| Objective:To investigate the impact of homocysteine on the expression of folate receptors.To observe the influence of the silence of folate receptor α on the apoptosis of human umbilical vein endothelial cells, and to study the interrelations of the influences above with the activation of PERK, a key protein in endoplasmic reticulum stress pathway.Methods:1.Homocysteine was used with different concentrations( 0μM/L, 50μM/L,100μM/L, 200μM/L, 500μM/L) to intervene HUVECs cultured in vitro. After24 hours, we used inverted microscope to observe the cellular morphological changes;CCK-8 to detect cell survival rate of each group; Real-time qPCR to detect the mRNA expression of five kinds of folate receptor gene FOLR1, FOLR2, FOLR3,SLC46A1, SLC19A1; Western blot was used to detect the expression of FRα.2.siRNAs of FRα were transfected into HUVECs with liposome.With fluoresce-ence microscope,we could observe the expression of green fluorescent protein in HUVECs. Then western blot was used to detect FRα protein expressionand select the best FRα-siRNA.3. Groups of the experiment were blank control group, negative control group,FRα-siRNA group. Annexin V + PI double staining flow cytometry was used to detectapoptosis rate and western blot was used to detect the expression of PERK and p-PERK.Results:1. With the increase of Hcy concentration, HUVECs gradually appear damage change in morphology, cell survival rate decreases gradually.When Hcy>50μM/L,after 24 hours, HUVECs survival were decreased compared with blank control group(P<0.05), and difference between each group was statistical significant(P<0.05).2.In human umbilical vein endothelial cells, the expression of FOLR2, FOLR3 especially FOLR2 mRNA was very low. With the increase of Hcy concentration, the mRNA expression of FOLR1 and SLC46A1 increased at first and decreasedsubsequently,increased at first and tended to be constant respectively. The relative mRNA expression of FOLR1, SLC46A1, SLC19A1 reached the peak in 50μM/L Hcy intervention group, and they were higher than the blank control group(P<0.05).The relative mRNA expression of FOLR1 in 200μM/L Hcy intervention group was decreased compared with the blank control group(P<0.05).3.With the increase of Hcy concentration, the protein expression level of FRαincreased at first and decreased subsequently, and reached the peak in 50μM/L Hcy intervention group. The protein expression of FRα in 50μM/L Hcy intervention group was higher than the blank control group(P<0.01), The protein expression of FRα in200μM/L Hcy intervention group was lower than the blank control group(P<0.05).The differences of protein expression of FRα between 100μM/L Hcy intervention group and blank control group was not statistically significant(P > 0.05).The differences between 50μM/L Hcy intervention group and 200μM/L Hcy intervention group, 100μM/L Hcy intervention group and 200μM/L Hcy intervention group was statistically significant(P<0.01).4.Green fluorescence was observed by inverted fluorescence microscope in HUVECs transfected with segments of negative control, three kinds of FRαinterference fragments FOLR1-homo-132, FOLR1-homo-272, FOLR1-homo-597.The transfection efficiency was 75% on average. Western blot detected the expression of FRα could be effectively inhibited after transfection, the result also showed that FOLR1-homo-132 interference segment with best suppression efficiency whose protein inhibition rate was 87.19%.5.Flow cytometry found that the cell apoptosis rates of blank control group,negative control group, FRα-siRNA group(%) were: 3.56±1.2, 5.12±2.1 and 2.1±3.0after 12 h transfection in HUVECs, the apoptosis rate of FRα-siRNA group and negative control group increased compared with blank control group(P < 0.01).6.Western blot measured a low level protein expression p-PERK in blank control group. The p-PERK/PERK ratio in FRα-siRNA group increased compared with blank control group and negative control group,the difference was statistically significant(p<0.01).Conclusions1.Homocysteine can make traumatic change in human umbilical vein endothelial cell morphogenesis and reduce human umbilical vein endothelial cell survival rate, which is dose-depended.2.The mRNA expression of FOLR2, FOLR3 especially FOLR2 in human umbilical vein endothelial cells are very low. The relative mRNA expression of FOLR1, SLC46A1, SLC19A1 in human umbilical vein endothelial cells can be promoted first and restrained aferwards by homocysteine.3. Homocysteine can affect gene expressive quantity of folate receptors represented by FRα.The mRNA and protein expression of FRα are promoted first and restrained aferwards. Its specific mechanism still need further exploration.4.Silence FRα can induce the apoptosis of human umbilical vein endothelial cells.5. PERK which is a marker of endoplasmic reticulum stress pathway may participate in the process of the apoptosis of human umbilical vein endothelial cells induced by FRα-siRNA.6. FRα may be an important link of human umbilical vein endothelial cell injury induced by homocysteine. |
PDF Full Text Request