Application of circCRIM1 in regulating cartilage cell differentiation and cartilage homeostasis

Abstract

The invention discloses application of circCRIM1 in regulation and control of cartilage cell differentiation and cartilage homeostasis, and relates to the technical field of gene engineering. The invention discloses an application of circCRIM1 in regulation and control of cartilage cell differentiation and cartilage homeostasis. A nucleotide sequence of the circCRIM1 is shown as SEQ. ID. NO.1; experiments prove that circCRIM1 is highly expressed in osteoarthritis cartilage, and the inhibition effect of circCRIM1 on MMP16 is relieved through specific binding of circCRIM1 to miR-320c, so that cartilage generation of human adipose-derived stem cells is inhibited, cartilage cell homeostasis is destroyed, and cartilage matrix degradation is promoted. Based on the mechanism, the invention can provide a new target and strategy for clinically and effectively delaying the progress of osteoarthritis and treating osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of circCRIM1 in regulating chondrocyte differentiation and cartilage homeostasis. Background Technology

[0002] Osteoarthritis (OA) is a degenerative disease characterized by progressive cartilage degeneration, accompanied by synovial inflammation and subchondral bone remodeling. Clinically, it manifests as joint pain, stiffness, and functional impairment, severely impacting patients' quality of life. Statistics show that in 2021, the number of OA patients among the global working-age population (15-64 years old) reached approximately 329 million, an increase of 123.11% compared to 147 million in 1990. However, the pathogenesis of OA is not yet fully understood, and there is a lack of disease-modifying therapies that can effectively halt or reverse cartilage degeneration. Current treatments primarily focus on symptom relief and cannot fundamentally slow disease progression. This unmet clinical need underscores the urgency of elucidating molecular mechanisms and developing targeted therapies.

[0003] Circular RNAs (circRNAs) are a class of covalently closed non-coding RNAs. The absence of a 5' cap and a 3' polyadenylated tail endows circRNAs with extremely high stability, allowing them to accumulate in blood and synovial fluid. This characteristic makes them promising diagnostic biomarkers and therapeutic vectors. Studies have shown that circRNAs play a crucial role in the pathogenesis of osteoarthritis (OA) by regulating chondrocyte metabolism, inflammatory responses, and extracellular matrix homeostasis. For example, circRNA_0005563 promotes chondrocyte apoptosis in synovial fluid, while mechanosensitive circular RNA-MSR contributes to stress-induced ECM degradation in degenerative cartilage. Based on genomic origin, circRNAs can be classified into exon circRNAs, circular intron RNAs, and exon-intron circRNAs. Functionally, circRNAs participate in the pathological process of OA through the following mechanisms: 1. Competitive endogenous RNA activity: acting as miRNA "sponges," they isolate miRNAs and regulate target genes. For example, circ-Foxo3 arrests the cell cycle by binding to CDK2 / p21; 2. Protein coding potential: Some circular RNAs translate functional peptides through rolling circle amplification mechanisms; 3. Transcriptional regulation: ci-ankrd52 promotes the transcription of its parent gene by enhancing RNA polymerase II activity.

[0004] In summary, circular RNAs regulate the pathological process of OA through complex networks. Their stability and targeted regulatory capabilities provide opportunities for the development of novel diagnostic biomarkers and treatments. Circular RNAs are becoming new targets for the diagnosis and treatment of OA. Summary of the Invention

[0005] The purpose of this invention is to provide an application of circCRIM1 in regulating chondrocyte differentiation and cartilage homeostasis. circCRIM1 can regulate the cartilage formation process and maintain cartilage homeostasis.

[0006] The objective of this invention can be achieved through the following technical solutions: An application of circCRIM1 in regulating chondrocyte differentiation and cartilage homeostasis, the nucleotide sequence of circCRIM1 is shown in SEQ.ID.NO.1.

[0007] As a further aspect of the present invention, the specific application is as follows: inhibiting the expression of circCRIM1 to promote the differentiation of human adipose-derived stem cells into chondrocytes; and promoting the expression of circCRIM1 to inhibit the differentiation of human adipose-derived stem cells into chondrocytes.

[0008] As a further aspect of the present invention: cartilage homeostasis is the balance between the synthesis and degradation of chondrocyte extracellular matrix; inhibiting the expression of circCRIM1, inhibiting the expression of matrix metalloproteinases MMP13 and MMP16 in chondrocytes, upregulating the expression of cartilage-specific genes COL2A1, AGGRECAN, and SOX9, and slowing down the degradation of chondrocyte extracellular matrix; promoting the expression of circCRIM1 promotes the transformation of chondrocytes to a hypertrophic-catabolic phenotype and accelerates the degradation of chondrocyte extracellular matrix.

[0009] As a further aspect of the present invention: a diagnostic method for the expression level of circCRIM1: a diagnostic reagent detects the expression of circCRIM1 in cartilage tissue or synovial fluid, and the expression level of circCRIM1 in osteoarthritis patient samples is significantly higher than that in healthy control samples; the diagnostic reagent includes any one of specific primers, probes or antibodies for detecting circCRIM1, and the specific primers include divergent primers targeting the circCRIM1 back splice site.

