Yanchuang Liang1,2, Yanzheng Song1, Fengju Zhang1, Mingshen Sun1, Ningli Wang1. 1. a Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University , Beijing Ophthalmology & Visual Sciences Key Lab , Beijing , China. 2. b Civil Aviation Medical Center , Civil Aviation Administration of China , Beijing , China.
Abstract
PURPOSE: To elucidate the role of the protein coding laminin α1 (LAMA1) gene in pathological myopia (PM) at the transcriptional level. To achieve this, the binding affinity of single nucleotide polymorphism (SNP) rs2089760-located on the LAMA1 promoter gene-to human fetal scleral fibroblast (HFSF) nucleoprotein was investigated and its effect on LAMA1 transcriptional initiation activity was analyzed. METHODS: Binding interactions of the HFSF nucleoprotein and biotin-labeled SNP rs2089760 probe were investigated by amplifying the LAMA1 promoter gene and performing overlap extension polymerase chain reaction (PCR) to obtain the G/A mutation of LAMA1 SNP rs2089760. Recombinant adenovirus vectors, Ad5f11p-pLAMA1SNPa-Luc2, Ad5f11p-pLAMA1SNPg-Luc2, and Ad5f11p-CMV-RLuc, were constructed. Fluorescence intensity ratios of firefly luciferase (FLuc) and renilla luciferase (RLuc) vectors were measured 48 h after HFSF infection. RESULTS: Both specific and mutant probes banded precisely with HFSF nucleoprotein. The intensity value of the mutant probe was significantly lower than that of the specific probe (p < 0.05). HFSFs were successfully infected by the recombinant adenoviruses. The FLuc/RLuc fluorescence intensity ratio of Ad5f11p-pLAMA1SNPa-Luc2 (0.0238 ± 0.0009) was significantly lower than that of Ad5f11p-pLAMA1SNPg-Luc2 (0.0281 ± 0.0015) (p < 0.05). CONCLUSIONS: It is highly likely that SNP rs2089760 in the LAMA1 promoter region is located at the transcription factor binding site. The SNP rs2089760 G > A mutation reduces transcription factor binding ability and transcriptional initiation activity, and negatively regulates gene transcription of LAMA1. We suggest that LAMA1 SNP rs2089760 plays an important role in the development of PM.
PURPOSE: To elucidate the role of the protein coding laminin α1 (LAMA1) gene in pathological myopia (PM) at the transcriptional level. To achieve this, the binding affinity of single nucleotide polymorphism (SNP) rs2089760-located on the LAMA1 promoter gene-to human fetal scleral fibroblast (HFSF) nucleoprotein was investigated and its effect on LAMA1 transcriptional initiation activity was analyzed. METHODS: Binding interactions of the HFSF nucleoprotein and biotin-labeled SNP rs2089760 probe were investigated by amplifying the LAMA1 promoter gene and performing overlap extension polymerase chain reaction (PCR) to obtain the G/A mutation of LAMA1 SNP rs2089760. Recombinant adenovirus vectors, Ad5f11p-pLAMA1SNPa-Luc2, Ad5f11p-pLAMA1SNPg-Luc2, and Ad5f11p-CMV-RLuc, were constructed. Fluorescence intensity ratios of firefly luciferase (FLuc) and renilla luciferase (RLuc) vectors were measured 48 h after HFSF infection. RESULTS: Both specific and mutant probes banded precisely with HFSF nucleoprotein. The intensity value of the mutant probe was significantly lower than that of the specific probe (p < 0.05). HFSFs were successfully infected by the recombinant adenoviruses. The FLuc/RLuc fluorescence intensity ratio of Ad5f11p-pLAMA1SNPa-Luc2 (0.0238 ± 0.0009) was significantly lower than that of Ad5f11p-pLAMA1SNPg-Luc2 (0.0281 ± 0.0015) (p < 0.05). CONCLUSIONS: It is highly likely that SNP rs2089760 in the LAMA1 promoter region is located at the transcription factor binding site. The SNP rs2089760 G > A mutation reduces transcription factor binding ability and transcriptional initiation activity, and negatively regulates gene transcription of LAMA1. We suggest that LAMA1 SNP rs2089760 plays an important role in the development of PM.
Entities:
Keywords:
Gene transcription; LAMA1 gene; human fetal scleral fibroblast; pathological myopia; single nucleotide polymorphisms