OA patients and healthy participants
The Fourth Affiliated Hospital of China Medical University Ethics Committee approved this study (No. FAHCMU20160245298, the investigation of lncRNA MCM3AP-AS1 in LPS-induced chondrocyte apoptosis). Research subjects of the present study were 70 cases of OA patients (stage III, 30 cases; stage IV, 40 cases; 26 males and 44 females; age range from 54 to 72 years’ old; mean age at 63.1 ± 6.0 years) and 70 healthy volunteers (26 males and 44 females, age range from 54 to 72 years’ old; mean age at 63.0 ± 6.1 years’ old), who were enrolled at aforementioned hospital between May 2016 and May 2019. Inclusion criteria of OA patients were the following: 1) no therapies performed within 100 days; 2) newly diagnosed OA. Exclusion criteria: 1) OA patients complicated with other clinical disorders; 2) recurrent OA. The OA patients were diagnosed by conventional techniques, such as joint fluid analysis and X-ray. The 70 healthy participants received routine physical examinations at the physical health center of aforementioned hospital. Among the 70 OA patients, lesions in knee were observed in 39 cases and lesions in hip were observed in remaining 31 cases. They were selected to match OA patients’ age and gender distributions. All OA patients (n = 70) and healthy participants (n = 70) were informed of experimental principle and informed consent was provided by all the 140 participants.
Synovial fluid
Synovial fluid (2 ml) was extracted from the affected sites of patients by performing arthrocentesis. To match the conditions of the OA group, synovial fluid extraction from knee was performed on 39 cases of healthy participants and extraction from hip was performed in 31 cases of healthy participants.
Primary chondrocytes and transient transfections
Primary chondrocytes (obtained from adult OA patients) from Sigma-Aldrich (402OA-05A, St. Louis, MO, USA) were used. Cells were cultivated in a 5% CO2 incubator at 37 °C with 95% humidity. The cell culture medium was Chondrocyte Growth Medium from PromoCell (Heidelberg, Germany). Following transfection experiments were performed using cells from passage 4–6. At passage 4–6, no significant changes in viability and morphology were observed.
The expression vector of MCM3AP-AS1 or HMGB1 was constructed using pcDNA3.1 vector as backbone. The vector construction service was provided by Genecopoeia (Guangzhou, China). Negative control (NC) miRNA and miR-142-3p mimic were synthesized by Beyotime Biotechnology (Shanghai, China). Transient transfections were mediated by lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). Chondrocytes (106) were transfected with 40 nM miRNA (NC miRNA as NC group) or 10 nM expression vector (empty vector as NC group) through the methods described by Invitrogen. In all cases, untransfected cells were control (C) cells. Subsequent experiments were performed using cells harvested at 24 h after transfections.
RNA-RNA interaction prediction
The interaction between MCM3AP-AS1 and miR-142-3p was predicted using an online program named IntaRNA (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp) [12]. MCM3AP-AS1 was used as the long sequence and miR-142-3p was the short sequence. Other parameters are the default.
Dual-luciferase reporter assay
psiCHECK-1 vector (Promega, Chicago, IL, USA) was used to construct the MCM3AP-AS1 expression vector. MCM3AP-AS1 expression vector combined with NC miRNA (NC group) or miR-142-3p mimic (miR-142-3p group) was transfected into 105 chondrocytes using aforementioned methods. Luciferase Reporter Assay Kit I (Firefly, PromoCell) was used to measure luciferase activity. Relative luciferase activity was calculated using firefly luminescence according to manufacturer’s instructions.
RNA extractions and digestion
TRIzol from Thermo Fisher Scientific (Cincinnati, OH, USA) was used to perform RNA extractions from both synovial fluid and chondrocytes. MiRNAs were harvested by precipitating RNAs using 85% ethanol. Genomic DNAs were removed by digesting RNA samples using gDNA Eraser (TaKaRa, Tokyo, Japan). In cases of lipopolysaccharide (LPS) treatment, chondrocytes were cultivated in medium containing 0, 500, 1000 and 2000 ng/ml LPS (Sigma-Aldrich) for 24 h under aforementioned methods to mimic OA conditions before use.
RT-PCR
SSRT IV system (Thermo Fisher Scientific) was used to reverse transcribe total RNAs into cDNAs with poly (T) as primer. To measure expression levels of MCM3AP-AS1 and HMGB1 mRNA, SensiFAST™ Real-Time PCR Kit (Bioline, Memphis, TN, USA) was used to perform quantitative PCR (qPCR) assays with Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) as endogenous control.
Expression levels of mature miR-142-3p were measured through the following steps: 1) addition of poly (A); 2) reverse transcription; 3) qPCR assays. All steps were performed using the All-in-OneTM miRNA qRT-PCR Detection Kit from GeneCopoeia. Ct values were determined using default threshold and 2−ΔΔCT method [13] was used for all data normalizations.
Western blot
At 24 h post-transfection, 105 chondrocytes were lysed using RIPA solution (Beyotime, Shanghai, China) to extract total proteins. The concentration of protein in each sample was measured by performing a BCA assay (Beyotime). Following denaturation in boiling water for 15 min, 10% SDS-PAGE gel was used to separate different proteins. Following gel transfer to (NC) membrane, PBS containing 5% fat-free milk was used to block membranes for 2 h at room temperature (RT). Then, primary antibodies including rabbit anti-GAPDH (ab38168, Abcam, London, UK) and anti-HMGB1 (ab18256, Abcam) were used to incubate with the membranes for 15 h at 4 °C, followed by incubating with HRP (IgG) secondary antibody (Goat Anti-Rabbit, ab6721, Abcam) for 2 h at RT. Western-Ready ECL Substrate Kit (BioLegend, San Diego, CA, USA) was used for signal production and ImageJ v.148 software was used for data normalization.
Cell apoptosis analysis
Cell apoptosis analysis after transfection was performed using flow cytometry. A 6-well cell culture plate was used to cultivate chondrocyte suspension with 2 ml (105 cells). Cell culture medium contained 2000 ng/ml LPS (Sigma-Aldrich). Cells were cultivated under aforementioned methods for 48 h and were trypsinized. After that, Annexin V-FITC (Thermo Fisher Scientific) and propidium iodide (PI, Thermo Fisher Scientific) staining was performed in dark for 20 min and apoptotic cells were detected by flow cytometry using BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences, San Jose, CA, USA).
Statistical analysis
Data from 3 independent biological replicates of each experiment were used to calculate mean values, which were used in the following data analysis. The unpaired t-test was used for data comparison between two groups. Data comparisons among multiple groups were performed by ANOVA (one-way) and Tukey test. p < 0.05 was statistically significant.
