Association Between Surfactant Protein B-C/A (-18)Polymorphism And Susceptibility To Hyperoxic Lung Injury

Part I Impact of SP-B-C/A(-18) promoter polymorphism on the transcriptional regulation of SP-B geneObjective (1) To construct SP-B gene promoter luciferase reporter plasmids(pGL3-basic/pGL4.17-C/A) and detect their transcriptional activities in H441cells;(2) To study the association between different genotypes of SP-B-C/A(-18) and SP-B mRNA levels of peripheral blood mononuclear cells (PBMs) in full-term infants. Methods (1) The fragments of SP-B promoter (-218/+435bp) were acquired by PCR ampliation from human genome DNA and then were inserted into pGM-T vector by T4DNA ligase. The vectors were transfected into TOP10E.coli. The positive clones were identified by DNA sequencing. Then the identified target SP-B promoter sequences with C and A in the-18location respectively were cloned into pGL3-basic vector to construct the pGL3-basic-C/A recombinant vectors and were identified by enzyme digestion and sequencing;(2) The pGL3-basic-C/A vectors were converted into pGL4.17-C/A vectors through enzyme digestion and were also identified by enzyme digestion and sequencing;(3) Identified recombinant vectors and control plasmid pRL-TK were transfected into H441cells by Lipofectamine2000and luciferase assays (firefly and renilla) were performed using the Dual-Luciferase Reporter Assay System;(4) Fifty blood samples collected from full-term newborns admitted into neonatal intensive care unit (NICU) in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from January2012to December2012. Genome DNA was extracted from PBMs and genotyping of SP-B-C/A (-18) was analyzed. Total RNA of PBMs was also extracted and real-time quantitative PCR was used to detect the SP-B mRNA level. Results (1) The sequences of SP-B promoter in the recombinant luciferase reporter plasmids were accordance to the one published on Genebank. The base of-18location in pGL3-basic/pGL4.17-C was C and the base of pGL3-basic/pGL4.17-A was A, respectively.(2) The firefly/renilla luciferase activity ratios of pGL3-basic-C and pGL3-basic-A was2.97±0.14,2.25±0.09, respectively, and they were all higher than that of pGL3-basic control plasmid (0.20±0.06)(P<0.01); The ratio of pGL3-basic-C was higher than that pGL3-basic-A (P<0.01);(3) The firefly/renilla luciferase activity ratios of pGL4.17-C and pGL4.17-A were66.46±3.83,47.50±3.06respectively, and they were all higher than that of pGL4.17control plasmid (4.30±0.44)(P<0.01); The ratio of pGL4.17-C was higher than that pGL4.17-A (P<0.01);(4) The level of SP-B mRNA in PBMs of full-term infants with CC genotype (SP-B-C/A(-18)) was significantly higher than that of AA genotype (P<0.01), and the AC genotype was also higher than AA (P<0.01). However, there was no difference between genotype CC and genotype AC (P>0.05). Conclusions (1) The pGL3-basic/pGL4.17-C/A vectors are successfully constructed with SP-B promoter activity in H441cells. The SP-B-C/A(-18) promoter polymorphism can influence the transcriptional activity of SP-B gene and the promoter activity of C allele was higher than A allele;(2) The luciferase activity of pGL4.17recombinant plasmid was higher than that of pGL3-basic recombinant plasmid, So pGL4.17recombinant vector is the better choice for further study;(3) SP-B-C/A (-18) polymorphism also affects the SP-B gene transcription in vivo. Part Ⅱ Relation between surfactant protein B-C/A (-18) polymorphism and hyperoxia-induced SP-B promoter activityObjective To explore the regulation of SP-B-C/A(-18) promoter polymorphism on SP-B promoter activity under hyperoxia and related mechanisms. Methods (1) H441cells were randomly divided into2groups:normoxia group (21%02/5%C02) and hyperoxia group (95%02/5%C02). Real-time quantitative PCR was used to measure the SP-B mRNA expression levels of H441cells(4h,8h,12h,24h) and Western blot was used to detect the SP-B protein expression levels of H441cells (12h,24h,48h); Western blot was also used to detect the transcription factor Spl expression levels of H441cells in whole-cell protein and nuclear extraction (6h,12h,24h);(2) The pGL4.17-C/A recombinant vectors and control plasmid pRL-TK were transfected into H441cells by Lipofectamine2000. The cells were randomly exposed to95%O2or21%O2the next day, then cells were lysed (4h,8h,24h) and luciferase activity (firefly/renilla ratio) were detected using the Dual-Luciferase Reporter Assay System;(3) The pGL4.17-C/A recombinant vectors, pRL-TK plasmid and different amounts of CMV-Spl plasmid (0,0.25μg,0.5μg) were transfected into H441cells and luciferase activities were detected24h afterwards. Results (1) Hyperoxia increased the SP-B mRNA expression levels of H441cells at8h,12h and24h (P<0.05); Hyperoxia increased SP-B protein expression levels of H441cells at12h and24h (P<0.05);(2) There was no significant difference of Spl expression levels in whole-cell protein between normoxia group and hyperoxia group (6h,12h and24h)(P>0.05), whereas hyperoxia increased the Sp1expression levels in nuclear extraction (6h and12h)(P<0.05);(3) Hyperoxia increased the luciferase activity of pGL4.17-C/A recombinant vectors at8h and24h (P<0.05); The degree of elevation of luciferase activity (hyperoxia/normoxic) of pGL4.17-C was higher than pGL4.17-A at8h and24h (P<0.05);(4) Overexpression of CMV-Sp1plasmid (0.25μg,0.5μg) increased the luciferase activity of pGL4.17-C/A recombinant vectors (P<0.05); There was no significant difference of elevation degree of luciferase activity of pGL4.17-C/A with0.25μg CMV-Sp1(P>0.05) whereas0.5μg CMV-Spl plasmid raised the luciferase activity of pGL4.17-C more effectively than pGL4.17-A (P<0.05). Conclusions (1) Hyperoxia can upregulate the expression level of SP-B mRNA and protein by enhancing the transcription of SP-B gene;(2) SP-B-C/A (-18) polymorphism affects the transcriptional regulation of hyperoxia on SP-B promoter activity;(3) Hyperoxia enhances the transcription of SP-B gene may through increasing the nuclear translocation of transcription factor Spl;(4) Transcription factor Spl plays a positive role in regulating the expression of SP-B gene, and SP-B-C/A (-18) polymorphism affects the transcriptional regulation of Spl on SP-B promoter activity.