[0010] As a further aspect of the present invention, a method for inhibiting circCRIM1 expression includes downregulating circCRIM1 expression using a circCRIM1 inhibitor; the circCRIM1 inhibitor includes any one of siRNA, shRNA, antisense oligonucleotide, or a viral vector carrying a circCRIM1 knockdown sequence targeting circCRIM1, wherein the viral vector includes adeno-associated virus. The circCRIM1 inhibitor downregulates circCRIM1 expression, restores miR-320c activity, inhibits MMP16 expression, and ultimately alleviates cartilage degeneration, reduces osteophyte formation, and delays the progression of osteoarthritis.

[0011] As a further aspect of the present invention, the circCRIM1 inhibitor also includes a carrier, which includes any one of physiological saline, buffer, excipient or stabilizer.

[0012] As a further aspect of the present invention, the carrier can be any pharmaceutically acceptable carrier.

[0013] As a further aspect of the present invention: the circCRIM1 inhibitor is administered via intra-articular injection.

[0014] The beneficial effects of this invention are: This application discloses the crucial role of circCRIM1 in chondrocyte differentiation and osteoarthritis (OA), revealing that circCRIM1 precisely regulates chondrocyte differentiation and degenerative changes by specifically binding to miR-320c to relieve the inhibition of matrix metalloproteinase 16 (MMP16). Significantly increased circCRIM1 expression inhibits the chondrogenic capacity of human adipose-derived stem cells (hADSCs) and disrupts chondrocyte homeostasis. Inhibiting circCRIM1 expression not only effectively prevents extracellular matrix (ECM) degradation but also significantly slows the progression of osteoarthritis. circCRIM1 can regulate chondrogenesis and maintain cartilage homeostasis.

[0015] (2) Diagnostic value: circCRIM1 is abnormally expressed in OA cartilage and synovial fluid and has high stability. It can be used as a specific diagnostic marker for OA and added to OA diagnostic drugs to improve the accuracy of OA diagnosis.

[0016] (3) Therapeutic potential: circCRIM1 inhibitors can be injected directly into the lesion site, which has strong targeting and can effectively delay the progression of OA, while avoiding systemic side effects, providing new drug candidates for the clinical treatment of OA; (4) Wide range of applications: circCRIM1 can be used not only as a diagnostic agent for OA and as a therapeutic drug, but also in cartilage tissue engineering to regulate the differentiation of stem cells into cartilage, and has broad application prospects. Attached Figure Description

[0017] The invention will now be further described with reference to the accompanying drawings.

[0018] Figure 1 The expression characteristics and identification of circCRIM1; Figure 2 It is circCRIM1 that regulates the chondrogenesis of hADSCs; Figure 3 It is the effect of circCRIM1 on chondrocyte homeostasis; Figure 4It is the interaction between circCRIM1 and miR-320c; Figure 5 This describes the function of miR-320c in OA cartilage; Figure 6 This is a rescue experiment on circCRIM1 regulating chondrogenesis and homeostasis via miR-320c; Figure 7 It is miR-320c that targets and regulates MMP16; Figure 8 This refers to the effect of circCRIM1 inhibitors in treating a mouse OA model; Figure 9 This refers to the expression of circCRIM1; Figure 10 This is a supplementary experiment on the effect of miR-320c on cartilage metabolism in chondrocytes; Figure 11 This is a supplementary experiment on the effect of miR-320c on cartilage metabolism in chondrocytes. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] I. Experiment 1.1 Sample collection, cell isolation and culture Fat samples were obtained from healthy subjects who underwent abdominal liposuction at the First Affiliated Hospital of Sun Yat-sen University. The obtained samples were placed in adipose-derived stem cells (ADSCs) obtained from Louisville, Missouri, USA. After separation, the ADSCs were mixed with 10% fetal bovine serum (FBS) to neutralize the type I collagenase. The cells were cultured in basal medium (α-modified Eagle's medium (α-MEM)) (Gibco Life Sciences, Grand Island, NY). After three generations of culture, chondrogenesis was induced in the third-generation ADSCs using a microsphere culture method.

[0021] The specific steps are as follows: Convert ADSCs to 2×10... 7Cells were aliquoted at a density of 10 cells / mL and added to incomplete chondrogenesis medium (97 mL of human adipose-derived stem cell chondrogenesis basal medium containing 10 μL dexamethasone, 300 μL ascorbic acid, 1 mL insulin-transferrin-selenoside combination supplement, 100 μL sodium pyruvate, and 100 μL proline). Cell droplets were prepared by Guangzhou Saijie Bioscience Co., Ltd. using the culture medium resuscitation method. The obtained cell droplets (12.5 μL) were then transferred to 24-well plates and incubated at 37°C for 90 min. 500 μL of transforming growth factor β3 (TGFβ3) was added to each well to prepare complete chondrogenesis induction medium—i.e., 20 ng TGFβ3 (Saijie Bioscience) was added to each mL of incomplete chondrogenesis medium. After chondrogenesis differentiation began, samples were collected on days 0, 3, 14, and 21 for experimental analysis.

