Sample collection and cells culture
The periodontal ligament (PDL) was collected from patients in the Shanghai East Hospital, Shanghai Jiao Tong University School of Medicine. The first or the second premolar teeth were collected from healthy patients ranging from 18 to 25 years old for orthodontic purposes. This study was approved by the ethics committee of Shanghai East Hospital Affiliated with Tongji University. All patients gave informed consent. All experiments were performed in accordance with approved guidelines and regulations from Shanghai East Hospital Affiliated with Tongji University of Medicine Ethics Committee.
The extracted teeth were cultured in 90% Dulbecco’s Modified Eagle Medium (DMEM) with 100 u/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS). The PDL tissue was obtained by scraping the root surface of teeth after washing 6–8 times with PBS (containing penicillin/streptomycin). The centrifugation of 5 min in 1000 r/min was performed for PDL tissue which was cut into small pieces of 1 mm3. Then, the PDL tissue was digested in 3mg/ml collagenase I (Roche, USA) and dispersed for 45 min at 37 °C. Then, added an equal volume of medium to terminate the digestion. The solution was centrifuge d at 1000 r/min for 5 min, and the supernatant was discarded. Next, the tissues were resuspended in 1 ml of medium and inoculated into 6-well plates and cultured in the environment of 37 °C and 5% CO2. After every 2 or 3 days, the medium would be changed until the cells appeared from the edge of the tissue after 3–8 days. Then the cells were cultured as Zhang et al. reported and four generations of poly-clonal PDLSCs were used in this study .
Induction of osteogenic differentiation in PDLSCs
PDLSCs were transferred into a 6 well plate, and per well has the density of 1×105 cells. Osteogenic differentiation induction medium (50 mg/mL of ascorbic acid (Sigma, St. Louis, MO, USA), 10 mmol/L of beta-glycerophosphate (BGP, Sigma, St. Louis, MO, USA) and 10 ng/mL of dexamethasone diluted in 10% FBSa-MEM) was given when cells reached 80% confluence. After every 3 days, the conditioned medium was changed. Cells cultured with standard culturing medium were designated as control group. After 21 days of culturing, the differentiated PDLSCs were formed.
The differential expressions of lncRNAs were identified by microarray between undifferentiated and differentiated PDLSCs. A total of six samples (three undifferentiated PDLSCs and three differentiated PDLSCs from six individuals), which each contained about 1 × 106 cells, were prepared, and TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA. And the RNA was amplified and transcribed into fluorescent cDNA. Human LncRNA Array was used to hybridize with labeled samples. Differentially expressed lncRNAs were identified through Volcano Plot filtering with the threshold of fold change > 2 and P < 0.05.
Quantitative real-time polymerase chain reaction (qRT-PCR)
For the extraction of total RNA, TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used. For quantitative analysis of RNA expression, 1 μg of total RNA was used to synthesize complementary DNA (cDNA) using PrimeScript RT reagent kit (TaKaRa), and the corresponding cDNA was used for quantitative PCR using SYBR green Master MIX (Applied Biosystem) and the ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The PCR program, comprising 1 cycle of 10 min at 95℃, 40 cycles of 30 s at 95℃, 30 s at 56℃, and 1 min at 72℃, and 1 cycle of 7 min at 72℃. For the endogenous control, GAPDH was used. The primers were shown in Table 1. The triple analysis was performed for each sample. Relative gene expression was calculated using the 2-ΔΔCt method.
Plasmid construction and cell transfection
The pcDNA3.1-DANCR and pCMV-sh-DANCR, as well as negative control plasmid were purchased in GenePharma (Shanghai, China). And a total of 3 × 105 cells were transferred into 6-well plates, after 24 hours, the negative control plasmid or pcDNA3.1-DANCR or pCMV-sh-DANCR were stably transfected into PDLSCs using Lipofectamine 2000 (Invitrogen, Canada).
The effect of lncRNA DANCR on cell proliferation was analyzed using an MTT assay. After 24 h of transfection in 100 mm2 culture dish, PDLSCs were digested and seeded in 96-well culture plate for another 0, 24, 48, 72 h. Then MTT solution was added and performed as Sun et al. reported .
Alkaline phosphatase (ALP) activity and ALP staining assay
PDLSCs were separately transferred into 96-well plates, and per well has a density of 1×105 cells. After 21 days of induction, ALP activity was measured using the ALP assay kit (Jiancheng Technology Co., Ltd., Nanjing, China). Cellular ALP was visualized by using Alkaline Phosphatase Color Development Kit (Beyotime, Shanghai, China) and the methodology is the same as the manufacturer’s protocol.
Alizarin red staining and quantification
PDLSCs were separately transferred into 24-well plates, and per well has a density of 1×105 cells. After transfection, the cells were then cultured for additional 21 days. We changed the induction medium every 3 days. Finally, the 15 min of alizarin red staining measurement was performed by using 40 mM Alizarin Red S (pH 4.4; Jiancheng Technology Co., Ltd.) at room temperature. After washing in PBS, the cells were observed using an inverted microscope. To measure the concentration of calcium deposits, the Alizarin Red dye in the PDLSCs was extracted with 400 µl of 10% (w/v) cetylpyridinium chloride in 10 mM sodium phosphate solution for 10 min, and then quantified on a UV-Vis spectrometer at 562 nm.
All the statistical analyses were performed by GraphPad Prism (vesion6.0) software. The form of means ± standard deviation (SD) was used for the results. Student t-test (paired t-test) was used to compare means between two different groups. And multiple comparisons were performed using the one-way analysis of variance (ANOVA) following the Tukey test. P values < 0.05 were considered as statistically significant.