Research subjects
This study was approved by The First Hospital of Fuyang Ethics Committee. From June 2018 to July 2019, this study enrolled both OS patients (n=64, 40 males and 24 females) and healthy controls (n=64, 40 males and 24 females) at this hospital. Age range of both groups was 42 to 68 years, and the mean age was 55.3 ± 5.8 years. It is known that initiated therapy and other clinical disorders can also affect gene expression, the 64 OS patients were excluded the ones with initiated therapy and the ones diagnosed with multiple clinical disorders. All the 64 OS patients were newly diagnosed OS cases. Range of T score was -2.83 to -4.99 in OS group. All healthy controls showed normal physiological functions in systemic physiological exam. Healthy controls with a history of OS were excluded. Range of T score was 3.19 to -0.47 in OS group. All participants signed informed consent.
Treatment, blood extraction and plasma preparation
Patients were treated with bisphosphonates (alendronate, risedronate, and zoledronic acid) and/or hormones (estrogen). The dosage was determined based on their conditions.
Prior to therapy, blood (3ml) was extracted under fasting from both OS patients and healthy controls. At 1 year after the initiation of therapy, fasting blood (3 ml) was also extracted from each patient. To prepare plasma samples, blood samples were mixed with citric acids to the ratio of 10:1, followed by centrifuging the mixture for 15 min at 1200g to collect the supernatant.
Osteoblasts
This study included primary osteoblasts from Sigma-Aldrich (USA) to perform in vitro cell experiments. Cells were cultivated in osteoblast growth medium (Promocell) at 37°C, 95% humidity and 5% CO2. Cells were cultivated to reach about 85% confluence prior to the subsequent assays. To study the effects of LPS treatment on gene expression, osteoblasts were cultivated in medium supplemented with 0, 2, 4, 6 and 8 μg/ml LPS for 48h prior to the subsequent assays.
Vector, miRNA and cell transfections
To overexpress circFADS2 and miR-16-5p, osteoblasts were transfected with either circFADS2 expression vector (1μg) or miRNA (40 nM) using Lipofectamine 2000 (Invitrogen). Expression vector of circFADS2 was constructed with pcDNA3.1 backbone vector (Invitrogen). Mimic of miR-16-5p and negative control (NC) miRNA were from Sigma-Aldrich. Empty vector- or NC miRNA-transfected cells (NC cells) and untransfected cells (Control cells, C) were included in each experiment. In fresh medium, cells were cultivated for further 48h after transfections prior to the subsequent assays.
RNA preparations
Total RNA extractions from plasma samples and osteoblasts were performed using Trizol reagent (Invitrogen). Genomic DNA removal was performed by incubating the RNA samples with DNase I (Invitrogen) at 37°C for 85 min. RNA integrity was analyzed using 5% urea-PAGE gel. OD 260/280 ratios were determined to analyze RNA purity.
QRT-PCR
SS-III-RT system (Invitrogen) was used to prepare cDNA samples through reverse transcriptions (RTs) with RNA samples with good integrity and an OD value close to 2.0 (pure RNA) as templates. To determine the expression of circFADS2, SYBR ® Green Realtime PCR Master Mix (Toyobo) was used to perform qPCRs with 18S rRNA as an internal control.
To determine the expression of miR-16-5p, poly (A) addition was performed, followed by using poly (T) as reverse primer to carry out RTs and qPCRs with U6 as an internal control. All steps were completed using All-in-OneTM miRNA qRT-PCR Detection Kit (Genecopoeia).
Ct values were normalized to corresponding internal controls using 2−ΔΔCt method.
Cell apoptosis assay
Osteoblasts with transfections were subjected to cell apoptosis assay using FITC/PI apoptosis kit (Life Technologies, USA). Briefly, osteoblasts were cultivated in medium containing 8 μg/ml LPS for further 48h, followed by washing with pre-cold PBS. After that, osteoblasts were resuspended in binding buffer, followed by staining with PI and Annexin-V for 20 min in dark. Finally, flow cytometry was performed to analyze cell apoptosis
Statistical analysis
Unpaired t test was used to compare OS and control groups. Paired t test was used to compare two time points. ANOVA Tukey’s test was used to compare multiple independent groups. Pearson’s correlation coefficient was used to analyze correlations. p<0.05 was statistically significant.