[0022] Pathological cartilage samples were obtained from patients who underwent total knee arthroplasty (TKA) and were affected by osteoarthritis (OA). Normal cartilage was obtained from patients with femoral neck fractures who underwent total hip arthroplasty (THA). All subjects had no history of OA or rheumatoid arthritis. Chondrocyte isolation and culture were then performed: the collected cartilage samples were separated from the subchondral bone and washed twice with PBS. The cartilage samples were mixed with modified Eagle's medium / nutrient mixture F12 (DMEM / F12) containing 4 mg / mL protease (containing 10% fetal bovine serum and 1% penicillin-streptomycin [PS]) and digested for 90 min at 37°C and 90 rpm in a constant temperature shaker. The digested cartilage samples were then washed twice with PBS and mixed with 0.25 mg / mL collagenase P (also dissolved in DMEM / F12 medium containing 10% fetal bovine serum and 1% PS) and digested continuously at 37°C and 50 rpm for 8 h. The supernatant was filtered through a 70 μm cell filter, and cells were collected by centrifugation. Cells were seeded at 75 cm⁻¹. 2 In the culture flask, DMEM medium containing 10% fetal bovine serum + 1% phosphate was used, and amplification was carried out in a 37°C, 5% CO2 incubator (Thermo Fisher Scientific, USA).

[0023] 1.2 RNA interference and overexpression PHCs preparation process: Mix 1µg pcDNA3-circCRIM1 plasmid (Hanbai Biotechnology, Shanghai) with Lipofectamine 3000 transfection reagent (Yingjie Life Technology Co., Ltd., USA), add cell culture medium and incubate for 4-6 hours, then change the culture medium.

[0024] Gene knockdown / overexpression: si-circCRIM1, si-MMP16 or miR-320c mimics / inhibitors (Ribbon Biotech, Guangzhou) were delivered using the RNAiMAX transfection system (Thermo Fisher Scientific, USA).

[0025] Human adipose-derived mesenchymal stem cells (hADSCs) were processed by transfecting monolayer cells with circCRIM1 siRNA. Cells were collected 3 days later for microsphere chondrogenesis experiments and were re-transfected with the same siRNA or miR-320c inhibitor on the same day as microsphere formation.

[0026] 1.3 RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis Total RNA was extracted using a one-step TRIzol reagent (Ingenieur Biosciences, Carlsbad, USA). Small RNA and mRNA were reverse transcribed using the Mir-X™ miRNA kit and RT premix (Takara Bio Inc., Japan), respectively. When genomic DNA amplification was required, the procedure was followed according to the Solarbio (Beijing) column chromatography kit: RNase A digestion for 10 min at 55°C, followed by proteinase K digestion for 30 min at 55°C, followed by ethanol precipitation and purification by column chromatography. cDNA amplification was performed on an ABI 7500 system (Applied Biosystems, Foster City, USA) using SYBR Green PCR premix (Takara Bio Inc.), and the use of divergent / polymeric primers was verified by the circCRIM1 circular structure. GAPDH was used as an internal control gene. Specificity and relative expression levels were evaluated using melting curves and the 2-ΔΔCt method. PCR products were confirmed by 2% agarose gel electrophoresis and Sanger sequencing.

[0027] 1.4 Western Blotting Western blotting was performed according to the previously described method. Total chondrocyte protein was extracted using RIPA lysis buffer (Beyotime Biotech) with a protease inhibitor added (1:100, Abogenetics, Cambridge, UK). Proteins were separated by SDS-PAGE electrophoresis (80V for 20 min, followed by 120V for 1 h), and then transferred to a PVDF membrane at 250 mA for 1.5 h. After blocking with protein-free blocking buffer (PS108, Shanghai Aipuxin), the membrane was incubated overnight at 4°C with primary antibody, followed by incubation at room temperature for 1 h with HRP-labeled secondary antibody (1:3000, Danfoss Cell Signaling Technologies, Massachusetts, USA). Finally, the bands were visualized using a ChemiDoc Touch imaging system (Bio-Rad Laboratories), and quantification was performed using ImageLab and ImageJ software (National Institutes of Health, USA).

[0028] 1.5 Immunofluorescence analysis Cells were seeded onto 12 mm glass coverslips pre-coated with 0.01% poly-L-lysine and fixed after reaching 70% confluence. Following experimental treatment, samples were fixed with freshly prepared 4% paraformaldehyde at room temperature for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 1% bovine serum albumin (BSA) for 30 min to reduce non-specific binding. Primary antibodies against MMP13, COL2A1, and SOX9 (all rabbit polyclonal antibodies, 1:200 dilution; Abcam) were added, and the samples were incubated overnight at 4°C in a humid environment. After three 5-min washes with PBS buffer, samples were incubated with Cy5-labeled secondary antibody (1:100 dilution; Abcam) in the dark for 1 h, followed by counterstaining with DAPI for 10 min. After the coverslips were mounted with anti-quenching mounting medium, they were imaged using a Zeiss LSM 880 confocal microscope with a 40× oil immersion lens. Five fields of view were randomly selected from each replicate sample for quantitative analysis of fluorescence intensity.

