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

Patient samples and ethics statement

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

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

Animal model

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

The establishment of animal models was performed according to previous literature22. Full thickness defects were created in adult Yunnan Xiaoer pigs (n = 6, male or female, average weight 15 kg). Briefly, pentobarbital sodium was injected into the ear vein for anaesthesia induction. The right joint was cut through a medial approach at the patellar tendon, and then, the patella was dislocated in an external position, exposing the joint cavity. A full thickness defect was created through the surface of the medial and lateral femoral cartilage with a drill (drilling: 7 mm in diameter, 4 mm in depth). After successful modelling, the Diannan Xiaoer pigs were kept in a controlled environment with free access to food and water. All animals were studied simultaneously at the same age (the average age was 9 months). At 30 days after surgery, blood (30 ml) was collected from the porcine anterior vena cava in a 5 ml vacuum collection tube containing heparin sodium. In addition, a 20 ml sterile syringe was used to puncture the medial side of the patellar ligament of the knee into the joint cavity at 45 degrees to collect the joint fluid.

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

The PBMSCs (80%-90% confluency) were digested with 0.25% trypsin containing EDTA and inoculated into six-well plates at a cell density of 2 × 104 cells/cm2. The differentiated PBMSCs were transfected with si-circRNA-ACCHC14, oe-circRNA-ACCHC143, si-GREM1, oe-GREM1, si-BMP2, oe-BMP2, miR-181a mimic, miR-181a inhibitor or their negative controls using 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.

RT‒qPCR

Total RNA was extracted from peripheral blood using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration was then measured using an ultratrace UV analyser. RNA was reverse transcribed into cDNA according to the Bestar qPCR RT Kit instructions. 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). The 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 on 24-well plates and induced using chondrogenic differentiation medium for 14 days. Cells were then fixed with 4% paraformaldehyde and stained with Alcian blue dye solution (Solarbio, China). Finally, the cells were photographed with an inverted optical microscope (Leica DMI 3000B, Germany).

Western blot analysis

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

Statistical analysis

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

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