The patients
This experiment was carried out in the hospital in China from September 2016 to January 2019. 49 patients with dry eye disease and 53 healthy people were admitted in the study. The basic information was recorded and the study was approved by the Tianjin Medical University General Hospital Ethics Committee and all patients had signed informed consent. The blood of patients with dry eye disease were collected as model group, then 53 healthy people’s blood were collected as control group. The expression of miRNA-146a-5p was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).
Bioinformatics Analysis
The target genes of miRNA-146a-5p were predicted by Targetscan [10] (http://www.targetscan.org/vert_72/), miRDB [11] (http://mirdb.org/), miRWalk [12] (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/), and PicTar [13] (https://pictar.mdc-berlin.de/), the results were admitted into draw venn diagram [14] and the results belonged to the 4 databases were selected. Then we get the sequence of miRNA-146a-5p and the target gene and look for the combining site to identify the relationship between them. Then we look for the relative miRNAs of the target gene in dry eye disease and visualized by Cytoscape3.6.1 [15].
Cell Culture
Adult SD rats (200–200 g) were purchased from the Shanghai xipul-bikai laboratory animal co., LTD. The study adheres to Association for Research in Vision and Ophthalmology statement for the use of animals in Ophthalmology and vision research was approved by the Ethics Committee of Tianjin Medical University General Hospital. The rats were anesthetized by 1% pentobarbital sodium and sacrificed, and then soaked in alcohol with a volume fraction of 75% for 15 min. The rat lacrimal glands were removed with ophthalmic scissors and washed 3 times with phosphate buffered saline (PBS). The upper fascia and surrounding yellow adipose tissue were peeled off, and the dendritic blood vessels and fibrous connective tissue were removed. After rinsing in the original solution, the tissue was cut into 1 ~ 2 mm3 pieces with an ophthalmic scissors. The prepared 2 g·L-1 type II collagenase application solution was added and shaken and digested for 25 min at 37 °C in an electrothermal oven to obtain a gland cell mass. The culture solution was added to terminate the digestion, and the gland cell pellet was thoroughly blown into a single cell suspension. Filter through a 200-mesh nylon mesh and centrifuge at 800 r·min− 1 for 5 min. The supernatant was added to a little D-Hank solution and centrifuged again at 800 r·min-1. The precipitate was taken, and the culture solution was added and pipetted into a single cell suspension, which was inoculated into a culture flask [16].
The cells were inoculated into a 50 mL plastic flask at 3 × 105 cm− 2, and the lacrimal gland epithelial cells were purified by repeated adherence for 3 times (15 to 20 min each), and cultured at 37 °C in a volume fraction of 5% CO2 incubator. After 36 h, the first half was changed, and after 48 h, the whole amount was changed once. Change the liquid once every 4 days. The first passage was performed after 10 days.
The concentration was 100 µmol / L H2O2 and the culture was continued for 60 min to induce cell death and recorded as a model group.
Luciferase Reporter Assay
Luciferase reporter assay was performed to identify the relationship between miRNA-146a-5p and IRAK1. In brief, cells at 80% confluence were co-transfected with wild-type or mutant IRAK1 3’-UTR reporters together with miRNA-146a-5p or negative control using lipo 2000 (Invitrogen, Carlsbad, CA, USA). The plasmid (Promega, Madison, WI, USA) encoding luciferase was used to control for transfection efficiency. Cells were lysed 24 h after transfection and tested for luciferase activities using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), according to the manufacturer's instructions.
Rt-pcr Analysis
The total RNA was isolated from mice cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and converted cDNA by OneScript Reverse Transcriptase cDNA Synthesis Kit (TaKaRa, Dalian, China). The bulge-loop TM miRNA reverse primer was used to replace Olige (dT). 25 µL Dream Taq PCR Master Mix (TaKaRa, Dalian, China), 1.5 µL primer (Ribobio, Guangzhou, China), 2 µL cDNA and 20 µL water nuclease free in amplification reaction mixture (50 µL), and the PCR condition were as follows: 95 °C (2 min, a cycle), 95 °C (30 s), 58 °C (30 s), 72 °C (1 min), 35 cycles in total. Finally, 72 °C (10 min, a cycle) [17]. GADPH served as the control of APP and U6 snRNA (U6) served as control of miR-146a-5p (El Fatimy et al., 2018), the primer sequences were as follows:
IRAK1
5’-ATCAGGCTTTTTCCCAGGCT-3’; 5’-GCACACTATGAGAACTTCCAAGC-3’
TNF-α
5’-ATAAGAGCAAGGCAGTGGG-3’; 5’-TCCAGCAGACTCAATACACA-3’
IL-6
5’-AGCCAGAGTCCTTCAGAGAG-3’;
5’-TCCTTAGCCACTCCTTCTGT-3’
miRNA-146a-5p
5’-CTGCCGCTGAGAACTGAATT-3’; 5’-CAGAGCAGGGTCCGAGGTA-3’
GADPH
5’-CCATGTTCGTCATGGGTGTGAACCA-3’; 5’-GCCAGTAGAGGCAGGGATGATGTTC-3’
calprotectin
5’-CCGGATCCAC-TAAGCTGGAAGATCACCTGGAGG-3’; 5’-CCAAGCTTTACTCTTTGTGGATATCTAT-GTGGGCTG-3’
Western Blot
Cells were lysed in radioimmuno precipitation assay buffer with the Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA), separated in sodium dodecyl sulfate polyacrylamide gels, and transferred to a polyvinylidene fluoride membrane. The membrane was incubated with anti-IRAK1, TNF-α, IL-6, CBP and anti-β-actin (Abcam, Cambridge, MA, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h. Bands were visualized with electrochemiluminescence (ECL).
Statistical Analyses
Mean ± standard deviation was used to present the data. Statistical comparisons were carried out using the Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.