[0029] 1.6 RNA Fluorescence In Situ Hybridization (FISH) Fluorescently labeled probes for human circCRIM1 and miR-320c were custom-developed by Guangzhou Ruibo Biotechnology Co., Ltd., while the mouse circCRIM1 probe was synthesized by Wuhan Saiwei Biotechnology Co., Ltd. For cultured primary human chondrocytes, fixation was first performed with 4% paraformaldehyde at room temperature for 15 min, followed by permeabilization for 10 min in a buffer containing 0.1% Triton X-100 and 10 mM VRC-CSK. After continuous dehydration, the probes were spotted onto glass slides and hybridized overnight in a humid environment at 37°C. Cartilage tissue sections were dewaxed, rehydrated with graded ethanol, and then permeabilized with 0.8% pepsin at 37°C for 15 min, followed by the same hybridization procedure. After three rigorous washes with high-salt buffer, cell nuclei were counterstained with DAPI for 10 min. Finally, fluorescence signals were acquired and analyzed using an Obercohen Zeiss LSM 880 confocal microscope.

[0030] 1.7 Pull-down experiment using circCRIM1 probe The biotin-labeled circular CRIM1-specific probe used in this study was custom-developed by SCbio (Guangzhou). SW1353 cells transfected with the circCRIM1 plasmid for 24 hours were collected, washed with ice-cold PBS, and then lysed with lysis buffer containing an RNase inhibitor. The lysis buffer was irradiated with 254 nm UV light to induce covalent cross-linking of the RNA-protein complex. The circCRIM1 probe was pre-coupled to streptavidin magnetic beads (Lifetech Corporation, USA) by rotating at 25°C for 2 hours to form a probe-magnetic bead complex. The cross-linked lysis buffer and the complex were gently incubated overnight at 4°C by rotating. After washing with high-salt buffer, low-salt buffer, and PBS buffer to remove non-specific binding, the specifically bound RNA was eluted with 100 μL of elution buffer at 65°C for 10 minutes. The purified RNA was immediately reverse transcribed to generate cDNA, which was then quantified by quantitative real-time PCR (qRT-PCR).

[0031] 1.8 RNA Immunoprecipitation We performed Ago2-RNA immunoprecipitation experiments using the Magna RIP™ kit (Merck, Inc., Billericca, Massachusetts, USA) according to the optimized protocol.

[0032] The specific operating procedure is as follows: First, SW1353 cells were washed twice with ice-cold PBS buffer, followed by lysis with RIP lysis buffer. The cells were then centrifuged at 12000g for 10 min at 4°C to remove the precipitate. 50µL of Protein A / G magnetic beads were aliquoted into two test tubes and incubated with 5µg of anti-Ago2 antibody or isotype control IgG (negative control) at 25°C for 30 min to complete conjugation. After three elutions to remove unbound antibodies, the pre-coated magnetic beads were resuspended in 200µL of clear lysis buffer and incubated overnight at 4°C to capture the endogenous Ago2-RNA complex. The next day, the magnetic bead-bound complex was rigorously washed with high-salt and low-salt RIP elution buffers, followed by extraction of the co-precipitated RNA using TRIzol reagent. Reverse transcription was immediately performed, and the relative enrichment levels of circCRIM1 and miR-320c were quantitatively detected using SYBR Green qRT-PCR. The fold change in gene expression was calculated using the IgG control group as a baseline.

[0033] 1.9 Luciferase reporter gene assay Wild-type (WT) and seed region mutant (MUT) fragments of circCRIM1, along with the 3' untranslated region (3'UTR) of MMP16, were directionally inserted into the Xba I site of the dual-luciferase reporter plasmid pSI-Check2 (Hanbai Biotechnology Co., Ltd., Shanghai). HEK293T cells were cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1000 g / well in 96-well plates. Once cell confluence reached 60%-70%, 50 ng of reporter plasmid and 20 nM miR-320c mimic or negative control (NC) were co-transfected using Lipofectamine 3000. Forty-eight hours after transfection, luciferase activity was measured using a Promega dual-luciferase reporter assay kit; Renilla signal was standardized using Firefly signal as a baseline. Four parallel samples were set up for each experimental condition, and results were validated through three independent experiments. Final data are expressed as mean ± standard deviation.

[0034] 1.10 OA's DMM Model Ten-week-old C57BL / 6J mice were purchased from an SPF-grade animal breeding facility in Jiangsu Province. All experimental protocols were approved by SYSU-IACUC-2024-002182. Osteoarthritis (OA) surgery was induced in the left knee by disrupting the medial meniscus to induce instability, while a pseudocapsulation was performed on the right knee. Four weeks post-surgery, the animals were divided into three groups (n=15 per group): a sham-operated group, a DMM combined with empty AAV group, and a DMM combined with AAV-circCRIM1 group. Under isoflurane anesthesia, intra-articular injections were administered via the patellar tendon: the first injection was 10 µL of empty vector (1.3 × 10⁻⁶). 11 pfu / mL -1 ) or AAV (2.2×10) encoded mmu_circ_0001704. 11 pfu / mL -1 The injection was repeated four weeks later. At week 12, the mice were euthanized, and samples from both knee joints were used for micro-CT reconstruction, Western blot analysis, and immunohistochemical detection to quantify the degree of cartilage damage and evaluate the therapeutic effect of circCRIM1.

