Objective: To study the effect of hydrogen peroxide (H2O2) in inducing apoptosis of type II alveolar epithelial cell (AECII) after overexpression by adenoviral transfection of micro RNA-21-5p (miR-21-5p), and to explore the mechanism of its anti-apoptosis.
Methods: Subculture AECII were randomly divided into four groups: normal control group (normal saline), H2O2 challenge group (0.5 mmol/L H2O2), miR-21-5p overexpression group (miR-21-5p adenovirus+0.5 mmol/L H2O2), miR-21-5p negative transfection group (adenovirus void+0.5 mmol/L H2O2). Transmission electron microscopy and flow cytometry were used to detect apoptotic morphology and early apoptotic rate. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-21-5p in AECII, and Western Blot was used to detect the protein expressions of Bcl-2, Bax, and caspase-3 at the highest transfection efficiency at different time points (6, 12, 24, 48 hours).
Results: (1) AECII identification: fluorescence microscopy showed the presence of characteristic structure of AECII, i.e. microvilli and osmiophilic lamellar bodies. (2) Apoptotic morphology: transmission electron microscopy showed cytoplasmic retraction, chromatin condensation, margination, lack of cell surface microvilli, and emptying of osmiophilic lamellar bodies in AECII. (3) The expression of miR-21-5p in AECII: the highest transfection efficiency was found at 48 hours. The expression of miR-21-5p in miR-21-5p overexpression group was significantly higher than that of the normal control group, H2O2 challenge group and miR-21-5p negative transfection group (A value: 1.54±0.02 vs. 1.02±0.02, 0.56±0.03, 0.58±0.02, all P<0.05). (4) The rate of early apoptosis: compared with normal control group, the early apoptotic rates in H2O2 challenge group, miR-21-5p negative transfection group and miR-21-5p overexpression group were gradually elevated with the prolongation of injury time. The early apoptotic rate in miR-21-5p overexpression group was significantly lower than that of the H2O2 challenge group and miR-21-5p negative transfection group at all time points except 6 hours [12 hours: (10.73±2.80)% vs. (16.26±0.59)%, (16.04±0.70)%; 24 hours: (16.00±3.44)% vs. (23.29±2.78)%, (23.58±2.31)%; 48 hours: (31.30±3.55)% vs. (50.53±2.17)%, (49.41±1.97)%, all P<0.05]. There was no significant difference in early apoptotic rate between miR-21-5p negative transfection group and H2O2 challenge group at each time point. (5) Protein expression: the expressions of Bax and caspase-3 in miR-21-5p overexpression group were significantly lower than those of the H2O2 challenge group and miR-21-5p negative transfection group [Bax (A value): 0.07±0.01 vs. 0.18±0.01, 0.13±0.01; caspase-3 (A value): 0.07±0.01 vs. 0.23±0.01, 0.12±0.01, all P<0.05], and Bcl-2 protein expression was significantly higher than that of the H2O2 challenge group and miR-21-5p negative transfection group (A value: 0.26±0.01 vs. 0.06±0.01, 0.10±0.01, both P<0.05).
Conclusions: (1) miR-21-5p has the function of anti-apoptosis of AECII. (2) Adenoviral vector is a successful gene transfer vector when transfected with AECII. (3) The anti-apoptosis of AECII by miR-21-5p may be associated with reduced Bax and caspase-3 protein levels and raised expression levels of Bcl-2 protein.