Subjects and specimens
The study involved 98 patients with osteoarthritis selected between January 2015 and January 2017 in Wei Nan Central Hospital. There were 54 males and 44 females, ranging in age from 32 to 69 years, with a mean age of 49 ± 8.7 years. In the same period, 76 healthy volunteers with similar age and gender distributions were selected to serve as a control group.
The healthy volunteers underwent comprehensive physical exams including laboratory tests, pulmonary function testing, urinalysis, chest x-rays, audiograms, full body CAT scanning, heart stress tests, EKGs, vascular age tests, and mammograms or prostate exams depending on gender. None of them showed any abnormities.
Whole blood (20 ml) was extracted from each participant on the day of admission to the study. Blood samples were kept at room temperature for 2 h, followed by centrifugation at 1200 g for 20 min to obtain serum. Serum samples were stored in liquid nitrogen before use.
This study was approved by the Ethics Committee of Wei Nan Central Hospital. All patients and healthy subjects signed written forms for informed consent.
Cell line and cell culture
Human chondrocyte cell line CHON-001 (ATCC CRL-2846) was obtained from ATCC. Cells were cultured in ATCC-formulated Dulbecco’s modified Eagle’s medium (DMEM, cat. no. 30–2002) supplemented with 0.1 mg/ml G-418 and 10% heat-inactivated fetal bovine serum in an incubator (37 °C, 5% CO2). Cells were harvested during the logarithmic phase for subsequent experiments.
Construction of cell line overexpressing lncRNA-ATB
LncRNA-ATB cDNA surrounded by NheI cutting sites was amplified via PCR and inserted into an NheI-linearized pEGFPC3 (Clontech) vector to establish the lncRNA-ATB expression vector. This vector has a CMV promoter.
Chondrocytes were cultured overnight to reach 70–80% confluence, and then 15 nM vectors were transfected into 4 × 105 cells using Lipofectamine 2000 reagent (cat. no. 11668–019, Invitrogen). Transfection of empty pEGFPC vector was used as a negative control.
Cells were harvested during the logarithmic phase to prepare a cell suspension using medium containing 10 mM tetraethylammonium (TEA). The final cell density was 4 × 104 cells/ml. Then 4 × 103 cells in 100 μl cell suspension were added to each well of a 96-well plate. Cells were cultured for 6 h and then 10 μl of MTT was added, followed by cell culture for an additional 4 h. A Fisherbrand accuSkan GO UV/Vis Microplate Spectrophotometer (Thermo Fisher Scientific) was used to measure absorbance at 570 nm.
Cell proliferation assay
Each well of a 96-well plate was filled with 100 μl of cell suspension containing 4 × 103 cells. Cells were cultured and 10 μl of CCK-8 solution (Sigma-Aldrich) was added 24, 48, 72 and 96 h later. Cells were cultured for another 4 h, and a Fisherbrand accuSkan GO UV/Vis Microplate Spectrophotometer was used to measure optical density at 450 nm (Fisher Scientific).
Quantitative real-time PCR
Trizol reagent (Invitrogen) was mixed with serum or in vitro cultured cells to extract total RNA. This extraction was repeated to make sure all RNA samples had an A260-to-A280 ratio between 1.8 and 2.0 (measured using a NanoDrop 2000 Spectrophotometer, Thermo Fisher Scientific).
After synthesis of cDNA, PCR was performed with these primers:
The PCR conditions were: 95 °C for 55 s, followed by 40 cycles of 95 °C for 20 s and 60 °C for 42 s. Data were processed using the 2-ΔΔCT method, and the relative expression level of lncRNA-ATB was normalized to endogenous control β-actin.
Total protein extraction was carried out using RIPA solution (Thermo Fisher Scientific) and the BCA assay was performed to measure protein concentration. After denaturing, protein samples (20 μg from each sample) were subjected to 12% SDS-PAGE gel electrophoresis, followed by gel transfer to PVDF membranes. Blocking was performed by incubating membranes with 5% skimmed milk for 1 h at room temperature.
After blocking, the membranes were incubated with rabbit anti- human primary antibodies of p-Akt (1:2000, ab38449, Abcam), Akt (1:2000, ab8805, Abcam) and GAPDH (1:1000, ab8245, Abcam) at 4 °C overnight. After that, membranes were washed 3 times for 5 min each time with phosphate-buffered saline (PBS). The membranes were further incubated with anti-rabbit IgG-HRP secondary antibody (1:1000, MBS435036, MyBioSource) at room temperature for 2 h. Finally, ECL (Sigma-Aldrich) was added and a MYECL Imager (Thermo Fisher Scientific) was used to detect signals. The relative expression levels of p-Akt and Akt were normalized to the endogenous control GAPDH using Image J software.
All data were processed using SPSS19.0 (SPSS Inc.). Count data were compared using the chi-square test. Student’s t test was used for comparisons of measurement data between two groups, and analysis of variance and the LSD test were used for comparisons of measurement data between multiple groups. p < 0.05 indicated a difference with statistical significance.