[0035] 1.11 MicroCT Analysis After fixing the entire knee joint in 4% paraformaldehyde for 48 hours, it was placed in the scanning chamber of a Zeiss Xradia 510 Versa miniature CT scanner and scanned for 115 minutes at 50kV, 30W, and 500µA to obtain an isotropic 8-µm voxel dataset. Reconstructed images were generated using the Feldkamp algorithm in Multimodal 3D Visualization software and exported as 8-bit grayscale volumetric data. In Mimics 5.0 software, two blinded operators manually delineated the periarticular osteophytes on the medial and lateral sides of the femoral and tibial condyles, highlighting them in red. The osteophyte volume was quantified for each condyle, and the average of the four locations was taken to obtain the total osteophyte volume of a single knee joint for subsequent statistical analysis.

[0036] 1.12 Immunohistochemistry Chondrogenic granules and articular cartilage explants derived from human umbilical cord mesenchymal stem cells (hADSC) were fixed in 4% paraformaldehyde for 24 h, treated with graded ethanol, and then embedded in paraffin. Serial 5 µm thick sections were prepared: granule sections were stained with Alsin blue to reveal sulfated glycosaminoglycans; cartilage sections, after dewaxing and hydration, were counterstained with hematoxylin-eosin to observe cell structural details, and proteoglycan distribution was labeled with Saxony O-Fast Green. The immunophenotypic analysis procedure was as follows: granule sections were incubated overnight at 4°C with anti-COL2A1 primary antibody, and cartilage sections with anti-MMP3 primary antibody, followed by incubation at room temperature with HRP-polymer secondary antibody for 1 h and DAB staining. Three non-overlapping high-power fields were selected for each sample, and the integrated optical density (IOD) of positive cells was quantitatively analyzed using Image-Pro Plus software, with the average value taken. Two blinded evaluators independently assessed the degree of cartilage degradation using the OARSI scoring system, and the average score was reported to reduce subjective bias.

[0037] 1.13 Statistical Analysis Statistical analyses were performed concurrently using GraphPad Prism 8 and SPSS 22.0 software. Before any comparisons, the normality of the data (Shapiro-Wilk test) and homogeneity of variance (Lvene test) were assessed. When the parametric assumptions were met, unpaired t-tests were used for continuous variables between two groups; if the parametric assumptions were not met, the Mann-Whitney U test was used instead. For three or more samples, if the data were normally distributed, significant differences were determined using one-way ANOVA combined with Bonferroni post-hoc correction; for nonparametric data, the Kruskal-Wallis test combined with Dunn's multiple comparison method was used. All results are expressed as mean ± standard deviation, and a two-tailed p-value <0.05 was considered statistically significant.

[0038] II. Test Results 2.1 Expression pattern of circCRIM1 in hADSC chondrogenesis and normal and OA cartilage Human adipose-derived stem cells (hADSCs) from passages 3 to 5 were continuously cultured in chondrogenic medium. Cell clumps were collected on day 14 (mature cartilage) and day 0 (undifferentiated control group) for circular RNA microarray analysis. Differentially expressed circular RNAs were screened based on a fold change ≥ 2 and an FDR < 0.05.

[0039] like Figure 1 As shown in Figure A, circCRIM1 is one of the most significantly upregulated transcripts. Figure 1 As shown in Figure B, time-series qRT-PCR detection at 0, 7, 14, 21, and 28 days revealed a biphasic expression pattern: circCRIM1 showed a slow increase similar to the control group from 0 to 7 days; a rapid increase from 7 to 14 days, reaching its first peak on day 14; however, it rapidly decreased from day 14 to 21, with the most significant decrease on day 21, showing a significantly lower expression level compared to the control group; subsequently, it rapidly rebounded from day 21 to 28, reaching an even higher peak on day 28, showing a more significant upregulation trend compared to the control group (P<0.01).

[0040] like Figure 1 As shown in the CE figure, cartilage specimens were obtained from patients with osteoarthritis (Kellgren-Lawrence grade 3-4) and traumatic amputation patients, whose cartilage remained intact to the naked eye. qRT-PCR analysis revealed that circCRIM1 expression was significantly downregulated in the osteoarthritis group compared to the control group, while linear CRIM1 expression levels showed no significant difference (P<0.01). Figure 1 As shown in Figure F, circCRIM1 is formed by the circularization of exons 3, 4, 5, 6, and 7 of the CRIM1 gene. Subsequent Sanger sequencing confirmed its head-to-tail splicing junction. Figure 1 As shown in Figure G, fluorescence in situ hybridization combined with confocal microscopy reveals that circCRIM1 in chondrocytes is mainly located in the cytoplasm, with extremely weak signal intensity in the nucleus. Figure 1 As shown in Figure H, homologous and reverse primers were designed to span the inverse splicing site: the homologous primers simultaneously amplified circCRIM1 and CRIM1 linear transcripts in both cDNA and gDNA; while the reverse primers only detected a circCRIM1-specific band in cDNA, with no product detected in either gDNA or CRIM1 linear transcripts. Figure 1 As shown in Figure I, after RNase R treatment, qRT-PCR and gel electrophoresis showed that circCRIM1 remained intact, while linear CRIM1 mRNA was almost completely degraded. Figure 1 As shown in Figure J, after transcriptional arrest using actinomycin, circCRIM1 exhibited a significantly longer half-life than its linear parent mRNA.

