Influence of the ARDS-associated 5′UTR variant rs2700408 on nmMYLK promoter activities
In our recent sequencing studies, rs2700408 was associated with sepsis induced-ARDS/ALI in AAs [41]. (A) Rs2700408A/G interrupted nmMYLK 5′UTR binding to TF GCM1. EMSA was utilized for studying TF protein–nmMYLK DNA interactions. Nuclear extract from HeLa cells for the TF pool was incubated with biotin-labeled MYLK DNA fragments containing rs2700408 A or G alleles, competed by an unlabeled DNA fragment containing GCM1 sequence. After electrophoresis, the DNA–protein complex was detected by HRP and ECL. Compared with rs2700408A, rs2700408A significantly increased GCM1 binding to MYLK DNA. (B) HPAECs were transfected with MYLK promoter-exon2 in pGL3 with rs2700408A or rs2700408G. Rs2700408G significantly increased promoter activities of MYLK, especially in response to LPS and 18% CS (*P<0.05 vs. rs2700408A, **P<0.01 vs. LPS/No SNP and 18% CS/No SNP, n=4 each).