Growth Hormone Strongly Induces hSMN2 Promoter Driving Construct Gene Expression in Mammalian Cells
1Marmara University School of Medicine, Institute of Health Sciences, Department of Medical Genetics, İstanbul, Türkiye
Objective:
Spinal muscular atrophy (SMA) is the most common neurodegenerative disease caused by the absence or insufficiency of the survival motor neuron (SMN) protein. Human SMN1 (hSMN1) produces fully functional SMN protein but hSMN2 produces only about 10% functional protein. Deletion or mutation in hSMN1 gene leads to SMA, while the hSMN2 copy number modifies disease severity. Increasing hSMN2 expression has emerged as a potential therapeutic approach. In this study, we investigated the effect of growth hormone (GH) on hSMN2 promoter activity using a reporter in Chinese hamster ovary (CHO) cells.
Methods:
Three different hSMN2 promoter regions (588 bp, 1036 bp and 1705 bp) were used to show the effect on gene expression of reporter response to GH in this study. Promoters were amplified by polymerase chain reaction (PCR) and cloned into the pGL3 luciferase reporter vector. The ligation reactions were transformed into DH5α cells and positive colonies containing specific hSMN2 promoter inserts were confirmed by PCR with hSMN2-primers. The plasmids carrying hSMN2 promoters were transfected into CHO cells. After transfection, the cells were treated with GH for 24 hours and luciferase activity was measured to assess promoter activity.
Results:
All hSMN2 promoter constructs responded to GH. The 1036 bp promoter construct showed the highest luciferase expression upon GH treatment. However, the 1705 bp promoter construct exhibited reduced gene expression compared to the control vector treated with GH.
Conclusion:
These findings suggest that GH can modulate hSMN2 expression in hSMN2 promoter dependent manner. GH may be a candidate hormone for SMA treatment by enhancing hSMN2 expression.
