Ethical declaration
All animal experiments were conducted under the protocols approved by and in accordance with the guidelines of the Institute Animal Care and Use Committee of the University (approval number: GY2018-096).
Induction of Pulpal Lesion on Mice
In this study, Pulpitis model was established as our previous work described [15]. In brief, C57BL/6 mice were acquired from the Guangdong Medical Laboratory Animal Center. Exposed pulp group (n=10) mice were anesthetized with intra-abdominal injection of pentobarbital sodium (50mg/kg). Their dental pulp of maxillary first molars were opened on the occlusal surface. This procedure was carried out with #1/4 dental round diamond burs on high-speed handpieces and operated under a surgical microscope. Mice without treatment were set to control group (n=10). Animals were sacrificed at time points of 1, 6, 12 and 24 hours after operation. Five samples were used for histopathological and immunohistochemical analysis, while the other 5 maxillae sections were prepared for analysis of IL-6, IL-1β and miR-155. Study of gene knockout animals was set with fifty miR-155-/- mice, which were purchased from the Jackson Laboratory, USA.
Cell culture and treatment
Mice odontoblast-like cell lines MDPC-23 cells (Cell Bio, Shanghai, China) were cultivated in Dulbecco's Modified Eagle's Medium (DMEM, Sigma, MO, USA) containing 10% fetus bovine serum (FBS) and 1% of penicillin/streptomycin mixture at 37ºC and 5% CO2. We observed the cells growth to cover 70-80% of the flask surface as an indication for LPS activation. The original medium was discarded and additional 1μg/mL LPS (Sigma) was used to stimulate cell inflammation for 3 hours, 6 hours, 12 hours, and 24 hours in different groups.
Cell transfection
MDPC-23 cells were transfected with miR-155 mimic, miR-155 inhibitor, miRNA negative control (miR-NC) and siRNA (RiboBio, Guangzhou, China) for 48hours using the riboFECT CP transfection reagent (RiboBio) following the manufacturer’s protocol. After transfection, the stably transfected cells were treated with medium containing 1 μg/mL LPS for 12 hours [16].
Histopathological Analysis
The murine maxillae were dissected and fixed in 4% phosphate-buffered paraformaldehyde for 48 hours at room temperature. Then the samples were rinsed with PBS and decalcified with ethylene diamine tetra acetic acid (EDTA) solution for two weeks. After that, the samples were embedded in paraffin and cut into 4mm thick serial sections. Representational sections of each group were stained with hematoxylin-eosin (H&E) for further analysis.
Immunohistochemical Analysis
Animal samples were performed to immunohistochemical analysis according to a previously described protocol [16]. The primary antibodies were anti-PI3K p85 (1:300, Abcam, MA, USA), anti-AKT (1:300, Abcam) and SHIP1(1:300, Abcam), following by biotinylated secondary antibody. In each section, we chose three random different visions (× 400 magnification). Acquired photographs were analyzed with Image Pro Plus 6.0 software.
RNA isolation and quantitative real time-PCR (qRT-PCR)
Total RNA was extracted from cells or tissues using the Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s protocol. Conversion into cDNA were performed with PrimeScript RT Master Mix and Mir-X miRNA First-Strand Synthesis Kit (Takara, Dalian, China) for mRNA and miRNA determination respectively. qRT-PCR analysis for mRNA and miRNA were conducted by using SYBR Premix Ex Taq II solution (Takara) with relative quantification method. The mRNA level of U6 and GAPDH were used as normalization controls.
The following are the primer sequences: miR-155: forward ACACTCCAGCTGGGTTAATGCTAATTGTG and reverse CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACCCCTAT; IL-6: forward TCACAGAAGGAGTGGCTAAGGACC and reverse ACGCACTAGGTTTGCCGAGTAGAT; IL-1β: forward ACGGCCTTCCCTACTTC and reverse GCTGGACTGTTTCTAATGC; SHIP1: forward GTACAACTTGCCGTCCTGGT and reverse TGTGACTCCTGCTTCAAACG; GADPH: forward AGAAGGTGGTGAAGCAGGCATC and reverse AGAAGGTGGTGAAGCAGGCATC; U6: forward CTCGCTTCGGCAGCACA and reverse AACGCTTCACGAATTTGCGT.
Western Blot
The proteins were extracted with RIPA lysis buffer (Thermo Fisher, IL, USA). Protein concentration was determined with bicinchoninic acid (BCA) quantitative detection reagent kit following manufacturer’s protocol (Epizyme Biotech, Shanghai, China). Western Blot was carried out in following steps: Proteins were separated with 10% SDS-PAGE electrophoresis and transferred on PVDF membranes, following manufacturer’s protocol (Epizyme). We then probed proteins with primary antibodies: anti-PI3K p85 (1:2000, Abcam), anti-AKT (1:2000, Abcam), anti-SHIP1 (1:1000, Abcam, Cambridge, UK) and GAPDH (1:6000, Abcam) in 4℃ overnight. Then the membranes were incubated with secondary antibody of goat anti-mouse IgG (Proteintech, IL, USA) carrying alkaline phosphatase for 1h at room temperature. We detected the membranes using BCIP/NBT kit. (Beyotime, Shanghai, China) following the protocol. Quantification was calculated with Image Pro Plus 6.0 software.
Luciferase reporter assay
The SHIP1 3′UTR target site and the luciferase reporter constructs carrying a putative miR-155-binding site were amplified by PCR (Promega, WI, USA). Cells were cultured on 48-well plates and co-transfected with psiCHECK-2 luciferase reporter plasmid, miR-155 control were transfected with double luciferase reporter vector plasmid. Plates were incubated in a CO2 incubator at 37°C for 24~96 hours. Dual Luciferase Reporter Assay (Promega) was performed to calculate relative luciferase activity after 24 h transfection following manufacturer’s protocol.
In brief, the pre-configured Luciferase Assay Reagent was added to the samples, we measured the luciferase reaction intensity (hLuc fluorescence value) and the internal reference Renilla fluorescence value (hRluc fluorescence value). Every sample was performed with three times repeat. The hLuc/hRluc ratio and the ratio of the control wells were statistically analyzed for verification of the accuracy of miRNAs' target sites. This procedure was set to determine whether the predicted binding site mutations will change the effects of miRNAs.
Enzyme-linked immunosorbent assay (ELISA)
The culture medium collected from the cells of different groups were measured directly by ELISA to quantify the production of IL-1β and IL-6 according to the manufacturer’s protocol (R&D Systems, MN, USA). The results were analyzed with an enzyme - labelled meter (Thermo Fisher Scientific, MA, USA)
Statistical analysis
Data is presented as mean ± standard deviation (SD) for at least three independent experiments. Differences between groups were subjected to t-test or one-way ANOVA using SPSS statistics 16.0 (SPSS Inc, Chicago, IL, USA). The values of p< 0.05 was considered to be statistically significant.