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    • br Materials and Methods br Results br Discussion The presen

    2018-10-30


    Materials and Methods
    Results
    Discussion The present study demonstrated that both rs6435156C>T and rs1048829G>T variant genotypes in the 3′UTR of BMPR2 were associated with increased risk of COPD in a southern Chinese population. COPD smokers carrying the T variant (CT and TT) of rs6435156 exhibited significantly reduced mRNA and protein expression of BMPR2 in PBMCs comparing with the CC carrier smokers. Moreover, we found that the rs6435156T variants significantly decreased the expression of BMPR2 both alone and in synergy with cigarette smoke exposure in lung epithelial cells. The miRNA hsa-miR-20a was responsible for decreased expression of BMPR2 in cells carrying rs6435156T genotype. Reduction of BMPR2 expression was observed in the lung tissue of COPD patients (Llinàs et al., 2011). However, none has been known about the association between BMPR2 mutation and COPD. As an identified risk factor, BMPR2 gene mutations have been shown to be related with the occurrence of heritable and idiopathic PAH. Deficiency of BMPR2 induced more obvious perivascular inflammation and muscularization characterized by increased thickness of small pulmonary flap inhibitor in BMPR2 mutants than in wild type mice with pulmonary hypertension (PH) (Song et al., 2008). Though PH is a common comorbidity of COPD, BMPR2 gene mutation has not been assessed as a risk factor for COPD. This may be due to the complex nature of COPD pathology, which has been recognized to involve interactions of multiple genes and environment factors. In this study, we identified that both rs6435156C>T and rs1048829G>T in 3′UTR of BMPR2 were significantly associated with increased COPD risk, when analyzed separately and in a joint manner. However, it was rs6435156T, but not rs1048829T variant found being associated with decreased BMPR2 expression in lung epithelial cells when assessed by luciferase reporter assay. The fact that both rs6435156T and rs1048829T variants were significant when included in the same regression model further suggests that the association of rs1048829 with COPD is not due to the LD with rs6435156. The apparent discrepancy between the above population study and in vitro functional study is likely due to the effects of rs1048829G>T on COPD risk is not strong enough to be monitored in our tested in vitro system. Consistently, we found rs6435156C>T took the main effect on COPD risk comparing to rs1048829G>T when regression analysis was performed with two of the SNPs included in the same regression model. In addition, it is also likely that rs1048829G>T functions by affecting rs6435156C>T, thus presenting a joint effect on COPD risk; yet, this assumption needs to be further investigated in the future study. In this study, we showed that rs6435156T variant genotype is associated with reduced BMPR2 expression in PBMCs from COPD non-smokers and with further reduction in COPD smokers. Moreover, consistent with these results from the in vivo study, we found that, although CSE decreased the luciferase reporter activity in cells carrying rs6435156C, it caused greater decrease of the reporter activity in cells carrying rs6435156T variant (Fig. 4D). According to bioinformatics analysis, we found the rs6435156C>T resides in the potential binding site of has-miR-20a. MicroRNA miR-20a was shown to regulate BMP signaling pathway through targeting on the binding sites of 3′UTR of BMP-2 and possibly BMPR2 gene in fish skeletogenesis (Tiago et al., 2014). Treatment of antagomiR-20a restored the functional levels of BMPR2 expression in pulmonary arteries (Brock et al., 2014). Interestingly, we found treatment of miR-20a mimics increased, and miR-20a inhibitor decreased the luciferase activity in cells carrying rs6435156TT variants. In contrast, both miR-20a mimics and inhibitor did not change the level of luciferase activity in cells carrying rs6435156CC genotypes. These results indicate that cigarette smoke normally could downregulate BMPR2 expression independent of has-miR-20a; however, under the presence of rs6435156T variant, it further decreases BMPR2 expression in has-miR-20a dependent manner; the rs6435156C>T mutation is responsible for downregulated BMPR2 expression likely by recruiting the binding of miR-20a to the 3′UTR of BMPR2. Consistently, Brock M. et al. identified that expression of BMPR2 was modulated by a STAT3-miR-17/92-BMPR2 pathway, in which miRNA cluster 17/92 (miR-17/92) encodes hsa-miR-20a. A highly conserved STAT3-binding site was found in the promoter region of the miR-17/92 gene and mediated IL-6 modulated BMPR2 expression (Brock et al., 2009). As a proinflammatory factor, IL-6 was upregulated by cigarette smoking in lung tissue from COPD patients (Llinas et al., 2011). Plasma IL-6 level was suggested to be a persistent and progressive parameter in evaluating systemic inflammatory process and mortality association in COPD patients (Ferrari et al., 2013). Based on the above evidence from us and others, we propose that the higher risk of COPD in cigarette smoking populations carrying rs6435156T variant is likely due to IL-6 and hsa-miR-20a mediated downregulation of BMPR2 expression.