PDL tissues collection
A total of 30 patients with periodontitis and 30 healthy control participated in the study. This study protocol was approved by the Ethics Committee of Beihua University Affiliated Hospital. Written informed consent was provided prior to the study. All participators were diagnosed with periodontitis or not by Beihua University Affiliated Hospital. None of them had infectious diseases, a history of smoking, and orthognathic surgery. At routine premolar or third molar extractions, the PDL tissues were separated from the middle 1/3 of the dental roots. Partial tissues were stored at -80°C until further use. All methods in the study were carried out in accordance with the Helsinki guidelines and declaration.
Cell culture and osteogenic induction
Other tissues were cut into 1 mm3 pieces and digested by 3mg/ml of collagenase type I (Sigma-Aldrich, USA) and 4 mg/ml of dispase (Corning, USA) at 37°C. The cell suspension was maintained in DMEM (Hyclone, USA) supplemented with 10% FBS (Solarbio, China) and 1% penicillin/streptomycin (Solarbio, China) at 37°C with 5% CO2. The medium was changed every 2-3 days until cell passage to the fifth generation. For osteogenic induction, PDLCs were seeded into 6-well plates until the confluency researched exceed 70%. DMEM supplemented with 10% FBS, 10 mM β-glycerophosphate (Sigma-Aldrich, USA), 50 μg/ml vitamin C (Aladdin, China), and 10 nM dexamethasone (Aladdin, China) was used as an osteogenic-induced medium. Two weeks post-incubation, PDLCs were harvested for testing osteogenic differentiation.
Alkaline phosphatase (ALP) activity analysis
ALP activity was assessed by the ALP Assay Kit (Beyotime, China). Briefly, PDLCs were lysed by lysis buffer and seeded into 96-well plates. The test buffer was added and incubated with cells at 37°C for 10 min. After stopping the reaction, the absorbance was measured at 405nm.
Dual-luciferase reporter assay.
The sequences of ANRIL containing the miR-7 potential binding sites were amplified and inserted into pGL3 vectors (Promega, USA) as ANRIL- WT group. The ANRIL-MUT group was obtained by targeted mutation. MiR-7 mimic and mimic negative control (NC)were purchased from GenePharma (Shanghai, China). HEK-293T cells were seeded into 24-well plates, and co-transfected with ANRIL-WT or ANRIL-MUT and miR-7 mimic or mimic NC using Lipofectamine 2000 (Invitrogen, USA). After 24 h, the relative luciferase activity (firefly activity normalized to Renilla activity) was measured by Dual-luciferase Reporter Assay Kit (Promega, USA).
Cell transfection
shRNA-NC, shRNA-ANRIL, miR-7 inhibitor, and inhibitor-NC were all acquired from GenePharma (Shanghai, China). PDLCs in the logarithmic growth phase were seeded into 6-well plates and transfection process was used Lipofectamine 2000 (Invitrogen, USA). After 48 h, transfection efficiency was detected.
qPCR
Total RNA was isolated from PDLCs by TRIzol reagent (Sigma-Aldrich, USA). After concentration and purity testing, RNA was reverse transcribed into cDNA using LnRcute lncRNA cDNA First Chain Synthetic Kit (Tiangen, China), and miRNA reverse transcription was conducted by miScript II RT Kit (Qiagen, Germany). INRcute lncRNA qCPR Detection Kit (SYBR Green) (Tiangen, China) was performed for qPCR of lncRNA with the following conditions: 95°C for 3min, 40 cycles of 95°C for 5 sec and 60°C for 15 sec. qPCR was used to measure miR-7 level by MicroRNAs qPCR Kit (SYBR Green Method) (Sango, China) with the conditions as 95°C for 30 sec, 95°C for 5 sec and 60°C for 30 sec (40 cycles). The level of mRNA was detected by Real Time One Step RT-qPCR (Tiangen, China) for reverse transcription and qPCR, and the conditions were: 50°C for 30 min, 95°C for 3 min, 40 cycles of 95°C for 15 sec and 60°C for 30 sec. The reaction instrument was ABI PRISM 7500 system (Applied Biosystems, USA). β-actin level was performed as the loading control. The results of relative expression were assessed by the 2−ΔΔCt.
Western blot
The transfected cells were collected, and pre-cooled RIPA lysate (Beyotime, China) was added to extract the total protein. After 10% SDS-PAGE, the protein was transferred to PVDF membranes (Millipore, USA) and blocked with 5% skim milk. Primary antibodies including anti-BMP2, anti-Osterix, anti-osteocalcin (OCN), anti-P65, anti-p-P65, anti-IκBα, and anti-p-IκBα were added and incubated with the membranes at 4°C overnight. After washing the membranes, the secondary antibody was added to incubate at room temperature for 1 h. The protein bands were developed by ECL Western Blotting Substrate (Pierce, USA) and then photographed. The gray analysis was performed by Image J software 1.48U (Bethesda, USA).
Statistical analysis
The results in this study were analyzed by GraphPad Prism 6.0 (GraphPad Software, USA) and presented as mean ± standard deviation (SD). Student’s t-test was used for multiple comparisons between two groups, and one-way ANOVA was used between three or more groups. P<0.05 was deemed significant differences.