Downregulation of miR-223 promotes HMGB2 expression and induces oxidative stress to activate JNK and promote autophagy in an in vitro model of acute lung injury

Cell culture and induction of cell injury using LPS

Human alveolar epithelial cells (A549, American Type Culture Collection (ATCC)) were cultured and maintained according to the manufacturer’s instructions and in the appropriate media. Under control conditions, A549 cells were treated with LPS (5, 10 and 20 µg/mL) for 6, 12, 24, 48 and 72 h, respectively. Then, A549 cells treated with different LPS concentrations for different times points were harvested for the MTT assay. Only A549 cells treated with LPS for 72 h were analysed with quantitative polymerase chain reaction (qPCR), western blot, and enzyme-linked immunosorbent assay (ELISA).

MTT assay

The viabilities of A549 cells treated with different LPS concentrations of LPS for different times and untreated A549 cells were evaluated by MTT assay according to the manufacturer’s instructions. Briefly, approximately 1 × 105 mL−1 LPS-treated A549 cells or untreated A549 cells were seeded in flat-bottomed 96-well polystyrene coated plates. After 24 h of incubation, 10 µL of MTT reagent was added to each well, followed by incubation for another 4 h. The plates were then read immediately in a microplate reader (BIO-RAD microplate reader-550) at 570 nm.

ELISA

ELISA kits for 8-hydroxy-2’-deoxyguanosine (8-OHdG), tumour necrosis factor α (TNFα), interleukin-1β (IL-1β) and interleukin 6 (IL-6) were utilized according to the manufacturer’s instructions. 100 µL of the 8-alveolar epithelial cells medium were tested per well. Values were assayed in triplicate and calibrated against an 8-OHdG standard.

Western blot

Proteins were isolated with 12 % SDS–PAGE and then transferred onto homopolymers and copolymer membranes (Schleicher & Schuell, Germany). The membranes were blocked in phosphate-buffered saline (PBS) that containing 10 % nonfat dry milk and 0.5 % Tween-20 overnight. Subsequently, the membranes were incubated with primary antibodies for 2 h. The following antibodies were used: anti- LC3B (Cell Signaling Technology, Danvers, MA, USA; #2775; 1:1000), anti-p62 (Cell Signaling Technology; #5114; 1:1000), anti-HMGB2(Abcam, Cambridge Science Park, UK; ab67282; 1:1000), p-JNK (Abcam, Cambridge Science Park, UK; ab47337; 1:1000), JNK (Abcam, Cambridge Science Park, UK; ab213521; 1:1000), and anti-β-actin (Abcam, Cambridge Science Park, UK; ab8226; 1:1000). The bands were visualized using a chemiluminescence detection system (CWBIO; Beijing, China) and were normalized to that of β-actin.

ROS assay

CellROX™ Deep Red (Invitrogen, USA) was used to measure intracellular ROS production. CellROX® Deep Red reagent was added at a final concentration of 5 µM to human alveolar epithelial cells, followed by incubation 37 °C for 30 min. The medium was removed, and the cells were washed three times with PBS. The fluorescence was measured at 644 nm excitation and 665 nm wavelengths utilizing a Bio-Tek Synergy HT-I plate reader (Bio-Tek Instruments, USA).

RT-qPCR

Total RNA was isolated from A549 cells using TRIzol (Invitrogen). Then RNA was reverse transcribed using a PrimeScript™ RT reagent kit (cat. No. RR037A; Takara Biotechnology Co. Ltd.). RT-qPCR was performed using RT-qPCR UltraMix(SYBR Green) (LMAI Bio) according to the manufacturer’s instructions. Each reaction was performed in triplicate. Relative expression levels were calculated by the 2−∆∆Ct method and normalized to those of the GAPDH gene and U6. The primers were as follows: HMGB2: Forward, 5’-GTGGCCTAGCTCGTCAAGTT-3’; Reverse, 5’-GCGTACGAGGACATTTTGCC-3’; miR-223: Forward, 5’-GGCGCTTGTCAGTTTGTCAAAT-3’; Reverse, 5’-GTCGTATCCAGTGCAGGGTCCG-3’; GAPDH: Forward, 5’-CCAGGTGGTCTCCTCTGA-3’; Reverse, 5’-CCGTGTTCCTACCCCCAATG-3’; U6: Forward, 5’-GCTTCGGCAGCACATATACTAAAAT-3’; Reverse, 5’-CGCTTCACGAATTTGCGTGTCAT-3’.

Flow cytometry

A549 cells were trypsinized and washed twice with PBS. The cells were stained with Annexin V and propidium iodide (PI) using an FITC Annexin V/PI Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) based on the manufacturer’s instructions. Briefly, cells were seeded in 24-well plates for approximately 24 h and then stained with the Annexin V-FITC Annexin V and PI solution for 15 min. Then, the cells were analysed using a FACS cytometer (BD Biosciences).

Construction of the plasmid vector and cell transfection

The HMGB2 gene and pcDNA3.1 vector were used to construct an RNA knockout vector. MiR-223 mimic and inhibitor and their negative control oligonucleotides were obtained from GenePharma (Shanghai, China). The JNK inhibitor, SP600125, was purchased from Calbiochem (San Diego, CA). According to the manufacturer’s instruction, cells were transfected using LipofectamineTM 2000 (Invitrogen, Carlsbad, USA). Briefly, 10 µg/mL alveolar epithelial cells were seeded to flasks until they were 70 % - 80 % confluent. Then, cells were transfected with 20 µL Lipofectamine followed by rinsing with the serum-free, antibiotic-free medium. After 6 h of incubation, the transfected cells were resuspended and cultured in the regular cell culture medium for 72 h before analyses. Then the cells were treated with SP600125 (20 µg/mL) or LPS (10 µg/mL) to induce injury.

Luciferase assay

Site-directed mutagenesis of the miR-223 target site at the 3’-UTR of HMGB2 was performed utilizing a QuickChange mutagenesis kit (Stratagene, Heidelberg, Germany). HMGB2 wild-type (wt-HMGB2) or mutant (mut-HMGB2) 3’-UTR nucleotide sequences were inserted into the pLuc luciferase vector (Ambion, Austen, Texas, USA). Alveolar epithelial cells, which were cultured in 24-well plates, were transfected with 100 ng of wt-HMGB2 or mut-HMGB2 and 50 nM of miR-223 mimic or miR-223 inhibitor utilizing Lipofectamine™ 2000 (Invitrogen). The cells were harvested following 48 h after the transfection. According to the manufacturer’s instructions, a Dual-Luciferase Reporter assay kit was used to measure luciferase activity (Promega, Madison, Wisconsin, USA). All transfections were performed in triplicates.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 5.0. Two-sample t-tests were used for comparisons between two groups. In cases of comparison among more than two groups, ANOVA was applied. Each experiment was performed at least three times independently with cells from three different passages. Data were presented as the mean ± standard deviation (SD). A value of P < 0.05 was considered statistically significant.

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