All protocols for treating periodontal ligament tissues were performed in accordance with relevant guidelines and regulations. This study was approved by the Research Ethics Committee of Shandong University (No. G201401601). Informed consent was obtained from the donors and their parents before treatment. The research methods of animal experiments in this study are consistent with routine, and the experimental design was confirmed with the principles of animal protection. This study was approved by the Committee on the Ethics of Animal Experiments of Shandong University (No. 20190503).
Cell isolation, culture, and identification
In this study, 46 premolars without caries or periodontitis from 33 donors aged 12-20 years were obtain for orthodontic purposes. The isolation and culture of PDLSCs were performed as previously reported (31, 32). The premolars were extracted at the Department of Oral Maxillofacial Surgery, School of Stomatology, Shandong University (Jinan, China). We used flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) to detect the cell surface markers including cluster of differentiation 31 (CD31), CD45, CD146, and Stro-1 (33).
Application of mechanical force in vitro
Mechanical force was applied using the Flexcell-FX-6000-Tension System (Flexcell International Corporation, Burlington, NC USA). PDLSCs were seeded onto flexcell Amino silicone-bottomed plates 6 well cell culture plates coated with collagen I solution (Collagen I, rat Tail, Corning, NY, USA) at a density of 2.0×106 cells per well. After the density reached ~80% confluence, the cells were serum deprived (2% serum) for 24 h before stretching. We imposed 10% stretch at 0.5 Hz. The control group was cultured in same silicone bottomed plates and the same culture environment without stretching.
Total RNA was extracted from the non-stretched and stretched groups of cells using RNAiso TM Plus (Takara, Shiga, Japan) according to the manufacturer’s protocols. Strand-specific cDNA libraries were constructed following a previously described protocol (34) and were sequenced on the Illumina HiSeq 2000/2500 sequencer (LC Biotech, Guangzhou, China). Sequencing was done according to the HiSeq 2000 User Guide with paired‐end program.
RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was isolated from PDLSCs using RNAiso TM Plus (Takara) according to the manufacturer’s protocol. The extracted total RNA was reverse-transcribed using the Prime Script RT Reagent Kit with gDNA Eraser (Takara). Relative RNA level was detected using the LightCycler-480 system (Roche Diagnostics GmbH, Mannheim, Germany) and TB Green Premix Ex Taq II (Takara). GAPDH and U6 were used as internal controls to quantify and normalize the results. The PCR reaction conditions were as follows: 95 ℃ for 30 s, then 55 cycles of 95 ℃ for 10s, 60 ℃ for 30 s. The 2-ΔΔCT value was used for comparative quantitation. The sequences of primers are shown in Table 1. All PCR processes were performed in triplicate.
SNHG8 and EZH2 knockdown was conducted via lentiviral transfection (Genechem, Shanghai, China). Regarding SNHG8, two lentiviral constructs designated sh-SNHG8-1# and sh-SNHG8-2# were generated based on different regions of the human SNHG8 sequence (NCBI Gene ID: 100093630). For EZH2 knockdown, we constructed sh-EZH2 based on previous researches (35). The negative control containing a nonspecific RNA oligonucleotide was constructed as previously described (36, 37). Cells were observed under a fluorescence microscope and an inverted phase contrast microscope (TH4-200; Olympus, Tokyo, Japan).
Induction of osteogenic and adipogenic differentiation.
We cultured hPDLSCs in osteogenic-inducing medium containing 100 μM ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 2 mM β-glycerophosphate (Sigma), 10 nM dexamethasone and 0.1 mg/mL penicillin-streptomycin (Biosharp, Hefei, China). Alkaline phosphatase (ALP) staining (Solarbio, Beijing, China) and 1% Alizarin Red S (Sigma) staining were used to evaluate the effect of osteogenic induction. For adipogenic-induction, we cultured hPDLSCs in medium containing of 1 μM dexamethasone, 200 μM indomethacin (Sigma), 10 μM insulin (Sigma), 0.5 mm isobutyl methylxanthine (Sigma), and 0.1 mg/mL Penicillin-Streptomycin Solution (Biosharp) for two weeks to achieve the adipogenic differentiation. Oil Red O staining (Solarbio) was used to identify lipid-laden fat cells.
