The Role Of FUT6 In TGF-β₁ Induced Epithelial-Mesenchymal Transition Of Renal Tubular Epithelial Cells

Objective:To study the role of α-1,3fucosyltransferase(FUT6)on the epithelial-mesenchymal transition(EMT) that induced by transforming growth factor-β1 in human renal proximal tubular epithelial cells(HK-2);to explore plausible mechanism of the activation of the TGF-β/Smad pathway in early stages of diabetic nephropathy(DN). This study is to provide information on the genetic etiology of DN.Methods:(1) We selected a type 2 Diabetic Nephropathy(DN) subject that with early activation of TGF-β/Smad signal pathway. Genome DNA was extracted from peripheral blood. Using Agilent SureSelect Human Kit chip for exome capturing, sequencing was performed on an Illumina HiSeq 2000 high-throughput sequencing platform after exon library was established.(2) Sequencing annotation:gene polymorphisms was filtered by public databases YH, dbSNP129, eight HapMap exomes and dbSNP thousands genome. Experssion profiles of mutant genes were checked by the UCSC Genome Bioinformatics(Genome.ucsc.edu).(3) Expression of FUT6 were evaluated by immunohistochmistry with Streptavidin-HRP(AP) for the renal biopsy of the DN patient.(4) Using fluorescent labeled siRNA transient transfected into HK-2 cells, using fluorescence microscopy to check transefection efficiency of siRNA. Slience-effect was assessed by western blot.(5) To evaluted EMT effects of siRNA interference, HK-2 cells were randomly divided into four groups: the NC grop, HK-2 cells were cultured in normal media, as the negative control group; the TGF group, HK-2 cells were cultured 48 h with TGF-β1 at concentration of 10ng/ml; the RNAi group, FUT6-siRNA(50nM) was transient transfected into HK-2 cells to knock-down the expression of FUT6; the iTGF group, 50 nM FUT6-siRNA was transient transfected into HK-2 cells, followed by a 48 h culture with 10ng/ml TGF-β1. Phase contrast microscopy was used to observe cell morphological changes; expressions of FUT6, TGF-βRⅠ, TGF-βRⅡ, Smad2/3 and p-Smad2/3 were determined by western blot; EMT indexes of correlation(including Snail,E-cadherin,N-cadherin and Vimentin) were determined by western blot; in addition, expressions of Fucα-1,3GlcNac that specifically conjoined with TGF-βRⅠand TGF-βRⅡ were detected by immunoprecipitation(IP) and lectin blotting analysis(IB).(6) TGF-β1 stimulating tests: HK-2 cells were cultured 1d, 2d and 3d with TGF-β1 at the concentration of 10ng/ml. Phase contrast microscopy was used to observe cell morphological changes; expressions of FUT6, TGF-βRⅠand TGF-βRⅡwere determined by western blot; expressions of Fucα-1,3GlcNac that specifically conjioned with TGF-βRⅠand TGF-βRⅡ were detected by IP and IB.Results:(1) Exome sequencing found 38914 single nucleotide variants. Through data filtering, there were 509 novel mutations were identified in the DN patient, including 497 missense mutations and 12 nonsense mutations. In 12 genes with stop codon mutations, ANKRD35, ACSM2 A, FUT6 and HAAO are highly expressed in kidney. A mutation of codon 315 TAA>TAC in the FUT6 gene, changed the amino acid sequencing from Tyr to stop codon. Immunohistochemistry confirmed the decreasing of FUT6 expression in renal biopsies of the DN patient.(2) Fluorescence microscopy and western blot proved that FUT6-siRNA at concentration of 50 nM and 100 nM could successfully knocked down the FUT6 expression by approximately 50%.(3) Phase contrast microscopy revealed: the morphology of NC group showed normal epithelial cells as the cobblestone pattern; the TGF group cells showed enlarged,elongated fusiform cells; compared with NC group,the morphology of RNAi group changed very little; the iTGF group cells changed to fusiform, but the morphological changes were less significan than those in the TGF group.(4) Compared with the NC group, the FUT6 expression reduced in the RNAi group, but up-grated in the TGF and the iTGF group, this illustrated FUT6 upgrated induced by TGF-β1,while siRNA transient transfection unable to inhibited increased expression of FUT6 induced by TGF-β1. Compared with the NC group, the expression of p-Smad2/3 were increased in the TGF,RNAi and iTGF groups. Expression of smad2/3 were increased in the RNAi and the iTGF groups while almost unchanged in the TGF group. Expressions of snail,N-cadherin and Vimentin expressed were elevated compared with NC group. The E-cadherin expression reduced, which indicated changing to fibroblasts of TGF group; the expression of Snail, E-cadherin,N-cadherin and Vimentin was almost not changed in the RNAi group; In the iTGF group, expression of Snail, N-cadherin and Vimentin reduced compared with the TGF group, the expression of E-cadherin was not changed. This may explain that FUT6-siRNA transfection could suppressed EMT which induced by TGF-β1.(5) IP combined with lectin blot revealed that the level of Fucα-1,3GlcNac expression conjoined with TGF-βRⅠ and TGF-βRⅡ. The NC group and the TGF group had very little change, but fucosylation were obviously down-regulated in the RNAi and the iTGF groups.(6) HK-2 cells were stimulated by TGF-β1 for different lengths of time. The cell shape changed to fusiform during the TGF-β1 simulation, the expression of FUT6 was first increased and then declined, same tendencies were observed for TGF-βRⅠand TGF-βRⅡ. IP combined with IB revealed that levels of fucostlation were not changed that conjoined with TGF-βRⅠ and TGF-βRⅡ.Conclusion:(1) The fucosylation on TGF-βRⅠand TGF-βRⅡwere regulated by FUT6.(2) After transient transfected by FUT6-siRNA, the TGF-β/Smad signaling pathway were activated.(3) Inhibition of fucosylation on TGF-βRⅠand TGF-βRⅡcould suppress EMT that induced by TGF-β1.Therefore FUT6 could regulated the TGF-β/Smad signaling pathway and the EMT of renal tubular epithelial cells by affected fucosylation on TGF-βRⅠand TGF-βRⅡ.