[0041] 2.2 circCRIM1 regulates hADSC chondrogenesis Following siRNA-mediated circCRIM1 knockdown, human adipose-derived mesenchymal stem cells (hADSCs) underwent 14 days of chondrogenic culture, and Alsin blue staining showed a significant increase in cartilage matrix deposition. Figure 2 As shown in the AD diagram, the expression levels of MMP13 and COL10A1 decreased sharply, while the transcripts of COL2A1, AGGRECAN, and SOX9 increased synchronously.

[0042] like Figure 2 As shown in the EH figure, overexpression of circCRIM1 inhibits the chondrogenic differentiation process of hADSCs, manifested by increased expression of MMP13 and COL10A1, while downregulation of COL2A1, AGGRECAN, and SOX9. These bidirectional regulatory results collectively indicate that circCRIM1 suppresses the differentiation process towards chondrogenicity in stem cells during differentiation.

[0043] 2.3 circCRIM1 induces degradation of human chondrocyte matrix circCRIM1 was silenced and overexpressed in human chondrocytes, respectively. Figure 3 As shown in the AD diagram, knocking down circCRIM1 causes cells to shift towards a synthetic repair phenotype, characterized by significant inhibition of the catabolic enzyme MMP13, while simultaneously upregulating cartilage-specific genes COL2A1 and SOX9. Figure 3 As shown in the EH figure, forced expression of circCRIM1 initiates a hypertrophy program, manifested by upregulation of MMP13 and corresponding downregulation of COL2A1 / SOX9. This confirms that circCRIM1 is a key regulator of chondrocyte matrix homeostasis and phenotypic fate.

[0044] 2.4 circCRIM1 as the sponge for miR-320c like Figure 4 As shown in Figure A, given that circCRIM1 is primarily located in the cytoplasm, candidate miRNAs were screened by integrating prediction results from four independent databases: TargetScan, CircBank, Starbase, and miRanda. Figure 4 As shown in Figure B, qRT-PCR detection combined with RNA pull-down assay revealed that miR-320c was the molecule with the highest enrichment. Figure 4As shown in Figure C, time-series analysis revealed a gradual increasing trend of miR-320c during the early, middle, and late stages of chondrogenesis in human adipose-derived stem cells (hADSCs). Figure 4 As shown in Figure D, the Ago2-RIP experiment, verified by PCR and gel electrophoresis, showed that both circCRIM1 and miR-320c were recruited to the Ago2-containing complex.

[0045] like Figure 4 As shown in the EF diagram, dual-luciferase reporter gene detection and FISH co-localization further confirm the direct interaction and cytoplasmic co-localization between the two. Figure 4 As shown in Figure G, 14 days after transfection with miR-320c mimic, matrix deposition in hADSCs was significantly enhanced; Figure 4 As shown in Figure H, sections of HADS-derived cartilage glomeruli were stained with alicin blue. Immunohistochemistry was used to observe the expression of COL2A1 in the cartilage glomeruli. Figure 4 As shown in Figure I, this is accompanied by MMP13 inhibition and increased expression of COL2A1 and SOX9. The combined data indicate that circCRIM1, by isolating miR-320c, weakens its promoting effect on cartilage genes, disrupts matrix homeostasis, and ultimately inhibits cartilage formation in hADSC.

[0046] like Figure 5 As shown in Figure A, qRT-PCR results from clinical cartilage samples revealed a significant decrease in miR-320c abundance in chondrocytes from osteoarthritis patients compared to healthy controls. This was followed by the introduction of miR-320c mimics and inhibitors into osteoarthritis-derived chondrocytes. Figure 5 As shown in the BF diagram, gain-of-function experiments indicated that MIMics significantly suppressed MMP13 transcription while upregulating the expression levels of COL2A1 and SOX9; however, loss-of-function experiments showed the opposite effect, such as... Figure 5 As shown in the GK plot, MMP13 expression was upregulated while COL2A1 and SOX9 expression decreased simultaneously. These results indicate that miR-320c still possesses the ability to inhibit catabolism and promote phenotypic synthesis in the osteoarthritic environment, and its decreased expression level may be one of the important inducing factors for osteoarthritis progression.

[0047] 2.5 circCRIM1 mediates hADSC chondrogenesis and PHC homeostasis through interaction with miR-320c. like Figure 6As shown in Figure AB, through a two-way rescue strategy, we found that ectopic overexpression of circCRIM1 significantly inhibited chondrogenesis of human adipose-derived stem cells (hADSCs) – this was evident from the decreased expression levels of collagen IIA and SOX9 and the increased expression of matrix metalloproteinase 13 (MMP13), while co-transfection with miR-320c mimics reversed these abnormalities. Figure 6 As shown in the mid-CD image, knockdown of circCRIM1 promotes chondrogenesis, an effect neutralized by miR-320c inhibition. Figure 6 As shown in the EJ figure, in human chondrocytes, overexpression of circCRIM1 induces catabolic extracellular matrix characteristics, while overexpression of miR-320c restores these characteristics; when both circCRIM1 and miR-320c are knocked out, the loss of miR-320c offsets the anabolic advantage brought about by the loss of circCRIM1.

