Construction of a lentiviral vector overexpressing fibronectin type III domain containing 5 to inhibit apoptosis of endothelial cells
Ou Hangjun, Zhao Guangjian, Pan Yujia, Gong Caiwei, Zhao Quanwei, Liu Danan
Department of Cardiology, Affiliated Hospital of Guizhou Medical University, Institute of Medical Sciences, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
Abstract: BACKGROUND: Fibronectin type III domain containing 5 (FNDC5) is a protein molecule that regulates glycolipid metabolism, regulates cell apoptosis and participates in the inhibition of atherosclerosis.
OBJECTIVE: By constructing a lentiviral vector overexpressing FNDC5 for transfecting human umbilical vein endothelial cells, to verify the effect of FNDC5 in regulating endothelial cell apoptosis.
METHODS: The lentiviral vector overexpressing FNDC5 was constructed. Human umbilical vein endothelial cells in logarithmic growth phase were cultured and divided into three groups: blank group was not transfected with lentivirus, control group was transfected with an empty vector lentivirus without FNDC5, and experimental group was transfected with the lentiviral vector overexpressing FNDC5. At 7 days after transfection, RT-PCR and western blot were used to detect the mRNA and protein expression of FNDC5. After 7 days of transfection, oxidized low-density lipoprotein solution was added to the three groups of cells, and the cell viability was determined by cell counting kit-8 method, and cell apoptosis was detected by Hoechst 33342/PI double staining and flow cytometry.
RESULTS AND CONCLUSION: The mRNA and protein expressions of FNDC5 in the experimental group were higher than those in the blank and control groups (P < 0.05). Cell counting kit-8 test results showed that the cell viability in the experimental group was higher than that in the blank and control group at 1 and 2 hours after addition of oxidized low-density lipoprotein (P < 0.05). However, there was no significant difference in the cell viability between the three groups at 4 hours after addition of oxidized low-density lipoprotein (P > 0.05). Flow cytometric results showed that the apoptotic rate was lower in the experimental group than the blank and control group at 24 hours after addition of oxidized low-density lipoprotein (P < 0.05). Hoechst 33342/PI double staining results showed that the number of apoptotic cells in experimental group was less than that in the blank and control groups. To conclude, overexpression of FNDC5 could inhibit the apoptosis of human umbilical vein endothelial cells.
Key words: fibronectin type III domain containing 5, lentivirus, transfection, human umbilical vein endothelial cells, apoptosis, atherosclerosis