TRIM46 aggravated high glucose-induced hyper permeability and inflammatory response in human retinal capillary endothelial cells by promoting IκBα ubiquitination

Cell culture

HRCECs bought from the Type Culture Collection of the Chinese Academy of Science were maintained in Dulbecco's Modified Eagle Medium (10566024, Thermo Scientific, USA) containing 10% fetal bovine serum (10099141, Thermo Scientific, USA) in a 37 °C culture incubator supplemented with 5% CO2.

High glucose-induced model

HRCECs (2 × 105/well) were plated in 6-well plates. The cells were then treated with 10, 15 or 25 mM glucose for 24 h. The control cells were treated using 5.5 mM glucose and mannitol (M2069, Sigma Aldrich, USA) to control osmotic pressure.

Quantitative real-time PCR (qRT-PCR)

TRIzol (10,296,010, Thermo Scientific, USA) was used to isolate the total RNA in HECECs following the manufacturer’s protocol. The qRT-PCR analysis was conducted using SYBR®Green PCR Master Mix (4309155, Thermo Scientific, USA) on the ABI 7300 instrument (Applied Biosystems) and β-actin was used as the reference gene. The relative expression level of a specific gene was calculated by employing 2−ΔΔCt method. The primers used are listed here:

TRIM46-Forward-5′-CTGCTTGAGAACCCCGACA-3′,

TRIM46-Reverse-5′GCTCGCTGGTCCTTGCTG-3′;

IκBα-Forward-5′-CACCAACCAGCCAGA AAT-3′,

IκBα-Reverse-5′-ACCCAAGGACACCAAAAG-3′;

β-actin-Forward-5′-TGGCATTGCCGACAGG-3′,

β-actin-Reverse-5′-GCATTTGCGGTGGACG-3′.

Western blot

Total protein was harvested using the radio-immunoprecipitation assay (RIPA) lysis and extraction buffer (89901, Thermo Scientific, USA) containing proteinase inhibitor cocktail (87786, Thermo Scientific, USA). The NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (78835, Thermo Fisher Scientific, USA) was then used to isolate cytoplasmic and nuclear proteins following manufacturer’s protocol. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred into polyvinylidene fluoride membrane (PVDF membrane, 88518, Thermo Fisher Scientific, USA). After blocking with 5% skim milk in phosphate buffer saline (PBS), specific primary antibodies were used to incubate with above PVDF membrane at 4 °C. After incubation overnight, the membrane was washed for 3 times using PBS and was incubated with appropriate Horseradish peroxidase (HRP)-conjugated secondary antibodies (A0208, Beyotime, China) for 2 h. The protein bands were detected using HRP chemiluminescence substrate (A38555, Thermo Scientific, USA) with the iBright imaging system (iBright CL1500, Invitrogen, USA). The primary antibodies are as follows: TRIM46 (21026-1-AP, Proteintech, USA, 1:1000), IκBα (Ab76429, Abcam, UK, 1:1000), zona occludens 1 (ZO-1) (Ab96587, Abcam, UK, 1:1000), Occludin (Ab167161, Abcam, UK, 1:1000), NF-κB (Ab16502, Abcam, UK, 1:1000), H3 (17168-1-AP, Proteintech, USA, 1:1000), and β-actin (66009-1-lg, Proteintech, USA, 1:1000).

Short hairpin RNA (shRNA)-mediated interference

ShRNA oligonucleotides paired with TRIM46 were cloned into the pLKO.1 plasmid (10878, Addgene, USA). The successful construction of plasmids was verified via DNA sequencing. The targeted sequences are as listed here:

shTRIM46-1-GGAGAGCAAGCUUCAAGAATT;

shTRIM46-2-CAUGGUUUAUAAACAAUAATT;

shTRIM46-3-GGGCUGUGCUGGAGGAGAATT.

Then, 293 T cells were transfected using the above plasmids containing shTRIM46-1, shTRIM46-2, or shTRIM46-3, and the packaging plasmids psPAX2 and pMD2G by employing Lipofectamine 2000 (11668019, Invitrogen, USA). After incubation for 48–72 h, the generated lentiviruses were harvested for subsequent assays.

