Application of circNFIX coding protein NFIX-219aa in preparation of cartilage degeneration inhibition drugs

Abstract

The invention discloses application of circNFIX encoding protein NFIX-219aa in preparation of a medicine for inhibiting cartilage degeneration, and relates to the technical field of biological medicine. The coding protein NFIX-219aa has an amino acid sequence as shown in SEQ ID NO: 2, the coding protein NFIX-219aa and inflammasome NLRP3 are subjected to protein-protein interaction, the effect of inhibiting chronic inflammation of cartilage cells is achieved, and then the effect of inhibiting cartilage degeneration is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of circNFIX-encoded protein NFIX-219aa in the preparation of a drug for inhibiting cartilage degeneration. Background Art

[0002] Osteoarthritis is a common chronic degenerative disease of bones and joints, with clinical manifestations such as joint pain, deformity, and limited range of motion. The increase in life expectancy and the aging of the population have led to an upward trend in the incidence of osteoarthritis. Currently, there are more than 250 million osteoarthritis patients worldwide. In my country, about 50% of the elderly over 60 years old who undergo X-ray examinations show osteoarthritis changes, of which 30%-50% have clinical symptoms. Osteoarthritis has become the primary cause of patients' loss of mobility. Although osteoarthritis can affect most joints in the human body, it is usually more common in the hip and knee joints. Therefore, osteoarthritis was once thought to be caused by excessive joint activity and load. However, recent studies have shown that the occurrence and progression of osteoarthritis is the result of a series of combined factors; among them, the destruction of the cartilage microenvironment caused by chronic low-grade inflammation is one of the most important driving factors.

[0003] Changes in the inflammatory microenvironment of osteoarthritis and the degeneration of chondrocytes after injury are mutually initiating factors, indicating that chronic inflammation of chondrocytes plays a vital role in early cartilage degeneration and the progression of osteoarthritis. Therefore, effectively controlling the inflammatory response of the cartilage microenvironment may bring new hope for the treatment of osteoarthritis. However, some of the existing early drugs and physical therapies that only target the "inflammation" of osteoarthritis are too broad and lack clear therapeutic effects. Exploring the deeper molecular mechanisms of disease progression and developing targeted small molecule therapeutic drugs based on this has become a hot topic in osteoarthritis research in recent years. With the advent of high-throughput and second-generation sequencing technologies, researchers have been able to open up a broader molecular world, including a new understanding of non-coding RNA. Current studies have confirmed that NLRP3, as a member of the NLR receptor family, contains NOD, LRR and pyrin domains, leading to inflammatory responses and even cell death, and exacerbating inflammatory responses and cartilage degeneration in the joint cavity microenvironment. After human chondrocytes overexpress NLRP3, COL2A1 is downregulated, MMP13 is upregulated, and the expression of YAP molecules in the Hippo pathway is promoted. YAP has a regulatory effect on the oxidative stress response in the chondrogenic differentiation of bone marrow mesenchymal stem cells. NLRP3 and YAP have a close interaction in bone marrow cells, which is crucial for the pro-inflammatory effect of NLRP3. In the previous study on the pathogenesis of osteoarthritis, the applicant found that non-coding RNA can participate in the regulation of cell function in the form of RNA itself or encoded protein, and non-coding RNA plays an important role in the progression of osteoarthritis and the regulation of cartilage homeostasis. Summary of the invention

[0004] The purpose of the present invention is to provide a use of circNFIX-encoded protein NFIX-219aa in the preparation of a drug for inhibiting cartilage degeneration, and to solve the following technical problems: Existing early-stage medications and physical therapies that only target the "inflammation" of osteoarthritis are too broad and lack clear therapeutic effects.

[0005] The purpose of the present invention can be achieved through the following technical solutions: Application of circNFIX-encoded protein NFIX-219aa in the preparation of drugs for inhibiting cartilage degeneration.

[0006] As a further embodiment of the present invention: the nucleotide sequence of circNFIX is shown in SEQ ID NO: 1.

[0007] As a further embodiment of the present invention: the active substance of the drug is protein NFIX-219aa or its encoding gene circNFIX.

[0008] As a further solution of the present invention: when the protein NFIX-219aa is used to prepare a drug for inhibiting cartilage degeneration, it is administered by in situ injection or infusion into a joint.

[0009] As a further embodiment of the present invention: when preparing a drug for inhibiting cartilage degeneration using the gene circNFIX encoding the protein NFIX-219aa, the drug is administered by highly expressing the gene circNFIX in chondrocytes.

[0010] As a further embodiment of the present invention: the gene circNFIX encoding the protein NFIX-219aa is introduced into chondrocytes via any one of a retroviral vector, a lentiviral vector, and an adenoviral vector.

[0011] As a further embodiment of the present invention: the encoded protein NFIX-219aa inhibits cartilage degeneration by inhibiting chronic inflammation of chondrocytes.

