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Published on: February 20, 2015
[Screening type II alveolar epithelial cell apoptosis related microRNA]
Hui Ji1, Miao Chen, Ming-jiang Qian
1Department of Critical Care Medicine, Affiliated Hospital of Zunyi Medical College, Zunyi 563000, Guizhou, China. Corresponding author: Chen Miao,
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.
Insights
This study identified microRNAs (miRNAs) linked to type II alveolar epithelial cell (AECII) apoptosis. These findings offer new therapeutic strategies for hyperoxia-induced acute lung injury (HALI).
Area of Science:
- Cell Biology
- Molecular Biology
- Pulmonary Medicine
Context:
- Hyperoxia-induced acute lung injury (HALI) is a critical condition affecting lung tissue.
- Type II alveolar epithelial cells (AECII) are crucial for lung function and susceptible to injury.
- MicroRNAs (miRNAs) are key regulators of cellular processes, including apoptosis.
Purpose:
- To screen apoptosis-related microRNAs (miRNAs) in type II alveolar epithelial cells (AECII) using gene chip technology.
- To establish an in vitro model of AECII apoptosis using hydrogen peroxide (H2O2) injury.
- To identify potential therapeutic targets for hyperoxia-induced acute lung injury (HALI).
Summary:
- Primary rat AECII were cultured and exposed to H2O2 to induce apoptosis, confirmed by Transmission Electron Microscopy (TEM) and Flow Cytometry (FACS).
- Gene chip technology identified several apoptosis-related miRNAs, including miR-34 family members and miR-21-5p.
- RT-PCR validated the expression changes of these miRNAs, confirming their association with AECII apoptosis.
Impact:
- Identified specific miRNAs (e.g., miR-34 family, miR-21-5p) involved in AECII apoptosis.
- Provides a foundation for developing novel miRNA-based therapeutic strategies for HALI.
- Enhances understanding of the molecular mechanisms underlying lung epithelial cell apoptosis.
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