Response of CREG1 promoter to serum starvation in human vascular cells

AIM: In order to explore the regulatory mechanisms of the human cellular repressor of E1A-stimulated genes (hCREG1) in vascular smooth muscle cells (VSMCs)-HITASY and in human umbilical vein endothelial cells (HUVECs), we clone and construct hCREG1 promoter vector to detect its expression in different vascular cells. METHODS: With the results of biological information, the fragments including -3 677 bp, -2 310 bp and -945 bp upstream sequences of hCREG1 were amplified respectively using human genomic DNA template by PCR. The products were inserted into pMD18-T vector, and then were subcloned into pEGFP-1 report vector to obtain the pEGFP-hCREG1-promoter vectors. The pEGFP-hCREG1-P3677, pEGFP-hCREG1-P2310 and pEGFP-hCREG1-P945 vectors were transfected into HITASY cells and HUVECs transiently and the expression of green fluorescent protein (GFP) was detected respectively by Western blotting and fluorescent microscope. RESULTS: It was confirmed that all three PCR fragments inserted into vectors were corrected by sequencing analysis. However, the expression of GFP was different significantly in both types of vascular cells. The expression of GFP in HUVECs was higher than that in HITASY cells. Meanwhile, the expression of GFP in HITASY cells with 0.5% FBS was increased obviously compared to that in HITASY cells with 10% FBS. CONCLUSION: The reporter vectors of hCREG1 promoter are constructed successfully in which the core promoter region might be located in -945 bp-0 bp of 5′ upstream sequences. This study will provide an experimental basis for exploring the regulation of hCREG1 expression.