Isolation and culture of hADSCs
Fresh adipose tissue was collected from 35-year-old healthy women provided by Guizhou Provincial People’s Hospital (Guizhou, China). The donor signed informed consent. The adipose tissue was put into the sterile flask with PBS and double antibody, and taken back to the laboratory within 2h. About 10g of adipose tissue was brought into the petri dish in the ultra-clean table and cleaned with PBS. Then, the blood vessels and connective tissue were cut off in advance, and the adipose tissue was cut into 1mm × 1mm fragments. Then, 0.1% Ⅰ type collagenase was added to double volume, and the upper liquid was absorbed aftershock digestion at 37℃ for 45min, and 1000r/min, centrifugation for 5min. The undigested adipose tissue was added with new collagenase for further digestion two times. The obtained cells were resuspended through a 200-mesh cell screen with H-DMEM medium (Thermo, USA) and then seeded into a 10cm culture dish. The cells were cultured in an incubator at 37℃ with 5% CO2. The liquid was changed for the first time after 24h.
Induce of chondrogenic differentiation of hADSCs by microsphere culture
The fourth generation of human adipose-derived stem cells was selected 1×106 cells taken into a 15mL centrifuge tube, centrifuged at 1000r/min for 5min, the supernatant was discarded, washed twice with PBS, and the PBS was removed. 3 ml chondrogenic induction medium (H-DMEM, 10 ng/ ml TGFβ1, 50 ug/ ml VC, and 10-7 M dexamethasone, 6.25ug/ ml insulin, 1% double antibody, 1%FBS) were added into the cells for induction for 21 days. 3-4 days half amount of liquid change.
Alcian blue staining
After induction of adipose-derived stem cells for 21 days, the medium was sucked, and 40g/L paraformaldehyde was added to the cell clumps and fixed for 30min. Dehydrate with ethanol gradient, embed with paraffin, and prepare slices with a thickness of 5um for use. Blot off the fixed solution and wash with PBS three times. The cell masses were washed with 0.1N HCl (0.9ml concentrated hydrochloric acid added to 100ml ultra-pure water) for 5min. 1% Alcian dye (1g Alcian powder added with 100ml of 3% glacial acetic acid) overnight. 0.1N HCl was washed for 3 times, 5min each time, and the background was removed. The results were recorded under the microscope.
Immunocytochemical
After induction of adipose-derived stem cells for 21 days, the medium was sucked, and 40g/L paraformaldehyde was added to the cell mass for fixation for 12h. Dehydrate with ethanol gradient, embed with paraffin, and prepare slices with a thickness of 5um for use. Normal goat serum was sealed for 30min. Rabbit anti-COLⅡ(1:1000) was added and stayed at 4℃ overnight. Rewarm at 37℃ and clean three times with PBS. Subsequently, the secondary antibody was added, incubated at room temperature for 1h, then washed 3 times with PBS. DAB color development lasted for 5-10min, and the color development was observed under the microscope.
Cell transfection
The LncRNA NONHSAT030515 expression was conducted by transfection of pcDNA3.1-NONHSAT030515 and sh-NONHSAT030515. The miR-490-5p expression was conducted by transfection of miR-490-5p mimics. They were designed and synthesized by GenePharma(Shanghai, China). The transfection results were performed using LipofectamineTM 2000 reagent (Life Technologies Corporation, USA) follow the manufacturer’s protocol.
Dual-luciferase reporter assay
The dual-luciferase assay was used to confirm whether NONHSAT030515 directly targeted miR-490-5p and whether miR-490-5p directly targeted BMPR2. HADSCs were co-transfected with NONHSAT030515 (BMPR2) wild or mutant plasmids, miR-490-5p control, or miR-490-5p mimics. Luciferase activity was assessed 48h after transfection using a dual-luciferase reporting system (Promega Company).
Real-time quantified PCR
First, total RNA was extracted from hADSCs using Trizol reagent (Invitrogen, USA). Then cDNA was synthesized using the PrimeScript RT reagent (Invitrogen, USA). RT-qPCR was performed by SYBR® Premix Ex Taq kit (Invitrogen, USA) using GAPDH as an internal control. The expression miRNA using U6 as an internal control. The related primers are shown in Table Ⅰ. The outcomes were analyzed by 2−ΔΔCt method.
Table Ⅰ Primers used in this study
Primer
|
Sequence(5’-3’)
|
miR-490-5p-RT
|
GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAC ACCCACC
|
U6-RT
|
AACGCTTCACGAATTTGCGTG
|
miR-490-5p-qPCR
|
Forward: TGCGGCCATGGATCTCCA
Reverse: CCAGTGCAGGGTCCGAGGT
|
U6-qPCR
|
Forward: GCTCGCTTCGGCAGCACA
Reverse: GAGGTATTCGCACCAGAGGA
|
Aggrecan-qPCR
|
Forward: AGTCCTCAAGCCTCCTGTA
Reverse: CCTCCTCACATACCTCCTG
|
SOX9-qPCR
|
Forward: ACCACCAGAACTCCAGCTCCT
Reverse: TCTGCGGGATGGAAGGGA
|
COL2A1-qPCR
|
Forward: GCTCCCAGAACATCACCTACC
Reverse: TGAACCTGCTATTGCCCTCT
|
NONHSAT030515-qPCR
|
Forward: TTGGTGTTGATATGGGTA
Reverse: CAAAGTTAGGAGGAAGAA
|
BMPR2-qPCR
|
Forward: AAATAGCCTGGCAGTGAG
Reverse: ATGTGACAGGTTGCGTTC
|
GAPDH-qPCR
|
Forward: GACCTGACCTGCCGTCTA
Reverse: AGGAGTGGGTGTCGCTGT
|
Western blot analysis
Total protein extract from hADSCs using a protein extraction kit (Bio-Rad, USA), and the concentration was determined by BCA kit (Tianjin, China). Subsequently, the protein (50ug) was separated by SDS-PAGE. Then, moved to PVDF (Millipo,USA), the membrane was incubated with blocking buffer at room temperature for 1h. Then, incubate the first antibody GAPDH (Abcam)、Aggrecan (Proteintech)、SOX9 (Proteintech)、COL2A1 (Proteintech)、BMPR2 (Abcam) overnight, followed by secondary antibody at room temperature for 1h. The GAPDH was used internal control. All western blot experiments were repeated at least three times.
Statistical analysis
In this experiment, all experimental data were expressed as mean ± standard deviation and were processed by SPSS21.0. An independent sample t-test was used to compare the two groups,and a one-way analysis of variance (ANOVA) was used to compare the groups. P <0.05 between groups was considered statistically significant. Each group was repeated at least 3 times independently.