIX-01294, chondrocytes were prepared and cultured for chondrogenic induction for 14 days before paraffin embedding and sectioning. Figure 5 D shows that the contents of proteoglycan and type II collagen increased significantly after the addition of BIX-01294.

[0062] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Use of an inhibitor of histone modification enzyme gene G9A in the preparation of a promoter for the chondrogenic differentiation of dental pulp mesenchymal stem cells, wherein the inhibitor is a specific small molecule inhibitor of G9A, BIX-01294.

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