Objective: To screen type II alveolar epithelial cell (AECII) apoptosis related microRNA (miRNA) with gene chip technology, provide a new strategy for the prevention and treatment of hyperoxia-induced acute lung injury(HALI).
Methods: AECII of male Sprague-Dawley (SD) rats was primarily cultured for 36 hours, then exposed to 0.5 mmol/L H2O2 to establish apoptosis model. Transmission electron microscope (TEM) was used to identify AECII and to observe apoptosis cell morphology. Before and after H2O2 injury for 2.5, 6, 12 and 24 hours, fluorescence-activated cell sorting (FACS) was employed to detect apoptosis rate. Additionally, gene chip technology and real time polymerase chain reaction (RT-PCR) were used to screen and verify apoptosis related miRNA respectively.
Results: Microvilli and osmiophilic multilamellar body were found under TEM, which were the characteristic structure of AECII. This proved that the cells cultured were AECII. After H2O2 injury for 24 hours, cytoplasmic retraction, chromatin condensation and margination, microvilli and osmiophilic multilamellar body disappearance could be found under TEM. Compared with the blank control group, the apoptosis rate of AECII was significantly increased after exposed to 0.5 mmol/L H2O2, and gradually increased with time [the early apoptosis rate before and after H2O2 injury for 2.5, 6, 12 and 24 hours were (9.43±1.02)%, (18.38±2.91)%, (28.57±1.18)%, (35.83±2.66)% and (57.68±2.22)%, respectively, all P<0.05]. Compared with before H2O2 injury, cells at 24 hours accompanied a significantly changed miRNA expression profiling, in which apoptosis related miRNA had been screened, they were rno-miR-449a-5p, rno-miR-34b/c-5p, rno-miR-200a/c-3p, rno-miR-146a-5p, rno-miR-141-3p, rno-miR-21-5p, rno-miR-375-3p, rno-miR-29b-3p, rno-miR-214-5p, rno-miR-210-5p and rno-miR-214-3p,and the results of RT-PCR were consistent with the gene chip results.
Conclusions: Screened out AECII apoptosis related miRNA, and miR-34 family may play a key role in AECII apoptosis regulation. Rno-miR-21-5p may be an important anti-apoptotic gene in AECII apoptosis regulation.