The Effect Of Human S100A13 Gene Lentiviral RNAi Vectors On The Release Of FGF-1 In Human Endothelial Cells

Object: To observe the possessing role of S100A13 in the release of FGF-1, we constructed the shRNA lentiviral RNAi vectors targeting S100A13 gene, and tested the silencing effect to S100A13 gene by the specific S100A13 shRNA in the human umbilical vein endothelial cells, then further studied whether the release of FGF-1 has changed after silencing of S100A13 gene and serum-deprivation.Method: We designed a pair of single stranded DNA oligonucleotide specially targeting S100A13 gene, top and bottom strand oligos generate the double-stranded oligos after annealing, which was cloned into linearized pENTRTM/U6 entry vector using Gateway technology. With the catalysis of LR clonase II, the U6 RNAi cassette in pENTRTM/U6 entry vector was transferred into pLenti6/BLOCK-iTTM-DEST Vector using the LR recombination reaction to form S100A13 shRNA pLenti6/BLOCK-iTTM expression clone. Human umbilical vein endothelial cells were transfected with shRNA lentiviral RNAi vectors for target gene S100A13 and for independent gene SR-PSOX, then immunofluorescence, real–time RT-PCR, western-blot were used to detect the suppression efficiency of S100A13. And HUVEC cells were treated with S100A13 gene inhibition and serum-deprivation, immunofluorescence and ELISA detected the change of release of FGF-1.Result: 1. The target sequence coding shRNA of the S100A13 gene inserted correctly to the pENTRTM/U6 entry vector, and connected with U6 promoter and PolIII terminator correctly to form U6 RNAi cassette.2. The potent suppression efficiency of S100A13 shRNA lentiviral RNAi vectors targeting specificly S100A13 gene reached 80% using real-time RT-PCR,western-blot,indirect immunofluorescence analysis, and independent gene(SR-PSOX) shRNA lentiviral RNAi vector did not inhibit the expression of S100A13 gene.3. Using indirect immunofluorescence analysis, The silence of expression of the S100A13 gene did not affect the expression and distribution of FGF-1 in HUVEC cells. While after cells were treated by serum-deprivation, FGF-1 in HUVEC reduced and redistributed mostly adjacent to cell membrane but not perinuclear region. After the expression of S100A13 was inhibited in HUVEC, cells was given by serum-deprivation stress, FGF-1 redistributed similarly to adjacent to cell membrane. Cell culture supernatant was analyzed by FGF-1 ELISA, in the supernatant of three cell groups of the control,S100A13 RNAi and treated by serum-deprivation after S100A13 RNAi, the density of FGF-1 was very lowed, and the difference was not significant; while in the supernatant of cells were given by serum-deprivation, the density of FGF-1 was increased obviously , compared to the forward groups, and the difference was significant (P<0.05).Conclution: 1. S100A13 gene shRNA lentiviral RNAi vectors(containing the entry and expression vector) was successfully constructed, and acquired the 80% suppression efficiency targeting specificly S100A13 gene. 2. S100A13 may contribute to the final export mechanism in the FGF-1 non classical release route as a key cargo protein .