[0048] In summary, these results indicate that circCRIM1 can regulate the expression of hADSCs and chondrocyte markers through a competitive endogenous RNA mechanism by adsorbing miR-320c.

[0049] 2.6 miR-320c inhibits the expression of downstream MMP16 and improves the ECM of chondrocytes. To identify the targets of miR-320c in the system, such as Figure 7 As shown in Figure A, integrating the prediction results from TargetScan, miRDB, and TarBase yielded a total of 258 overlapping candidate target points. Figure 7 As shown in Figure B, the 3' untranslated region of MMP16 contains a site that matches the miR-320c seed sequence. Luciferase reporter gene assays confirmed that miR-320c directly binds to this site and significantly reduces reporter gene activity. Figure 7 As shown in the CD diagram, qRT-PCR and immunofluorescence assays revealed elevated MMP16 levels in osteoarthritis cartilage compared to the non-osteoarthritis control group. Figure 7 As shown in the EF diagram, functionally, forced expression of MMP16 promotes the transformation of chondrocytes to a mast-catabolism phenotype, manifested by decreased COL2A1 and SOX9 and increased MMP13; while siRNA-mediated knockdown of MMP16 produces the opposite effect. Figure 7 As shown in the mid-GH plot, overexpression of miR-320c can repair MMP16-induced matrix damage: co-transfection restores COL2A1 and SOX9 to near-baseline levels and inhibits MMP13. Conversely, simultaneous inhibition of MMP16 and miR-320c produces a mirror effect. Overall, the data suggest that miR-320c maintains cartilage matrix integrity by targeting MMP16.

[0050] 2.7 Injection of circCRIM1-si can alleviate OA in a mouse model. An osteoarthritis model was established in 12-week-old male C57BL / 6J mice via DMM surgery. Postoperatively, the AAv-circCRIM1-si viral vector was injected into the joint cavity weekly, with empty AAV serving as a control group. Figure 8 As shown in Figure A, after four weeks of continuous injections to ensure transduction efficiency, the animals were kept at 20 weeks of age and euthanized for sampling. Figure 8 Figure B shows the expression levels of circCRIM1 in chondrocytes of DMM mice and IL-1β-stimulated mice; Figure 8 As shown in Figure C, the DMM+carrier group exhibited typical fibrosis on the cartilage surface. HE staining and hematoxylin O / fast green staining results showed significantly increased OARSI and synovitis scores. In contrast, the circCRIM1 knockdown group maintained intact cartilage structure, restored proteoglycan content, and showed significantly decreased OARSI and synovitis scores. Figure 8 As shown in the DE figure, qRT-PCR and immunofluorescence experiments confirmed that knockdown of circCRIM1 significantly downregulated the mRNA and protein levels of the catabolism marker MMP13, while significantly upregulated the synthesis markers COL2A1 and SOX9. The downregulation of MMP16 confirmed the existence of the circCRIM1-miR-320c-MMP16 regulatory axis. Figure 8 As shown in Figure F, micro-CT three-dimensional reconstruction further revealed that inhibiting circCRIM1 significantly reduced the volume and score of osteophytes at the tibial plateau and medial / lateral femoral condyles, suggesting that the joint remodeling process is regulated.

[0051] 2.8 Relevant Expression of circCRIM1 like Figure 9 Figure A shows the expression of circCRIM1 24 hours after IL-1β induction. Figure 9 Figure B shows the normalized expression of overexpression of circCRIM1.

[0052] 2.9 Supplementary experiment on the effects of miR-320c on cartilage metabolism in chondrocytes. like Figure 10 As shown in Figure A, Ago2-RIP confirms that both circCRIM1 and miR-320c are recruited in Ago2-containing complexes. Figure 10 Figure B shows the expression of miR-320c after transfection of stem cells with a miR-320c mimic. Figure 10As shown in Figure C, chondrocyte sections derived from HADSC were stained with alixin blue after transfection with the miR-320c inhibitor. Simultaneously, immunohistochemistry was used to observe the expression of COL2A1 in chondrocytes. After transfection with the miR-320c inhibitor, Western blotting was used to detect the expression of COL2A1, SOX9, and MMP13.

[0053] 2.10 Supplementary experiments on the effects of MMP16 and miR-320c on human chondrocyte metabolism like Figure 11 As shown in Figure AB, human chondrocytes were transfected with si-MMP16 alone or co-transfected with a miR-320c inhibitor; then the expression levels of COL2A1, SOX9, MMP13 and MMP16 were analyzed by RT-qPCR and IF.

[0054] Therefore, intra-articular administration of AAV-circCRIM1-si can effectively delay the pathological progression of OA in DMM mice.

[0055] III. Conclusion The results showed that circCRIM1 could inhibit cartilage differentiation and promote the cartilage matrix towards hypertrophic decomposition, specifically manifested by downregulation of COL2A1 and SOX9 expression, while MMP13 was significantly upregulated.

