J Cancer 2019; 10(4):903-910. doi:10.7150/jca.28169

Research Paper

Regulatory sequence analysis of semaphorin 4D 5' non-coding region

Lijuan Qiu1,2,3*, Hongchao Jiang2*, Jia Luo1,3,4, Juemin Xi1,3, Xiaodan Wang1,3, Yue Pan1,3, Junying Chen1,3, Yujiao Zhao1,3, Qiangming Sun1,3✉

1. Institute of Medical Biology, Chinese Academy of Medical Sciences, and Peking Union Medical College, Kunming 650118, PR China
2. Kunming Children's Hospital (The Affiliated Children's Hospital of Kunming Medical University), Institute of Yunnan Pediatric Disease Research, Kunming 650228, PR China
3. Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming 650118, PR China
4. Kunming Medical University Haiyuan College, Kunming 650106, PR China
*These authors contributed equally to this work.

Abstract

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.

Keywords: Hypoxia Inducible Factor 1α, Semaphorin 4D, 5' non-coding Region

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How to cite this article:
Qiu L, Jiang H, Luo J, Xi J, Wang X, Pan Y, Chen J, Zhao Y, Sun Q. Regulatory sequence analysis of semaphorin 4D 5' non-coding region. J Cancer 2019; 10(4):903-910. doi:10.7150/jca.28169. Available from http://www.jcancer.org/v10p0903.htm