1. Clinical samples
Samples of retinal fibrovascular membrane (FVM) were obtained from twenty DR patients who were admitted to Hainan West Central Hospital from September of 2020 to September of 2021. Epiretinal membranes (EM) from twenty patients who underwent vitrectomy for rhegmatogenous retinal detachment without proliferative vitreoretinopathy were assigned to the control group. All specimens were stored in 80˚C until being used. Informed consent was provided by all patients. Approval was obtained from the Ethics Committee of Hainan West Central Hospital.
2. Cell culture and DR cell model
Human retinal microvascular endothelial cells (HRMECs) were provided by Procell (Wuhan, China), which were later cultivated within endothelial cell medium (ECM, Gibco, Grand Island, NY, USA) based on the concentration of 10% fetal bovine serum (FBS, Gibco) under the conditions of 5% CO2 and 37°C. To construct DR cell model, HRMECs were exposed to different glucose concentrations, namely, normal-glucose (5.5 M, Normal), high-glucose (HG, 30 mM) or osmotic control group (Mannitol group, 5.5 mM HG supplemented with 24.5 mM mannitol).
3. Cell transfection
The shRNAs specifically targeting ELAVL1 (sh-ELAVL1) and negative control were provided by GenePharma (Shanghai, China). ELAVL1-overexpressing plasmid (oe-ELAVL1) and PI3Kδ-overexpressing plasmid (oe-PI3Kδ) were constructed by GenePharma (Shanghai, China). The miR-192-5p mimic, miR-192-5p inhibitor and negative controls were bought from GenePharma (Shanghai, China). Cells were seeded in 6-well plates. At 70% confluence, cell transfection was carried out by utilizing Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instruments. 48 h later, cells were harvested for following experiments.
4. Western blotting assay
This work utilized RIPA buffer (Beyotime, Shanghai, China) for extracting total cellular proteins. BCA method (Keygen Biotech, Nanjing, China) was applied in determining protein content. After separating protein aliquots through SDS-PAGE, this study transferred on PVDF membranes (Millipore, MA, USA). Later, 5% defatted milk was later utilized to block membranes for a 1-h period. Blots were cultivated with anti ELAVL1 antibody (#12582, 1:1000, Cell Signaling Technology, Danvers, MA, USA) as well as PI3Kδ antibody (ab1678, 1:1000, Abcam, Cambridge, MA, USA) overnight under the temperature of 4˚C. Subsequently, this study adopted secondary antibody (Cell Signaling Technology) for additional 1-h incubation under ambient temperature. Signals were detected by enhanced chemiluminescence (Millipore). β-actin was used as internal reference.
5. Real-time quantitative PCR (RT-qPCR)
In order to perform the extraction of total cellular and tissue RNAs, TRIzol reagent (Invitrogen) was used. Isolated mRNA was reversed with PrimeScript RT reagent kit (Takara, Dalian, China). Apart from that, the Mir-XTM miRNA First Strand Synthesis Kit (Takara) was applied in miRNA transcription. SYBR Green detection system (Takara) was used to quantify gene expression. The designed primers used in this study were: miR-192-5p F: 5’-GGCGCCTGACCTATGAATTG-3’, R: 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCTGT-3’; ELAVL1 F: 5’-CGCAGAGATTCAGGTTCTCC-3’, R: 5’- CCAAACCCTTTGCACTTGTT-3’; PIK3CD F: 5’-TAAGTTTGAGGGCAGCGAGG-3’, R: 5’-ACCTGCAGCGTGTAGTCTTC-3’;. U6 and GAPDH were employed as the internal control. Results were analyzed with the standard 2−∆∆Ct method.
6. Cell viability assay
The detection of HRMECs cell viability was made by applying Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). After transfection, this work inoculated cells in the 96-well plates (5000 cells/well). Twenty-four hours after seeding, the supplementation of each well was made with 10 µL CCK-8 solution. At 2-h post-incubation, we evaluated cell viability by measuring the absorbance (OD) (Bio Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm.
7. Transwell assay
3×103 cells/well were added into top Transwell chambers (8 µm, Millipore) using serum-free medium, whereas medium that contained 10% FBS was added to bottom chamber. At 24-h post-cultivation, 4% paraformaldehyde (PFA) was utilized to fix migrating cells, followed by staining using the 0.1% crystal violet. Olympus microscope (Tokyo, Japan) was adopted for taking cell images. At the same time, cell number was calculated from 5 random fields.
8. Tube formation assay
Matrigel (50 µL/well, BD Biosciences, Franklin Lakes, NJ) was added to 96-well plates and allowed to polymerize for 60 min at 37°C before seeding HRMECs on Matrigel-coated wells (7 × 103/well). Eight hours after seeding, capillary-like structures were captured using microscopy (Olympus). Tube formation was quantified by selecting five different fields randomly using ImageJ software (NIH, USA).
9. Dual-luciferase reporter assay
This work cloned mutant (MUT) or wild-type (WT) 3'UTR in ELAVL1 to pmirGLO vector (Promega, Madison, WI, USA). Then, HRMECs were subjected to co-transfection using NC mimic or miR-192-5p mimic and vectors carrying ELAVL1-WT or ELAVL1-MUT with Lipofectamine 3000 (Invitrogen). 2 days later, the dual-luciferase reporter assay system (Promega) was used for making the measurement of luciferase activities.
10. RNA immunoprecipitation (RIP)
EZ-Magna RIP kit (Millipore) was utilized for RIP assay. Transfected cells were lysed using RIP lysis buffer. The extract was added to magnetic beads and exposed to ELAVL1 antibody (Santa Cruz, USA), Ago2 antibody (Abcam) or anti-IgG (Sigma) at 4°C overnight. Finally, the relative enrichment of genes was calculated by RT-qPCR.
11. Statistical analysis
Results were represented by mean ± SD and explored with Graphpad Prism 8 (San Diego, CA, USA). Each experiment was conducted in 3 replicates. We used the Student’s t-test for making the comparison between 2 groups, while One-way ANOVA among several groups. In this study, p < 0.05 stood for statistical significance.