Western blot analysis
PDLSCs were collected and lysed in RIPA reagent (Solarbio) containing 1% PMSF. After heating, the protein samples were separated in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to a 0.2 μm polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). After blocking in 5% skimmed milk, membranes were incubated overnight at 4 ℃ with the following primary antibodies: rabbit anti-RUNX2 (1:1000, Lot # 8486S; Cell Signaling Technology, MA, USA), rabbit anti-ALP (1:1000, 11187-1-AP; Proteintech, Rosemont, IL, USA), rabbit anti-EZH2 (1:500, Lot # 2477979; Millipore), rabbit anti-embryonic ectoderm development (EED) (1:500, 16818-1-AP; Proteintech), rabbit anti-suppressor of zeste 12 (SUZ12) (1:500, 20366-1-AP; Proteintech) and rabbit anti-GAPDH (1:2000, 10494-1-AP, Proteintech, USA). After washing in Tris-buffered saline solution (TBS-T, Solarbio) with Tween-20, the membranes were incubated at room temperature for 1 h with peroxidase-conjugated anti-rabbit IgG (SA00001-2; Proteintech). ECL chromogenic substrate (Millipore) was used to detect the immunoreactive bands, and ImageJ software (National Institutes of Health, Bethesda, USA) was used to quantify the densitometry using GAPDH as the control.
Construction of tooth movement model in vivo
Twenty five 6-week-old male wistar rats (Charles River, Beijing, China) were used for the construction of tooth movement model in vivo. All rats were fostered 12/12 h day/night cycle to simulate common environment, and were adaptively fed for 3 days before experiment. The maxillary left first molar and the upper incisors were ligatured by 0.25 mm stainless steel with a nickel-titanium closed-coil spring (TOMY, Japan) in between. The nickel-titanium spring provide a force of approximate 20 g. In order to fix the structure, we drilled grooves at the left upper incisor tooth cervix, and fixed with light-curing resin. To avoid the individual differences of rats, each rat was performed tooth movement operation on dentition of the left maxillary, and the right side was not operated as a self-control. After retained the structure for 3 days, 7 days, 14 days, and 21 days, the periodontal tissues (include alveolar bone and periodontal ligament) were isolated for qRT-PCR.
Ectopic osteogenesis in vivo
The osteogenic differentiation potential of PDLSCs with different SNHG8 expression levels were tested by in vivo ectopic bone formation analysis. Briefly, untransfected hPDLSCs (Control group), hPDLSCs transfected with empty plasmids (sh-NC group), and hPDLSCs transfected with effective lentivirus (sh-SNHG8 group) were transferred subcutaneously to 5-week-old nude mice (Charles River) with osteo-inductive calcium phosphate bioceramic material (TH/P 1020, Sichuan University, China). After 10 weeks of fostering, the nude mice were executed and the ectopic bone formation under the skin was harvested. After decalcification treatment, HE staining (Solarbio), Masson’s trichrome staining (LEAGENE, Beijing, China) and SafraninO-staining (Solarbio) were performed according to the manufacturer’s instructions.
Isolation of nuclear and cytoplasmic RNA
The nucleus and cytoplasm of PDLSCs were separated using the Ambion® PARIS™ Kit (Life Technologies, Frederick, MD, USA) according to the manufacturer’s instructions. We lysed approximately 5.0×106 cells in ice-cold cell fractionation buffer, and separated the cytoplasmic fraction from the nuclear fraction by low-speed centrifugation. Then we lysed the nuclear fraction in cell disruption buffer. Two kinds of fraction were mixed with lysis/binding solution separately, washed with washing solution, and eluted with preheated elution solution. For qRT-PCR, GAPDH was used as the control for the nuclear fractions and U6 was the control for the cytoplasmic fractions.
RNA fluorescence in situ hybridization
The fluorescence in situ hybridization (FISH) assay was performed using a Fluorescence In Situ Hybridization Kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. After fixed in 4% paraformaldehyde, PDLSCs were washed with phosphate-buffered saline containing 0.5% Trition X-100 to increase cell permeability. We observed PDLSCs with an inverted phase contrast microscope (DMi8; Leica, Germany) after incubating PDLSCs overnight at 37 ℃ with hybridization solution containing the SNHG8, U6 and 18S probes. The excitation wavelengths were 405 nm for DAPI and 488 nm for the probes.
All statistical calculations were performed using SPSS19.0 (SPSS Inc., Chicago, IL, USA). All data are normally distributed and presented as the mean±standard deviation of three to five independent samples. Differences between the results obtained from various experimental groups were analyzed by the Student’s t-test or one-way analysis of variance. P < 0.05 was considered statistically significant.