Gingival tissues collection and cell culture
Gingival tissues were obtained from patients in the clinic. HGFs were isolated from healthy gingival tissues collected from teeth during tooth extraction. The obtained gingival tissue was rinsed with PBS solution at least three times. After cutting up the tissues in EP tubes, they were digested with Type I collagenase (Sigma, USA) in a 37 ℃ water bath for 30 min and were shaken every five minutes. Followed by centrifugation, precipitated tissue blocks were evenly spread on Petri dish for 12 h before adding culture medium. Cell culture medium of HGFs was composed of 89% (v/v) α-MEM (Gbico, Shanghai, China), 10% (v/v) fetal bovine serum (FBS, VivaCell, ShangHai, China), and 1% (v/v) penicillin/streptomycin (Hyclone, USA). Human embryonic kidney (293T) were cultured in DMEM (Gbico, Shanghai, China) with 10% (v/v) FBS (VivaCell, Shanghai, China) and 1% (v/v) penicillin/streptomycin (Hyclone, USA). Both 293T cells and HGFs were cultured at 37 ℃ with 21% O2.
The establishment of cell inflammation model
For establishing a model of inflammatory HGFs, a complete culture medium containing a 1 ng/mL concentration of lipopolysaccharide (LPS) (Sigma, USA) was used as the inflammatory medium. The inflammatory medium was added to the 6-well plate when cell density reaches about 60%. Before harvesting, HGFs were cultured with inflammatory medium for 1, 2, and 3 days.
The silencing of LINC01126 in HGFs
Three diffierent siRNAs (si–LINC01126_001, si–LINC01126_002 and si–LINC01126_003) of LINC01126 were designed and synthesized by Guangzhou RiboBio Co.,LTD (Table S1). Transfection experiments were performed in 6-well plate when cell density reached 80%. HGFs were transfected with siRNA or si-NC at a concentration of 50 nM using lipo3000 (Thermo Fisher Scientific, USA). HGFs were harvested 24 h later after transfection of synthesized sequences. LINC01126 silencing was verified using quantitative real-time PCR.
Cell transfection
HGFs were seeded in 6-well plates with 2×105 cells per well. HGFs were transfected with small interfering RNA from negative control (si-NC) or screened small interfering RNA from LINC01126 (si-LINC01126). MiR-655-3p mimics or mimics-NC (RiboBio, Shanghai, China) were transfected in the same manner with a concentration of 50 nM. And complete medium was substituted by an inflammatory medium 24 h later after cell transfection. Cell samples were harvested at the appointed time.
Cell viability assay
Cell Counting Kit-8 (CCK-8) kit (Bioss, Beijing, China) was utilized to perform cell viability assay. HGFs were seeded in 96-well plates with 103 cells per well. Following cell attachment, the selected si-LINC01126 sequence was utilized to transfect HGFs with a concentration of 50 nM. Cell viability was measured at 0, 1, 3, and 5 days after transfection, and OD values were measured at 450 nm using a SpectraMax ID5 Multi-Mode Microplate Reader (Molecular Devices, USA).
Wound healing assay
2×105 cells were seeded in 6-well plates. Briefly, simulated wounds were created with a 200 µ sterile tip on a single layer of HGFs when cells reached approximately 90% confluency. Following that, HGFs were rinsed with sterile PBS three times to rinse off the scraped cells. Finally, HGFs were cultured with fresh opti-MEM medium (Gbico, Shanghai, China). After 24 h, the healing of scratches was observed and photographed for analysis.
Flow cytometry
When the density reached approximately 80%, cell transfection was performed as before. After 24 h of si-LINC01126 transfection, HGFs were cultured with inflammatory medium for 48 h. HGFs were then collected for apoptosis detection through flow cytometry. All operations were performed following manufacturer’s instructions.