[0012] Beneficial effects of the present invention: This application uses the 219aa polypeptide NFIX-219aa encoded by the circular non-coding RNA (circNFIX) that is highly expressed in normal chondrocytes but lowly expressed in OA chondrocytes as an active ingredient to prepare a drug that inhibits cartilage degeneration. Among them, the circNFIX-encoded protein NFIX-219aa interacts with the inflammasome NLRP3 through protein-protein interaction. The encoded protein NFIX-219aa inhibits the inflammatory microenvironment and chondrocyte degeneration by regulating the NLRP3 / YAP axis, plays a significant protective regulatory role in the process of chondrocyte degeneration, inhibits the chronic inflammation of chondrocytes, and thus achieves the effect of inhibiting chondrocyte degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present invention will be further described below in conjunction with the accompanying drawings.

[0014] Figure 1 This is a diagram of the sequencing results of circRNAs sequencing of the specimen in Example 1; Figure 2 This is a schematic diagram of verifying the protein that circNFIX can encode in Example 2; Figure 2 A is a schematic diagram of circNFIX coding; Figure 2 B is the predicted amino acid sequence of the protein encoded by circNFIX; Figure 2 C is the expression of FLAG and NFIX-219aa detected by Western-blot after HEK293T cells were transfected with circNFIX-ORF-FLAG; Figure 2 D is immunofluorescence staining showing transfected HEK293T cells; Figure 3 This is a diagram showing the functional results of circNFIX in chondrocytes verified in Example 3; Figure 3 A and B are schematic diagrams of the effects of overexpression of circNFIX on COL2A1 and MMP13 verified by Western-blot and qRT-PCR; Figure 3 C and D are schematic diagrams of Western-blot and qRT-PCR verification of the effect of inhibiting circNFIX on COL2A1; Figure 4 is a schematic diagram of the expression of cartilage-related proteins after co-culture of different groups with normal chondrocytes by Western Blot in Example 3; Figure 5 This is a schematic diagram of verifying the effect of NFIX-219aa in inhibiting cartilage degeneration in Example 4. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Example 1 See also Figure 1 , preliminary screening of differentially expressed circRNAs during cartilage degeneration: CircRNAs sequencing was performed on cartilage samples from three groups of healthy subjects and three groups of OA patients (3NAs vs 3OAs). Figure 1 Figure A, sequencing results using bioinformatics analysis revealed a large number of differentially expressed circRNAs; see Figure 1 Figure B: PCR experiments verified that circNFIX was lowly expressed in OA samples.

[0017] Example 2 See also Figure 2 , experimental circNFIX encodes a new protein NFIX-219aa: (1) Please refer to Figure 2 Figures A and B show that circNFIX has ORF and IRES sequence elements, and has a high m6A level, which is suitable for translation. Based on the assumed ORF sequence, it is predicted that it can translate a 219aa protein polypeptide (NFIX-219aa); (2) Insert the FLAG nucleic acid sequence after circNFIX-ORF, use pcDNA as the backbone to construct a synthetic plasmid circNFIX-FLAG and a mutant plasmid circNFIX-FLAG-mut (circNFIX-ORF start codon ATG mutated to CTG), and transfect HEK293T cells with LIPO3000. Figure 2 C, FLAG antibody detection revealed that circNFIX can express a polypeptide of about 219aa, which is consistent with the predicted size. Figure 2 D, circNFIX-ORF fusion with FLAG was observed under immunofluorescence, indicating that the ORF of circNFIX is translatable and the promoter ATG is active.

[0018] Example 3 See also Figure 3-4 , verify the role of circNFIX and NFIX-219aa in chondrocytes: (1) Please refer to Figure 3 , verifying the role of circNFIX in chondrocytes: ① Acquisition and preparation of cartilage specimens: approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, with the Ethics Committee approval numbers

[2013] C-260 and

[2021] 334; obtain normal adult cartilage and degenerated cartilage from OA patients. Obtain femoral condyles and tibial plateaus from adults (over 40 years old, with a history of knee OA) (when undergoing knee replacement surgery due to severe knee OA) and young people (under 25 years old, without a history of knee OA, smooth articular cartilage surface, no joint degeneration) (when undergoing lower limb amputation surgery due to severe damage). Cut an appropriate amount of surface articular cartilage into 1mm 3The size of the chondrocytes was determined by digestion and centrifugation with collagenase P (Roche). The remaining articular cartilage was subjected to paraffin sectioning and the cartilage matrix was evaluated by HE staining, toluidine blue staining, Masson staining, and safranin-fast green staining.

[0019] ②Total RNA was extracted from OA and NA tissues, circNFIX primers were designed, and qRT-PCR was used to verify the expression level of circNFIX. Twenty pairs of fresh OA and NA tissue specimens were obtained, embedded in paraffin and sliced, and immunohistochemistry was used to detect the difference in NFIX-219aa in OA and NA.

