Research subjects
This study was approved by the Ethics Committee of Shanghai Yangpu Shidong Hospital. All experiments on human subjects were conducted in accordance with the Declaration of Helsinki (http://www.wma.net). Research subjects of this study included 30 periodontitis patients (18 males and 12 females, age range of 34 to 44 years old, mean age of 39.3 ± 2.1 years old) and 30 healthy controls (18 males and 12 females, age range of 33 to 44 years, mean age of 39.2 ± 2.0 years old) who were admitted at aforementioned hospital between June 2017 and June 2019. Patients’ inclusion criteria: 1) newly diagnosed cases; 2) no therapies for any diseases were initiated within 3 months before this study; 3) no other systemic diseases diagnosed; 4) the patients received tooth extraction at aforementioned hospital. Exclusion criteria: 1) recurrent cases; 2) multiple chronic diseases were diagnosed. The 30 patients underwent tooth extraction due to chronic periodontitis, while the 30 healthy controls received extraction during orthodontic treatment. All patients were informed of the experimental principle of this project and signed the informed consent.
Cell culture
Periodontal ligament (PDL) tissues were extracted from one tooth of each patient and healthy controls during tooth extraction. PDL was used to make small pieces and were kept in minimal essential medium supplemented with 0.292 mg/mL glutamine, 100 µM/l ascorbic acid, 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. Insolation of periodontal ligament stem cells (PDLSCs) was performed according to the methods described by Li et al [16].
Vectors and cell transfection
Two cases of periodontitis affected-PDLSCs with average expression of LOXL1-AS was selected to do transfection experiments. LOXL1-AS expression vector was constructed using pcDNA3.1 vector (Invitrogen) as backbone. Small interfering RNAs targeting the human IL-1β gene were designed by the Shanghai GeneChem Co., Ltd., China. The optimal sequence of small interfering RNAs against human IL-1β (5′-TCAAAGGAAAGAATCTATA-3′) was then cloned into the plasmid pGCL-GFP. IL-1β lentivirus shRNA was purchased from Shanghai GeneChem Co., Ltd., China. The negative control lentivirus shRNA was constructed by a similar process (5′-TTCTCCGAACGTGTCACGT-3′). PDLSCs were harvested at 75 - 85% confluence and were counted, followed by the transfection of 10 nM vector into 106 cells using lipofectamine 3000 (Thermo Fisher Scientific). Control (C) cells were untransfected cells. Negative control (NC) cells were transfected with empty pcDNA3.1 vector. The following experiments were performed using cells harvested at 48 h post-transfection.
RNA preparations and RT-qPCR
RNeasy Mini Kit (QIAGEN) was used to extract total RNAs from PDLSCs. LookOut® DNA Erase (Sigma-Aldrich) was used to digest all RNA samples to remove genomic DNAs. In cases of IL-1β treatment, PDLSCs were treated with 0, 10 and 50 ng/ml IL-1β for 48 h before the following RNA extractions. NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific) was applied to measure the concentration of RNA samples. With 1000 ng total RNAs as template, RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific) was used to perform reverse transcriptions to synthesize cDNA. With cDNA samples as template, QuantiTect SYBR Green PCR Kit (Qiagen) was used to prepare all qPCR reaction systems. The expression levels of LOXL1-AS and IL-1β mRNA were measured with GAPDH as endogenous control. All PCR reactions were repeated 3 times. Fold changes of gene expression levels of LOXL1-AS and IL-1β mRNA were calculated using 2-∆∆CT method. The primers sequences were as follows: forward: 5’-TTCCCATTTACCTGCCCGAAG-3’ (forward) and 5’-GTCAGCAAACACATGGCAAC-3’ (reverse) for LOXL1‐AS; 5’-ATAAGCCCACTCTACACCT-3’ (forward) and 5’-ATTGGCCCTGAAAGGAGAGA-3’ (reverse) for IL-1β; 5’- ATGGGTGTGAACCATGAGAA-3’ (forward) and 5’- GTGCTAAGCAGTTGGTGGTG-3’ (reverse) for GAPDH.Cell proliferation assay
PDLSCs were harvested at 48 h post-transfection and CCK-8 assay was performed to analyze the effects of transfection or infection on the proliferation of PDLSCs. Each well of one 96-well plate was filled with 104 PDLSCs in 0.1 ml cell suspension, followed by cell culture at 37 °C in a 5 % CO2 incubator. Three replicate wells were set for each experiment. CCK-8 solution (Sigma-Aldrich) was added into each well to reach the final concentration of 10% at 4 h before the end of cell culture. Cells were harvested every 24 h until 96 h. OD values of cell culture medium were measured at 450 nm.
Western blots analysis
PDLSCs were harvested at 48 h post-transfection and RIPA solution (Sigma-Aldrich) was used to resuspend cell pellets containing 105 PDLSCs to extract total proteins. BCA assay (Sigma-Aldrich) was performed to measure protein concentrations. RNA samples were incubated in boiling water for 10 min to reach protein denaturation. Electrophoresis was then conducted using 10 % SDS-PAGE gel to separate protein molecules, followed by gel transfer to PVDF membranes. After blocking in TBST containing 5% skim milk, the membranes were incubated with rabbit primary antibodies of GAPDH (ab9845, Abcam) and IL-1β (ab9722, Abcam) at 4 °C for 18 h, followed by incubation with secondary antibody of HRP Goat Anti-Rabbit (IgG) (ab6721, Abcam) at 24 °C for 2 h. Signals were produced using ECL (Sigma-Aldrich, USA) and data were processed using Quantity One software.
Statistical analysis
Three biological replicates were included in each experiment. Mean values of 3 replicates were calculated and were used for all data analysis. Unpaired t-test was used to explore differences between two groups. Differences among multiple groups were explored by ANOVA (one-way), followed by Tukey’s post-hoc test. Correlations were analyzed by Pearson’s correlation coefficient. P < 0.05 was considered as statistically significant.