[0056] Studies have found that miR-320c is a high-affinity target of circCRIM1, and miR-146a is significantly upregulated in the early stages of osteoarthritis. This is achieved by inhibiting the transcription of MMP13 and ADAMS-5 and reducing matrix catabolism levels. Intra-articular injection of miR-101 inhibitors can inhibit DNMT3B-mediated integrin α1 silencing, thereby preventing IL-1β-induced downregulation of COL2A1 and ACAN, and significantly delaying cartilage destruction in traumatic OA mice. Studies on miR-320c have shown that exon miR-320c, derived from synovial mesenchymal stem cells (MSCS), can enhance cartilage formation by targeting ADAM19. Furthermore, studies have shown that miR-320c can delay the development of osteoarthritis by inhibiting the classical Wnt signaling pathway, and intra-articular injection of miR-320c can effectively alleviate disease progression in a mouse model of osteoarthritis. This application proposes for the first time that circRM1, acting as a molecular sponge, influences miR-320c, thereby further regulating related metabolic processes in human adipose stem cells and human chondrocytes.

[0057] This study confirms that miR-320c exhibits a gradual upregulation trend during chondrogenesis in human adipose-derived mesenchymal stem cells (hADSCs) and plays a protective role in osteoarthritis chondrocytes. Gain-of-function experiments showed that miR-320c enhances the transcriptional activity of cartilage-specific genes (COL2A1, SOX9) while inhibiting the expression of the catabolism marker MMP13; conversely, inhibiting this miRNA produces the opposite effect, indicating that this miRNA is a positive regulator of the chondrogenesis process. Mechanistic studies further revealed that MMP16 is a direct target of miR-320c, and its 3′-UTR region contains a functional binding site that mediates post-transcriptional silencing.

[0058] MMP16 is a membrane-anchored matrix metalloproteinase, and its expression is significantly upregulated in osteoarthritis (OA): in a naturally occurring metacarpal / metatarsal OA model, the transcriptional level of MMP16 in cartilage increases in tandem with pathological grading. IL-1β, a key inflammatory cytokine in osteoarthritis, can induce MMP16 expression in cells, thereby regulating cell growth, extracellular matrix degradation, and the secretion of inflammatory cytokines. Therefore, MMP16 serves as a key collagenase in the progression of osteoarthritis and provides a novel molecular node for targeted intervention.

[0059] Overexpression of miR-320c rescued MMP16-induced matrix damage, as miR-320c directly binds to MMP16 and affects its expression. Co-transfection restored COL2A1 and SOX9 to near-baseline levels and suppressed MMP13.

[0060] Therefore, the competitive endogenous RNA network composed of circCRIM1 acts as a sponge for miR-320c to relieve its inhibitory effect on MMP16 expression. This mechanism leads to impaired chondrogenesis in human chondrocytes and enhanced extracellular matrix catabolism, thereby promoting the progression of osteoarthritis. In subsequent mouse experiments, we used an intra-articular injection method. Intra-articular injection has the advantages of direct targeting and avoiding systemic side effects, thus providing an attractive approach for molecular therapy of OA. Simultaneously, inhibition of circCRIM1 downregulates MMP16 expression levels, and the existence of the circcrim1-Mir-320C-MMP16 regulatory axis was also confirmed in the DMM model.

[0061] In summary, this application reveals the disruptive role of circCRIM1 in the pathogenesis of osteoarthritis and demonstrates that targeting the circcrim1-Mir-320C-MMP16 axis is a potentially effective strategy for treating osteoarthritis.

[0062] The foregoing has provided a detailed description of one embodiment of the present invention, but this description is merely a preferred embodiment and should not be construed as limiting the scope of the invention. All equivalent variations and modifications made within the scope of the claims of this invention should still fall within the patent coverage of this invention.

Claims

1. Use of circCRIM1 in modulating chondrocyte differentiation and cartilage homeostasis, characterized in that, The nucleotide sequence of the circCRIM1 is shown as SEQ.ID.NO.

1.

2. Use according to claim 1, characterized in that, The specific content of the application is: inhibiting the expression of circCRIM1 to promote the differentiation of human adipose-derived stem cells into chondrocytes; promoting the expression of circCRIM1 to inhibit the differentiation of human adipose-derived stem cells into chondrocytes.

3. The use according to claim 1, characterized in that, The cartilage homeostasis is the balance between the synthesis and degradation of cartilage extracellular matrix; inhibiting the expression of circCRIM1 to slow down the degradation of cartilage extracellular matrix; promoting the expression of circCRIM1 to accelerate the degradation of cartilage extracellular matrix.

4. Use according to any one of claims 2-3, characterized in that, The diagnostic method of the expression level of circCRIM1: the diagnostic reagent detects the expression of circCRIM1 in cartilage tissue or synovial fluid; the diagnostic reagent includes any one of specific primers, probes or antibodies for detecting circCRIM1.

5. Use according to any one of claims 2-3, characterized in that, The method for inhibiting the expression of circCRIM1: including down-regulating the expression of circCRIM1 by using circCRIM1 inhibitor; the circCRIM1 inhibitor includes any one of siRNA, shRNA, antisense oligonucleotide targeting circCRIM1, viral vector carrying circCRIM1 knockdown sequence.

6. Use according to claim 5, characterized in that, The circCRIM1 inhibitor further includes a carrier, and the carrier includes any one of physiological saline, buffer, excipient or stabilizer.

7. Use according to claim 6, characterized in that, The administration mode of the circCRIM1 inhibitor is intra-articular injection.

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