Western blot (WB) assay
For the cell samples, the cells were lysed on ice by RIPA containing 1% PMSF (Beyotime, Shanghai, China). For the tissue samples, the tissues were grinded in Freezer Mixer (Jingxin, Shanghai, China) with RIPA. After protein transmembrane and blocking, the PVDF membranes were incubated with primary antibodies overnight, including GAPDH (Zenbio, Chengdu, China, #200306-7E4), IL-6 (Zenbio, Chengdu, China, #347023), STAT3 (ABclonal, Wuhan, China, #A1192), phospho-STAT3 (Zenbio, Chengdu, China, #310019), JAK2 (ABclonal, Wuhan, China, #A11497), and phospho-JAK2 (ABclonal, Wuhan, China, #AP0631). The immunoblots were detected by chemiluminescence (Beyotime, Shanghai, China).
Quantitative real-time PCR (qRT-PCR) analysis
The purity and concentration of total RNA were detected by NanoDrop-2000 after the extraction of total RNA using RNAiso plus (Takara, Japan). Then, reverse transcription of mRNA and microRNA was performed by using PrimeScript™ Master Mix (Takara, Japan) and miRNA First Strand cDNA Synthesis (Tailing Reaction) (Sangon biotech, China), respectively. GoTaq® qPCR Master Mix (Promega, America) was used for qPCR according to the experiment instruction. The expression level was evaluated using a method of 2−ΔΔCT. The primers of the target genes were shown in Table S2.
Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization Kit specific for LINC01126 was designed and synthesized by Guangzhou RiboBio Co., LTD. HGFs were washed with PBS for 5 min and fixed in 4% paraformaldehyde for 10 min when their cell density reached about 60–70%. HGFs were then permeabilized by sterile PBS containing 0.25% Triton X-100 for 10 min under 4 ℃, and rinsed with sterile PBS at room temperature. Following that, pre-hybridization was performed under 37 ℃ for 30 min. After that, the probes for LINC01126, U6, and 18S were added into a hybridization solution, and the cells were cultured under 37 ℃ overnight in the dark. Finally, the nuclei were stained with DAPI, and photographs were taken using a fluorescence microscope. U6 and 18S were acted as internal references for nucleus and cytoplasm, respectively.
Dual-luciferase reporter assay
293T cells were used in this experiment. The wide or mutant type of 3’-UTR of LINC01126 and IL-6 was cloned into pmiR-RB-Report™ vector (Figure S1) (Ribo, China), a vector specifically designed for detecting the combining capacity of miRNA. The plasmids, mimics NC or miR-655-3p mimics, were transfected into 293T cells using Lipo3000 reagent (Thermo Fisher Scientific, USA). Plasmids and mimics NC or miR-655-3p mimics were transfected at 2.5 µg/mL and 50 nM, respectively. The luciferase activity was measured by using Dual-Luciferase Reporter Assay System (Promega, USA) after 48 h. Finally, the activity of firefly luciferase gene (hRluc) and renilla luciferase (hLuc+) was detected. The ratio of hRluc activity to hLuc + activity is the relative luciferase activity.
H&E staining and Immunohistochemistry staining
Periodontitis tissues and healthy controls were obtained from department of oral and maxillofacial surgery of Stomatological Hospital affiliated to Chongqing Medical University. After fixed in 4% paraformaldehyde for 24 h, gradient dehydration and paraffin embedding were performed. Following that, serial sections were performed and used for H&E and IHC staining. Hematoxylin-Eosin/HE Staining Kit (Solarbio, Beijing, China) was utilized for H&E staining. Immunohistochemistry (IHC) staining was conducted with antibodies specific for IL-6 (1:200, Zenbio, Chengdu, China, #347023), phospho-STAT3 (1:100, Zenbio, Chengdu, China, #310019), STAT3 (1:200, ABclonal, Wuhan, China, #A11497), phospho-JAK2 (1:200, ABclonal, Wuhan, China, #AP0631), and JAK2 (1:100, ABclonal, Wuhan, China, #A11497). H&E and IHC staining were performed according to the protocol.
Statistical analysis
Experimental results were analyzed and processed using CytExpert, Image Lab, GraphPad Prism (8.0), Adobe Photoshop and Image J. One-way ANOVA or the independent-sample T-test was used to compare the differences between groups. Data were presented as mean ± SEM compared with the control groups. And p < 0.05 was regarded as statistically significant. All experiments were repeated at least three times.