Mechanism Of Surfactant Protein B Intron5Polymorphism On Bronchopulmonary Dysplasia

Part I Association between surfactant protein B polymorphisms and bronchopulmonary dysplasia in Chinese Han infantsObjective This study aimed to investigate the association between surfactant protein B (SP-B) polymorphisms and bronchopulmonary dysplasia (BPD) in Chinese Han infants.Methods We performed a case-control study including86infants with BPD and156matched controls. Genotyping and allele distribution of four polymorphisms (rs2077079, rs11030866, rs7316and SP-B intron5) were performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing methods. Haplotypes were reconstructed by the fastPHASE software.Results significant differences were detected in the genotype distribution of rs2077079and intron5polymorphisms of SP-B gene between cases and controls, no differences were detected in the genotype distribution of rs1130866and rs7316between the two groups. The frequencies of intron5del allele were10.5%in BPD group and4.8%in control group, the C allele of rs2077079were45.3%in BPD group and59.6%in control group, the two alleles frequencies were markedly different between BPD group and control group (p<0.05). The frequency of A-del-C-A haplotype was significantly higher in BPD group than in control group (0.12compared to0.05, P=0.003), whereas the frequency of C-inv-C-A haplotype was higher in control group than in BPD group (0.19compared to0.05, P=0.000).Conclusion Polymorphisms of SP-B intron5and rs2077079are associated with BPD in Chinese Han infants, but on association was observed between polymorphisms of rs1130866and rs7316with BPD. The del allele of intron5could be a risk factor for BPD and C allele of rs2077079can reduce the risk of BPD. Haplotype analysis indicated that the four polymorphisms might be in partial linkage disequilibrium Part II Construction of pIRES2-EGFP-SP-B-intron5eukaryotic expressing plasmidsObjective To construct the wide type and deletion type of SP-B-intron5eukaryotic expressing plasmids.Methods The target genes contained SP-B intron5polymorphisms were obtained from genomic DNA by PCR. Then the target genes and pIRES2-EGFP vectors were digested with restriction enzymes Xho I and Hind III. The digested products were ligated by T4DNA ligase. The ligated products were transfected into escherichia coli and kanamycin was used for screening positive bacterial colonies. The wild type pIRES2-EGFP-SP-B-intron5-inv plasmid and deletion type pIRES2-EGFP-SP-B-intron5-inv plasmid were identified by PCR, restriction analysis and sequencing.Results The wild type plasmid contained SP-B intron5invariant(inv) target gene and the deletion type plasmid contained SP-B intron5deletion (del) target gene.Conclusion The pIRES2-EGFP-SP-B-intron5eukaryotic expressing plasmids were successfully constructed, which provides a foundation for further SP-B intron5polymorphisms study. Part III Mechanism of SP-B intron5variations on SP-B gene expressionObjective To detect the SP-B gene expression of pIRES2-EGFP-SP-B-intron5transfected 293T cells and H441cells, and explore mechanism of SP-B intron5polymorphisms on SP-B gene expression.Methods Two types of pIRES2-EGFP-SP-B-intron5plasmids were transfected into293T and H441cells using Lipofectamine2000. Expression of EGFP gene in293T cells was observed by fluorescence microscopy. Limiting dilution method was used for selecting monoclonal H441cells. SP-B RNA splicing and protein expression were detected by RT-PCR (reverse transcription-polymerase chain reaction) and western blot.Results The transfection efficiency of recombined plasmids in293T cells were more than50%. Size of RT-PCR products amplified by primer pair zx and fxl was about300bp in the pIRES2-EGFP-SP-B-intron5-inv transfected293T cells, while in the pIRES2-EGFP-SP-B-intron5-del cells the sizes were approximately300bp and950bp. With primer pair zx and fx2, the sizes of RT-PCR products were both320bp in the wild and deletion type plasmid transfected293T cells. With primer pair zx and fx3, the sizes of RT-PCR products were about730bp in wild type plasmid transfected293T cells and530bp in the deletion type plasmid transfected293T cells. Proteins in transfected293T and H441cells were detected with antibody against the surfactant protein B intermediate (DTB). In293T cells, size of proteins was about30kDa and the protein expression was significantly higher in wild type transfected cells than in deletion type (p<0.05). In H441cells, there were two bands of protein expression, one was42-kDa SP-B precursor protein and the other was25-kDa SP-B intermediate, both expression were markedly higher in wild type compared with deletion type (p<0.05).Conclusion In our study, the SP-B intron5deletion variation affected RNA splicing and resulted in incompleted mRNA and decreased expressions of normal SP-B mRNA and protein. This mechanism may be associated with SP-B intron5del allele increasing risk for BPD.