DR cells model construction
Human retinal microvascular endothelial cells (HRMECs) were obtained from Procell Life Science Technology Co,.Ltd. (Wuhan, China). HRMECs was cultured in Endothelial medium (ECM, Solarbio, Beijing, China) with 5% fetal bovine serum (FBS, Beyotime, Shanghai, China), 1% endothelial cell growth supplement (Beyotime) and 1% penicillin/streptomycin solution (Beyotime) in 37 oC and 5% CO2. HRMECs in the logarithmic growth phase were taken for subsequent experimentations. HRMECs in high glucose (HG) group were cultured in 30 mmol/L glucose medium for 48 h, and the cells in the Normal group were cultured in a 5.5 mmol/L glucose medium. In the Mannitol group, cells were cultured in 5.5 mmol/L glucose + 24.5 mmol/L mannitol medium.
Short hairpin RNA USP14 (sh-USP14), short hairpin RNA ATF2 (sh-ATF2), PcDNA3.1-USP14 (oe-USP14) plasmid, PcDNA3.1-ATF2 (oe-ATF2) plasmid, PcDNA3.1-PIK3CD (oe-PIK3CD) plasmid and corresponding negative control (NC) were obtained from Santa Cruz Biotechnology (Shanghai) Co., Ltd (Shanghai, China). On the authority of the directions of Lipofectamine 3000 transfection reagent (Invitrogen, NY, USA), above plasmids were transfected into cells for 48 h.
HRMECs were lysed with RIPA lysis buffer (Beyotime) to obtain the total protein, along with protein concentration was measured with BCA protein quantitative kit (Beyotime). Appropriate protein dosages were disunited by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis along with moved to PVDF membrane. 5% skim milk was sealed for 2 h, and primary antibody diluent of corresponding protein was incubated in 4 oC overnight. Thenceforth, the secondary antibody dilution was nurtured at ambient temperature for 2 h. Thenceforth the bands were nurtured with ECL luminescence solution (Thermo Fisher, MA, USA) and placed in chemiluminescence imaging system for exposure development. The relative expression of each protein was deconstructed by ImageJ software. The antibody information was as demonstrated: USP14 (ab246010, 1/2000), ATF2 (ab32160, 1/1000), PIK3CD (ab32401, 1/500), and GADPH (ab8245, 1/1000).
Quantitative real-time PCR
Total RNA of HRMECs was obtained on the basis of the standard procedure described in the instruction of TRIzol reagent (Invitrogen, MA, USA). RNA concentration were confirmed by ultraviolet spectrophotometer. Subsequently, the first strand cDNA was synthesized by reverse transcription accordant with the guidelines of the reverse transcription kit (Biosystems, Shanghai, China). Real-time PCR was manipulated utilizing PCR SYBR (TaKaRa, Tokyo, Japan) Green method. The gene expression was evaluated by 2−∆∆Ct method. The primers information is as demonstrated:
USP14: (F) 5’-TATGCAGGTGCCTTGAGAGC-3’,
ATF2: (F) 5’-TTATCCCCTCTTGCAACACC-3’,
PIK3CD: (F) 5ʹ-CATATGTGCTGGGCATTGGC-3’,
GAPDH: (F) 5’-CTGACTTCAACAGCGACACC-3’,
Cell Counting Kit-8 (CCK-8) assay
HRMECs were grown logarithmically at a cell density of 5 × 103 cells per well in 96-well culture plates. The 96-well plates were incubated in a 37 oC incubator with 5% CO2 for 24 h. Therewith, adding 10 µL of CCK-8 solution (Beyotime) to each well, incubating for 4 h, and recording the absorbance at 450 nm wavelength with enzyme labeling instrumen (BioTek, VT, USA).
HRMECs was lysed with RIPA lysis solution (Beyotime). The cleavage products were centrifuged in 4 oC, and the cleavage products were collected. Appropriate amount of cleavage products were directly detected by Western blot to ascertain the expression of USP14 and ATF2 proteins as input, and the rest of the cleavage products were incubated overnight with anti-USP14 or anti-ATF2 antibodies. After incubation, protein G beads (Santa Cruz) were added to incubate for 4 h, and then the precipitated protein G beads were collected by centrifugation. Proteins on the beads were eluted, and the ATF2 and USP14 proteins were detected by western blot.
HRMECs (2×105 cells) in logarithmic growth phase were suspended in 200 µL serum-free medium, and the cell suspension was added to upper chamber, and 600 µL medium with FBS was added to lower chamber. After 24 h of culture, the HRMECs were fixed with 4% paraformaldehyde and stained with crystal violet solution. Randomly select 5 visible points under the microscope to take pictures and count. The average number of cells that penetrated into lower chamber was used as the experimental result.
Tube formation assay
200 µL of Matrigel matrix glue (BD, NJ, USA) was added to the 24-well plate and put into 37 oC, 5% CO2 incubator for solidification. The cells cultured for 48 h in each group were prepared into 2 × 105 / mL suspension with ECM medium and added to the 24-well plate containing Matrigel matrix glue according to 1 mL/ well. The cells were cultured in 37 oC, 5% CO2 incubator. After 8 h, the capillary structure was photographed. ImageJ software was manipulated to examine the capillary-like structures.
Dual-luciferase reporter gene assay
Jaspar (https://jaspar.genereg.net/) was used to speculate the binding of ATF2 to PIK3CD. The 3'-UTR cDNA sequences of PIK3CD-WT and PIK3CD-MUT were cloned into the pGL3 luciferase reporter gene (Geneharma, Shanghai, China). The corresponding luciferase reporter gene vector was co-transfected with oe-ATF2 or oe-NC. Luciferase activity was determined according to the procedures of luciferase report Analysis Kit (Promega, WI, USA) after 48 h.
Chromatin immunoprecipitation (ChIP) assay
HRMECs were lysed using RIPA lysis buffer (Beyotime) and 100 µL of lysate was incubated with RIPA lysis buffer containing magnetic beads conjugated to anti-ATF2 or IgG. RNA was isolated and purified, and the content of RNA was detected by RT-qPCR.
GraphPad prism 7 software (IBM SPSS software, IL, USA) was utilized to examine the statistic. The statistic were expressed as mean ± standard deviation (all experiments were repeated for 3 times). The comparison of the two groups was evaluated by student’s t-test. Furthermore, one-way ANOVA was manipulated to test the discrepancy between multiple groups. P < 0.05 was esteemed statistically noteworthy.