The diagnostic value and pathogenetic role of lncRNA-ATB in patients with osteoarthritis

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.

MTT assay

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).

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.

Western blot

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.

Statistical analysis

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.

Expression of lncRNA-ATB in the serum of osteoarthritis patients

The expressions of lncRNA-ATB in the serum of osteoarthritis patients and healthy subjects was detected using quantitative Real Time-PCR. See Additional file 1 for the data normalization using the 2^-ΔΔCT method. As shown in Fig. 1, the serum levels of lncRNA-ATB were significantly lower in osteoarthritis patients than in the healthy controls. These data suggest that downregulation of lncRNA-ATB is very likely to be involved in the development of osteoarthritis.

Diagnostic values of lncRNA-ATB for osteoarthritis

ROC curve analysis was performed to evaluate the diagnostic value of lncRNA-ATB for osteoarthritis. In this analysis, the patients with osteoarthritis were used as true negative samples and the healthy controls were used as true negative samples. As shown in Fig. 2, the area under the curve (AUC) was 0.8902 (higher than 0.65), with a 95% confidence interval of 0.8430 to 0.9373 (p <  0.0001), indicating that serum lncRNA-ATB may serve as a diagnostic biomarker for osteoarthritis.

Correlation between the serum levels of lncRNA-ATB and the clinical data of osteoarthritis patients

LncRNA-ATB overexpression promoted the phosphorylation of Akt in cells of human chondrocyte cell line CHON-001

Akt signaling has been proven to be involved in the pathogenesis of osteoarthritis [11]. In our study, lncRNA-ATB overexpression showed no significant effect on the expression level of Akt in the cells of human chondrocyte cell line CHON-001 (Fig. 3). However, compared with the control cells and negative control cells (empty vector transfection), the phosphorylation level of Akt was significantly higher in cells with lncRNA-ATB overexpression (p < 0.05, Fig. 4). These data suggest that lncRNA-ATB overexpression can activate Akt signaling in chondrocytes.

Effects of lncRNA-ATB overexpression on the proliferation and viability of chondrocytes

As shown in Fig. 4a, lncRNA-ATB overexpression significantly promoted the proliferation of chondrocytes (p < 0.05), while treatment with the Akt signaling inhibitor SH-5 (10 nM, Santa Cruz Biotechnology) significantly reduced the enhancing effects of lncRNA-ATB overexpression on the proliferation of chondrocytes (p < 0.05).

In addition, lncRNA-ATB overexpression significantly increased the viability of chondrocytes, while treatment with the Akt signaling inhibitor SH-5 significantly reduced the enhancing effects of lncRNA-ATB overexpression on the viability of chondrocytes (Fig. 4b, p < 0.05).

These data suggest that lncRNA-ATB overexpression can promote the proliferation and increase the viability of chondrocytes by activating Akt signaling.