Duan Jun, Zhang Xiaoying, Zhang Sheng, Hua Shaodong, Feng Zhichun
Department of Pediatrics, BaYi Children's Hospital Affiliated to Clinical Medical College in Beijing Military General Hospital of Southern Medical University, Beijing 100700, P.R. China.
Exp Ther Med. 2017 Jun;13(6):3203-3208. doi: 10.3892/etm.2017.4430. Epub 2017 May 5.
Bronchopulmonary dysplasia (BPD) is a syndrome of respiratory distress caused by chronic lung injury, primarily in preterm infants. miR-206 and fibronectin 1 (FN1) are associated with the development of BPD. The present study used rat type II alveolar epithelial cells (AECII) to investigate the underlying mechanisms of BPD. AECII were isolated using a primary cell culture prior to alkaline phosphatase staining and immunofluorescence of surfactant protein C (SP-C). These were used to verify the presence of AECII. AECII were then divided into four groups, which were transfected with four different plasmids. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the relative expression of miR-206 in the each group. The gene and protein expression level of FN1 was detected by RT-qPCR and immunofluorescence. The proliferation of AECII in each of the four groups was evaluated using an MTT assay 48 h following transfection. The percentage of apoptotic cells was determined by flow cytometric analysis. The present study demonstrated that upregulation of miR-206 decreased the expression of FN1 (P<0.05) and low levels of miR-206 led to increased expression of FN1 (P<0.05) in AECII. Furthermore, the forced expression of miR-206 suppressed proliferation and promoted apoptosis of AECII while downregulation of miR-206 had the opposite effect (P<0.05). The results of the current study provide valuable insights into the prevention of BPD and suggest that miR-206 may be used as a potential molecular target for BPD therapy in the future.
支气管肺发育不良(BPD)是一种主要发生在早产儿中的由慢性肺损伤引起的呼吸窘迫综合征。miR-206和纤连蛋白1(FN1)与BPD的发生发展相关。本研究使用大鼠II型肺泡上皮细胞(AECII)来探究BPD的潜在机制。在进行碱性磷酸酶染色和表面活性蛋白C(SP-C)免疫荧光检测之前,采用原代细胞培养法分离AECII。这些方法用于验证AECII的存在。然后将AECII分为四组,分别用四种不同的质粒进行转染。采用逆转录-定量聚合酶链反应(RT-qPCR)来测定每组中miR-206的相对表达量。通过RT-qPCR和免疫荧光检测FN1的基因和蛋白表达水平。转染48小时后,采用MTT法评估四组中AECII的增殖情况。通过流式细胞术分析确定凋亡细胞的百分比。本研究表明,miR-206的上调降低了AECII中FN1的表达(P<0.05),而低水平的miR-206导致FN1表达增加(P<0.05)。此外,miR-206的强制表达抑制了AECII的增殖并促进了其凋亡,而miR-206的下调则产生相反的效果(P<0.05)。本研究结果为BPD的预防提供了有价值的见解,并表明miR-206未来可能作为BPD治疗的潜在分子靶点。