State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China; Department of Respiration, Inner Mongolia Autonomous Region People's Hospital, Hohhot 010017, Inner Mongolia, China; Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
EBioMedicine. 2016 Feb 5;5:167-74. doi: 10.1016/j.ebiom.2016.02.004. eCollection 2016 Mar.
Bone morphogenetic protein receptor type 2 (BMPR2) signaling is anti-inflammatory. Decreased BMPR2 expression was seen in lung tissue from chronic obstructive pulmonary disease (COPD) patients.
The selected single nucleotide polymorphisms (SNPs) in BMPR2 were genotyped with polymerase chain reaction (PCR) ligase detection reaction. The effects of SNPs on gene expression were analyzed with luciferase assays. The mRNA and protein expression levels of BMPR2 in peripheral blood mononuclear cells (PBMCs) from COPD patients were determined by quantitative PCR and western blotting, respectively.
Two SNPs, rs6435156C > T and rs1048829G > T in the 3'-untranslated region (3'UTR) of BMPR2 were selected and genotyped in COPD case and healthy control subjects from southern Chinese population. Both of them were found associated with significantly increased COPD risk (adjusted odds ratio [OR] = 1.58 with 95% confidence interval [CI] = 1.14-2.15, P = 0.0056 for rs6435156C > T; adjusted OR = 1.47 and 95% CI = 1.10-1.97, P = 0.0092 for rs1048829G > T). Older age, cigarette smoking, family history of cancer and COPD were all factors that interacted with rs6435156C > T and rs1048829G > T causing increased COPD risk. Cigarette smokers with rs6435156 (CT + TT) or rs1048829 (GT + TT) were more susceptible to COPD than that with the rs6435156CC or rs1048829GG genotypes. In A549 human alveolar epithelial cells, luciferase reporter assays revealed that introduction of 3'UTR of BMPR2 plasmids carrying rs6435156T allele but not rs1048829T led to lower luciferase activity than the wild-type C or G alleles. Comparing to rs6435156CC, treatment with hsa-miR-20a mimics deceased whereas hsa-miR-20a inhibitor restored the luciferase reporter activity in cells transfected with constructs carrying rs6435156TT. BMPR2 mRNA and protein expressions were significantly lower in PBMCs from COPD smokers than that in non-smokers. COPD patients carrying rs6435156T allele had less BMPR2 expression in PBMCs.
This study demonstrated that both rs6435156C > T and rs1048829G > T variants in BMPR2 contributed to increased susceptibility to COPD. The T variants of rs6435156 increased COPD risk likely by binding with hsa-miR-20a, thus leading to downregulated BMPR2 expression in lung epithelial and immune cells.
骨形成蛋白受体 2 型（BMPR2）信号具有抗炎作用。慢性阻塞性肺疾病（COPD）患者的肺组织中 BMPR2 的表达减少。
采用聚合酶链反应（PCR）连接酶检测反应对 BMPR2 中的选定单核苷酸多态性（SNP）进行基因分型。通过荧光素酶测定分析 SNP 对基因表达的影响。采用定量 PCR 和 Western blot 法分别检测 COPD 患者外周血单个核细胞（PBMCs）中 BMPR2 的 mRNA 和蛋白表达水平。
从中国南方人群中选择 BMPR2 3'非翻译区（3'UTR）中的两个 SNP（rs6435156C > T 和 rs1048829G > T）进行 COPD 病例和健康对照的基因分型。这两个 SNP 均与 COPD 风险显著增加相关（校正后的优势比[OR]为 1.58，95%置信区间[CI]为 1.14-2.15，P = 0.0056 用于 rs6435156C > T；校正后的 OR 为 1.47 和 95%CI = 1.10-1.97，P = 0.0092 用于 rs1048829G > T）。年龄较大、吸烟、癌症和 COPD 家族史均为与 rs6435156C > T 和 rs1048829G > T 相互作用导致 COPD 风险增加的因素。与 rs6435156CC 或 rs1048829GG 基因型相比，携带 rs6435156（CT + TT）或 rs1048829（GT + TT）的吸烟者更易患 COPD。在 A549 人肺泡上皮细胞中，荧光素酶报告基因分析显示，携带 rs6435156T 等位基因而不是 rs1048829T 等位基因的 BMPR2 3'UTR 质粒的引入导致荧光素酶活性低于野生型 C 或 G 等位基因。与 rs6435156CC 相比，hsa-miR-20a 模拟物处理降低了转染携带 rs6435156TT 构建体的细胞中的荧光素酶报告活性，而 hsa-miR-20a 抑制剂则恢复了该活性。与非吸烟者相比，COPD 吸烟者的 PBMCs 中 BMPR2 mRNA 和蛋白表达明显降低。COPD 患者携带 rs6435156T 等位基因时，PBMCs 中的 BMPR2 表达减少。
本研究表明，BMPR2 中的 rs6435156C > T 和 rs1048829G > T 变体均导致 COPD 易感性增加。rs6435156 的 T 变体可能通过与 hsa-miR-20a 结合增加 COPD 风险，从而导致肺上皮和免疫细胞中 BMPR2 的表达下调。
