Zhu J Y, Ma L Q, Zhang J
Department of Critical Care Medicine, General Hospital of Ningxia Medical University, Yinchuan 750004, China.
Department of Pathology, General Hospital of Ningxia Medical University, Yinchuan 750004, China.
Zhonghua Yi Xue Za Zhi. 2020 Aug 25;100(32):2481-2487. doi: 10.3760/cma.j.cn112137-20200326-00945.
To explore the relationship between family with sequence similarity 13 member A (FAM13A) gene and small airway remodeling in chronic obstructive pulmonary disease (COPD), and the effect of interference with FAM13A gene expression on the apoptosis and proliferation phenotype of human airway epithelial cells (16HBE). From January 2018 to January 2020, 74 patients in the Department of Thoracic Surgery of General Hospital of Ningxia Medical University were treated by surgery for lung tumors or pulmonary bullae. According to the lung function and smoking history, the 74 patients were divided into four groups: non-smoking group with normal lung function (normal group, 23 patients), smoking group with normal lung function (smoking group, 24 patients), non-smoking group with COPD (11 patients) and smoking group with COPD (16 patients). The expression of FAM13A in small airway of each group was detected by immunohistochemistry, and the correlation between FAM13A and the airflow restriction indexes by pulmonary function was analyzed. The shRNA fragment of FAM13A gene was designed, and the shRNA lentivirus vector of FAM13A gene was constructed and packaged. The expression level of FAM13A gene was detected by real-time fluorescent quantitative PCR (qRT-PCR) and Western blot, and the best shRNA sequence was screened. Flow cytometry was used to detect apoptosis rate and the fluorescence intensity of proliferation marker Ki-67 in 16HBE cells. FAM13A was mainly expressed in the cytoplasm of small airway epithelial cells. The levels of FAM13A absorbance () of small airway epithelial cells in non-smoking group and smoking group with COPD were higher than those in normal group and smoking group (0.365±0.026, 0.412±0.053 to 0.113±0.018, 0.105±0.009, all 0.05), and they were negatively correlated with forced expiratory volume in 1s/forced vital capacity (FEV(1)/FVC) and FEV(1)% pre (-0.48 and -0.40, all 0.05). The FAM13A shRNA lentiviral vector was successfully constructed, and FAM13A interference was successfully achieved in the 16HBE cell line. After infection of 16HBE cells, the results of qRT-PCR and Western blot showed that the expression of FAM13A in shRNA-target-2 group decreased (all 0.01). Compared with the negative control group (shRNA-NC), the apoptosis rate of FAM13A shRNA group decreased (0.023), and the fluorescence intensity of Ki-67 also decreased (0.042). FAM13A gene expression is increased in COPD small airway epithelial cells, and it is related to COPD airflow limitation. FAM13A gene may participate in the process of COPD remodeling by affecting the apoptosis and proliferation of human airway epithelial cells.
探讨家族序列相似性13成员A（FAM13A）基因与慢性阻塞性肺疾病（COPD）小气道重塑的关系，以及干扰FAM13A基因表达对人气道上皮细胞（16HBE）凋亡和增殖表型的影响。2018年1月至2020年1月，宁夏医科大学总医院胸外科74例因肺部肿瘤或肺大疱接受手术治疗的患者。根据肺功能和吸烟史，将74例患者分为四组：肺功能正常的非吸烟组（正常组，23例）、肺功能正常的吸烟组（吸烟组，24例）、患COPD的非吸烟组（11例）和患COPD的吸烟组（16例）。采用免疫组织化学法检测各组小气道中FAM13A的表达，并分析FAM13A与肺功能气流受限指标的相关性。设计FAM13A基因的shRNA片段，构建并包装FAM13A基因的shRNA慢病毒载体。采用实时荧光定量PCR（qRT-PCR）和Western blot检测FAM13A基因的表达水平，筛选出最佳的shRNA序列。采用流式细胞术检测16HBE细胞的凋亡率及增殖标志物Ki-67的荧光强度。FAM13A主要表达于小气道上皮细胞的细胞质中。患COPD的非吸烟组和吸烟组小气道上皮细胞的FAM13A吸光度（）水平高于正常组和吸烟组（0.365±0.026，0.412±0.053对0.113±0.018，0.105±0.009，均P<0.05），且与第1秒用力呼气容积/用力肺活量（FEV(1)/FVC）和FEV(1)%预计值呈负相关（分别为-0.48和-0.40，均P<0.05）。成功构建了FAM13A shRNA慢病毒载体，并在16HBE细胞系中成功实现了FAM13A干扰。16HBE细胞感染后，qRT-PCR和Western blot结果显示shRNA-target-2组FAM13A表达降低（均P<0.01）。与阴性对照组（shRNA-NC）相比，FAM13A shRNA组的凋亡率降低（P<0.023），Ki-67荧光强度也降低（P<0.042）。COPD小气道上皮细胞中FAM13A基因表达增加，且与COPD气流受限有关。FAM13A基因可能通过影响人气道上皮细胞的凋亡和增殖参与COPD重塑过程。
