Semaphorin 4D (
Sema4D) has been proven to be one of the
hypoxia effectors regulated by
hypoxia inducible factor (HIF-1) in multiple cells, and play a role in angiogenesis like
VEGF. However, the regulatory sequence characteristics of the
Sema4D are not clarified. The possible
hypoxia response element (HRE) sequences in 5' non-coding Region before ATG
start codon of
Sema4D were screened, followed by point mutagenesis and
luciferase assay analysis. Sequencing and alignment of this region in 11
cancer cell lines and 4 normal cell lines were also performed, followed by cloning, mutation and
luciferase assay analysis. The results showed that there were four possible HREs (HRE1-4) sequences in 1275bp range before ATG
start codon. Among HRE1-4, HRE2 and HRE4 were functional HIF-1α binding sites. In addition, these two binding sites play different roles in the regulation of
Sema4D expression in HUVEC and Caco-2 cells. There were three
nucleotide variants (T471C/A600G/C862T) frequently detected in
cancer cell lines. The site variation rates of T471C/A600G/C862T were 72.7%, 18.2%, and 72.7% in
cancer cells respectively.
Luciferase assays showed that T471C and C862T could significantly increase the expression efficiency of downstream target genes. Furthermore, secondary structure prediction showed that mutations at T471C and C862T apparently lead to change of the gene structure. Our study describes the sequence characteristics of 5' non-coding region of
Sema4D, enhances our understanding of the regulatory mechanism of
Sema4D and benefits the development of a possible anti-angiogenesis therapeutic strategy for
malignancies.