circRNA-ZCCHC14 affects the chondrogenic differentiation ability of peripheral blood mesenchymal stem cells by regulating GREM1 through miR-181a

Patient Samples and Ethics Statement

Peripheral blood samples from 10 OA patients and 7 healthy subjects were collected from the Second Affiliated Hospital of Kunming Medical University. The characteristics of the subjects participating in the OA study are shown in Table 1. The diagnosis of patients with OA was based on the guidelines of the American College of Rheumatology. This research program follows the ethical principles of the Declaration of Helsinki and has been approved by the Clinical Research Ethics Committee of the Second Affiliated Hospital of Kunming Medical University (Shen-PJ-Ke-2022-64). All selected patients signed an informed consent form.

Table 1 Characteristics of subjects enrolled in the osteoarthritis study. animal model

All surgical procedures and protocols were performed in accordance with the Guide to the Care and Use of Experimental Animals and approved by the Animal Experimentation Ethics Review Committee of Kunming Medical University (kmmu20221858). All animal methods are reported according to ARRIVE guidelines.

Animal models were established based on previous literature22. Full-thickness defects were produced in adult Yunnan Xiaoer pigs (n=6, male or female, average weight 15 kg). Briefly, pentobarbital sodium was injected into the ear vein to induce anesthesia. The right joint was transected through a medial approach at the patellar tendon, and then the patella was dislocated externally, exposing the joint cavity. A full-thickness defect was created through the surface of the medial and lateral femoral cartilage with a drill (hole: 7 mm diameter, 4 mm depth). After successful modelling, the Diannan Xiaoer pigs were kept in a controlled environment with free access to feed and water. All animals were examined simultaneously at the same age (mean age was 9 months). Thirty days after the operation, blood (30 ml) was collected from the pig’s anterior vena cava into a 5 ml vacuum collection tube containing sodium heparin. In addition, a 20 mL sterile syringe was used to puncture the medial side of the patellar ligament of the knee at 45 degrees into the joint cavity to collect the synovial fluid.

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Isolation and culture of PBMSCs

PBMSCs were isolated and cultured according to a previous study17. Briefly, the peripheral blood of Xiaoer pigs was collected, diluted with D-Hanks solution and subjected to Ficoll density gradient centrifugation to directly separate and purify peripheral blood mononuclear cells. Mononuclear cells were collected and cultured in serum-free medium and placed in an incubator at 37°C with 5% CO2.

cell transfection

PBMSCs (80%-90% confluency) were digested with 0.25% trypsin containing EDTA and inoculated into six-well plates at a cell density of 2 x 10 4 cells/cm 2 . The differentiated PBMSCs were treated with si-circRNA-ACCHC14, oe-circRNA-ACCHC143, si-GREM1, oe-GREM1, si-BMP2, oe-BMP2, miR-181a mimetic, miR-181a inhibitor or their negative controls using transfected by Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). After transfection, the cells were cultured in an incubator at 37°C with 5% CO2 for 48 h.


Total RNA was extracted from peripheral blood using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration was then measured using an ultratrace UV analyzer. RNA was reverse transcribed into cDNA according to the instructions of the Bestar qPCR RT Kit. PCR amplification was then performed with DBI Bestar® SYBR Green qPCR Master Mix (DBI Bioscience, Shanghai, China) using a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems). Expression levels of miRNAs and circRNAs were normalized against U6 or GAPDH expression and relative quantification was performed using the 2-ΔΔCt method. Primers are shown in Table 2.

Table 2 Primer sequences. Alcian blue staining

PBMSCs were seeded in 24-well plates and induced using chondrogenic differentiation medium for 14 days. The cells were then fixed with 4% paraformaldehyde and stained with Alcian blue stain solution (Solarbio, China). Finally, the cells were photographed with an inverted optical microscope (Leica DMI 3000B, Germany).

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Western blot analysis

Total protein was extracted using RIPA buffer (Beyotime Biotechnology) and its concentration was quantitated using BCA protein assay kit (Thermo). Equal amounts of protein were subjected to 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin (BSA) (Amresco, USA) and followed with specific primary antibodies (anti-GAPDH (Abcam), anti-BMP2 (Abcam), anti-COL2A1 (Abcam) and anti-AGR (Abcam)). from incubation with HRP-conjugated secondary immunoglobulin antibodies (booster).After enhanced chemiluminescence (ECL) color development, gel imager images were acquired.Finally, protein bands were quantitatively analyzed using ImageJ software.

Statistical analysis

Data tables were analyzed with GraphPad Prism 6.0 (GraphPad, USA). For a normal distribution, at-test was used to assess differences between two groups, while one-way analysis of variance (ANOVA) was used for comparisons between three or more groups. P<0.05 was considered statistically significant.