Overexpression of TRIM46 and IκBα

The complementary DNA (cDNA) of TRIM46 or IκBα was constructed into pcDNA3.1 vector (Life Technologies). The successful construction of plasmids was verified via DNA sequencing. Then, HRCECs were transfected with the plasmids using Lipofectamine 2000 (11668019, Invitrogen, USA).

Cell counting kit-8 (CCK-8) assay

Briefly, 10 µL CCK-8 solution (96992, Sigma-Aldrich, USA) was utilized to incubate with HRCECs as described in the manufacturer’s protocol for 4 h. Finally, a Multiskan™ FC microplate tester (51119180ET, Thermo Scientific, USA) was used to read the absorbance at 450 nm.

Cell cycle analysis

HRCECs treated as indicated were collected and fixed with ice-cold ethanol at 4 °C overnight. After labelling with propidium iodine (PI, Sigma-Aldrich), cell cycle was analyzed with flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s instructions.

Determination of TNF-α, IL-6 and IL-1β

The protein levels of TNF-α, IL-6 and IL-1β secreted via HRCECs was measured using specific enzyme-linked immunosorbent assay (ELISA) kits (BMS223HS, BMS213-2 and BMS224-2, Invitrogen, USA) according to the manufacturer's protocol.

Transepithelial electrical resistance (TEER) assay and fluorescein isothiocyanate (FITC)-dextran assay

HRCECs (1 × 104 cells/well) were cultured in Transwell filters (pore size, 0.4 µm, CLS3396, Corning, USA) in a cell incubator at 37 °C supplemented with 5% CO2. After the cells grew to confluence, and were treated as indicated, TEER was determined utilizing the Millicell-ERS2 Volt-Ohm Meter (Millipore, USA) according to the manufacturer’s instructions. The TEER value (Ω cm2) was obtained by removing the resistance of the base filter and correcting for the surface area (0.6 cm2). Normalized TEER was determined as the ratio of the treated group’s TEER to the control group’s TEER.

After the cells grew to confluence, the cells were treated as indicated for 0 and 24 h. FITC-conjugated dextran (1 μg/μL, MW 70 000; 53471, Sigma-Aldrich, USA) was added to the upper chamber for 2 h. Then, a 100 μL mixture in the lower chamber was harvested and were detected using a spectrofluorometer (970CRT, YiTian, China). A permeability coefficient (PC) for FITC-dextran was determined as follows: PC (cm/min) = V/(SA × C0) × (Ct/T) [23], where V is the volume of medium in the lower chamber, SA is the surface area (0.6 cm2), C0 is the concentration of FITC-conjugated dextran in the upper chamber at time 0, and Ct is the concentration of FITC-conjugated dextran in the lower chamber at sampling time T.

Co-immunoprecipitation (Co-IP)

Cell lysates were incubated with antibodies TRIM46 (21026-1-AP, Proteintech, USA, 1:500), IκBα (Ab76429, Abcam, UK, 1:500) or IgG (sc-69786, Santa Cruz Biotech, USA, 1:500) for 1 h. The mixture was then incubated with Pierce Protein A/G Plus Agarose (20423, Thermo Scientific, USA) for another 3 h. All incubation processes were carried out at 4 °C. Finally, the precipitate was washed using washing buffer for three times and then analyzed via Western blotting.

Dual-luciferase reporter gene assay

The sequence of the TRIM46 promoter was cloned into a pGL3 vector (Promega, USA). HRCECs were transfected with the pGL3-TRIN46 promoter and pRL-SV40 vector using Lipofectamine 2000, and then exposed to high glucose (HG, 25 mM) and 10 μM NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) (HY-18738, MedChemExpress, USA) for 24 h. Luciferase activity was detected using the Luciferase Reporter Gene Detection Kit (LUC1, Sigma-Aldrich, USA) and a Multiskan™ FC microplate tester (51119180ET, Thermo Scientific, USA).

Statistical analysis

Statistical data analysis was performed using GraphPad Prism 8 (GraphPad, USA). All data are presented in mean ± standard deviation (SD) and were compared utilizing one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 represent statistically significant differences. All experiments were conducted in at 3 biological replicates.

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