Experimental lung research · 2019
miR-221 participates in the airway epithelial cells injury in asthma via targeting SIRT1.
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Zhang Hong, Sun Yuanchun, Rong Wei, Fan Linxia, Cai Yufeng, Qu Qiang, Gao Yun, Zhao Hongxia
Аннотация
AIM OF THE STUDY: To investigate the role of microRNA-221 (miR-221) in the airway epithelial cell injury in asthma and delineate the underlying mechanism that may involve with SIRT1.
MATERIALS AND METHOD: Bronchial epithelial cells from asthma patients and healthy controls were obtained by bronchoscopic brushing. The miR-221 and SIRT1 mRNA level in collected cells were detected by qRT-CPR. BEAS2B cell lines were cultured in vitro. In order to up-regulate miR-221 and SIRT1, miR-221 mimic and pcDNA3.1-SIRT1 vector was transfected into BEAS2B cells, respectively. The expression changes of miR-221 and SIRT1 after transfection was observed by qRT-PCR and Western blot. The target relationship between miR-221 and SIRT1 was confirmed using dual-luciferase reporter assay.The cell viability changes after transfection was measured using cellTiter-blue reagent. The apoptosis rate was detected by flow cytometry.
RESULT: Compared with healthy controls, miR-221 expression significantly increased in bronchial epithelial cells from patients subjects. In contrast, the level of SIRT1 mRNA reduced in the bronchial epithelial cell from asthma patients. In vitro, up-regulation of miR-221 could inhibit the expression of SIRT1 both at mRNA and protein level in BEAS2B cells. A negative correlation between miR-221 and SIRT1 mRNA in samples from patients was confirmed and dual-luciferase reporter assay showed that miR-221 directly binds to the 3'UTR of SIRT1 mRNA. Overexpression of miR-221 or SIRT1 knockdown could inhibit proliferation but induce apoptosis in BEAS2B cells. Moreover, up-regulation of SIRT1 could antagonize miR-221's inhibitory effect.
CONCLUSION: miR-221 may participate in the airway epithelial cells injury in asthma via targeting SIRT1.
MATERIALS AND METHOD: Bronchial epithelial cells from asthma patients and healthy controls were obtained by bronchoscopic brushing. The miR-221 and SIRT1 mRNA level in collected cells were detected by qRT-CPR. BEAS2B cell lines were cultured in vitro. In order to up-regulate miR-221 and SIRT1, miR-221 mimic and pcDNA3.1-SIRT1 vector was transfected into BEAS2B cells, respectively. The expression changes of miR-221 and SIRT1 after transfection was observed by qRT-PCR and Western blot. The target relationship between miR-221 and SIRT1 was confirmed using dual-luciferase reporter assay.The cell viability changes after transfection was measured using cellTiter-blue reagent. The apoptosis rate was detected by flow cytometry.
RESULT: Compared with healthy controls, miR-221 expression significantly increased in bronchial epithelial cells from patients subjects. In contrast, the level of SIRT1 mRNA reduced in the bronchial epithelial cell from asthma patients. In vitro, up-regulation of miR-221 could inhibit the expression of SIRT1 both at mRNA and protein level in BEAS2B cells. A negative correlation between miR-221 and SIRT1 mRNA in samples from patients was confirmed and dual-luciferase reporter assay showed that miR-221 directly binds to the 3'UTR of SIRT1 mRNA. Overexpression of miR-221 or SIRT1 knockdown could inhibit proliferation but induce apoptosis in BEAS2B cells. Moreover, up-regulation of SIRT1 could antagonize miR-221's inhibitory effect.
CONCLUSION: miR-221 may participate in the airway epithelial cells injury in asthma via targeting SIRT1.