[0020] ③ In vitro validation: circNFIX-ORF, circNFIX and circNFIX-ORFmut lentivirus were constructed. Human chondrocytes were transfected with empty lentiviral vectors and target vectors for 48 hours, total RNA was extracted, and changes in cartilage markers were detected by qRT-PCR; total protein was extracted and Western blot was used to detect changes in cartilage markers. Human chondrocytes were plated on six-well plates, transfected with empty lentiviral vectors and target vectors for 48 hours, and changes in cartilage markers were detected by immunofluorescence (same steps as above); human chondrocytes were transfected with empty lentiviral vectors and target vectors for 48 hours, and cells were collected and resuspended in PBS for flow cytometry to detect chondrocyte apoptosis.

[0021] ④ In vivo validation: Adeno-associated viruses (AAV-vector, AAV-ORF, AAV-circNFIX, AAV-circNFIXmut) were injected into the joint cavity of OA model mice. Ten weeks later, the knee joints of mice were grossly observed and scored, and the knee joints were scanned by micro-CT to observe the articular surface and osteophytes. Then the mice were killed, and the knee joint specimens of mice were collected for histological staining and Markin scoring, and sGAG optical density was quantified under a microscope. The content of NFIX-219aa and cartilage characteristic indicators was detected by IHC and WB. GAG quantification and hydroxyproline quantification were used to detect the synthesis of cartilage matrix.

[0022] The function of circNFIX was verified by plasmid transfection of primary human chondrocytes. Figure 3 A, B, Overexpression of circNFIX in chondrocytes significantly increased COL2A1 expression and downregulated MMP13 expression. Figure 3 C, D, After interference inhibition of circNFIX, the expression of COL2A1 and MMP13 was significantly reversed, indicating that circNFIX may have an inhibitory effect on the inflammatory response of chondrocytes.

[0023] (2) Please refer to Figure 4, verifying the effect of NFIX-219aa in chondrocytes: Three groups of plasmids were constructed: NFIX-219aa, circNFIX and circNFIXmut (ORF promoter ATG mutated to CTG) overexpression plasmids, and human primary chondrocytes were transfected with empty vectors. Western-blot was used to detect the expression of MMP13 and COL2A1 in chondrocytes. The results showed that the NFIX-219aa and circNFIX overexpression groups showed obvious cell protection effects, while the circNFIXmut group showed increased expression of inflammation-related genes. This indicates that the new protein NFIX-219aa encoded by circNFIX, rather than circNFIX itself, inhibits the inflammatory response and degeneration of cartilage.

[0024] Example 4 See also Figure 5 , verify the effect of NFIX-219aa in inhibiting cartilage degeneration Construction of NFIX-219aa knock-in mouse (NFIX-219aa-OE). Figure 5 A, Whole skeleton staining shows that the bones of knock-in mice are significantly larger than those of wild-type mice. Figure 5 B, hyaline cartilage of knock-in mice is relatively thicker. We constructed DMM mouse osteoarthritis model with 10-week-old wild-type and knock-in mice and observed them for 8 weeks. Figure 5 C, D, the results show that the OA progression of NFIX-219aa knock-in mice is significantly milder than that of wild-type mice, as shown by lower OARSI scores and relatively less osteophyte formation around the knee joint. This indicates that NFIX-219aa can promote cartilage development and inhibit cartilage degeneration and OA progression in vivo.

[0025] The above is a detailed description of an embodiment of the present invention, but the content is only a preferred embodiment of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. Application of circNFIX-encoded protein NFIX-219aa in the preparation of drugs for inhibiting cartilage degeneration.

2. The use of a circNFIX-encoded protein NFIX-219aa according to claim 1 in the preparation of a drug for inhibiting cartilage degeneration, characterized in that: The nucleotide sequence of circNFIX is shown in SEQ ID NO:

1.

3. The use of a circNFIX-encoded protein NFIX-219aa according to claim 1 in the preparation of a drug for inhibiting cartilage degeneration, characterized in that: The active substance of the drug is protein NFIX-219aa or its encoding gene circNFIX.

4. The use of a circNFIX-encoded protein NFIX-219aa according to claim 3 in the preparation of a drug for inhibiting cartilage degeneration, characterized in that: When protein NFIX-219aa is used to prepare drugs for inhibiting cartilage degeneration, the drugs are administered by in situ injection or infusion into the joint.

5. The use of a circNFIX-encoded protein NFIX-219aa according to claim 3 in the preparation of a drug for inhibiting cartilage degeneration, characterized in that: When preparing a drug for inhibiting cartilage degeneration, the coding gene circNFIX of the protein NFIX-219aa is highly expressed in chondrocytes and administered.

6. The use of a circNFIX-encoded protein NFIX-219aa according to claim 3 in the preparation of a drug for inhibiting cartilage degeneration, characterized in that: The gene circNFIX encoding the protein NFIX-219aa is introduced into chondrocytes via any of retroviral vectors, lentiviral vectors, and adenoviral vectors.

7. The use of a circNFIX-encoded protein NFIX-219aa according to claim 3 in the preparation of a drug for inhibiting cartilage degeneration, characterized in that: The encoded protein NFIX-219aa inhibits cartilage degeneration by suppressing chronic inflammation of chondrocytes.

Images