FUT8-siRNA Mitigate Renal Tubular Epithelial Mesenchymal Transition Induced By Transforming Growth Factor-beta1 In Vitro

Objective: To investigate the effect ofα-1,6 fucosyltransferase (FUT8) on the epithelial mesenchymal transition(EMT) in human renal proximal tubular cells(HK-2) induced by transforming growth factor-β1 (TGF-β1).Methods: The cultured HK-2 cells were randomly divided into three groups: (CON group) as a negative control group, HK-2 cell cultured in normal media; (TGF group) treated HK-2 cells with TGF-β1 at different concentration of (5,10,20,40)ng/ml; (TGFF group) in this group FUT8 was silenced using siRNA methodology subsequently exposed to 20ng/ml TGF-β1. Frist of all, a fluorescent dye labeled FUT8-siRNA was syn- thesized and transient transfected into HK-2 cells. Employ fluorescence microscope to assess transefect-effect. RT-PCR and immunofluorescence were used to test FUT8 inhibition at mRNA and protein level. The changes in morphology of HK-2 cells were inspected 72 hours after adding the stimulating factors. Expressions of FUT8 and its substrate—α-1,6 fucose were evaluated by western blot and immunofluorescence respectively. In addition, expressions ofα-1,6 fucose especially binding to TGF-βtype I receptor activin-like kinase5(ALK-5) and type II receptor (TGF-βRII) were analyzed by immunoprecipitation(IP) combining lectin blot. Location of ALK5 and FUT8 was confirmed using immunofluorescent co-stain. In the meantime, phosphorylated smad2/3 and alpha-smooth muscle actin (α-SMA) were evaluated by immunocytochemistry stain.Results: RT-PCR and Immunofluorescence proved that FUT8 siRNA at concentration of 100nM knocked down FUT8 expression successfully with transefect-effect of 90%. Cells of CON group showed nomral epithelial moprhological state and ceobblestone pattern; In contrast, cells of TGF group showed marked hypertrophy , elongated shpae , which indicated changing to fibroblasts; whreras, FUT8 siRNA mitigate moprhological changes indueced by TGF-β1. Compared with CON group, FUT8 up- regulated in TGF group(P<0.05), but its substrate—α-1,6 fucose had no dramatic difference(P>0.05); in TGFF group both FUT8(P<0.01) and its substrate—α-1,6 fucose(P<0.05) were depressing compared with TGF group. IP combining lectin blot showed level of fucosylation binging TGF-βRII and ALK5 were up-regulated in TGF group compared with CON group (P<0.05), and in TGFF group they were obviously down-regulated com- pared with TGF group(P<0.01); while the whole TGF-βRII and ALK5 were no differences in the measured indexes(P>0.05). Compared CON group P-smad2/3 was significantly up-regulated by TGF-β1 in a doze-dependent manner in TGF group(P<0.05), FUT8 siRNA can inhibit TGF-β1 effects of above in TGFF group(P<0.05).Conclusion: I FUT8-siRNA down-graduates level of fucosylation on ALK5 and TGF-βRII. II Suppress level of fucosylation on ALK5 and TGF-βRII can supress activation of P-smad2/3 and its mediated renal tubular EMT. III Fucosylation of TGF-βRI,RII is crucial element for TGF-β1 reduced renal tubular EMT.