Effect Of Over-expression Of E-cadherin On Epithelial-mesenchymal-transition In HK-2Cell Induced By High Glucose

Objective:To observe the effect of over-expression of E-cadherin (E-cad) by transfecting with cDNA plasmid of E-cad on epithelial-mesenchymal-transition induced by high glucose and the expression of extracellular matrix in human renal tubular epithelial cell(HK-2).Methods:(1) Using E. coli DH5a to amplify the E-cad cDNA plasmid and blank plasmid, the plasmid was identified by enzyme digestion and DNA sequencing analysis.(2) HK-2were cultured and divided into3groups:normal control group(NG), mannitol control group(MG), high glucose group(HG). The morphological changes of cell were observed by inverted microscope; The level of mRNA and protein expression in Oh,24h,48h,72h about E-cad and a-SMA were assessed by Real time PCR and Western blot. COL-Ⅰ, COL-Ⅲ, and FN synthesis were detected by enzyme-linked immunosorbent assay (ELISA).(3) HK-2were cultured and divided into3groups:NG, E-cad group, blank plasmid group. The plasmids were transient transfected into HK-2using Lipofectamine2000reagent. The level of mRNA and protein expression in48h about E-cad and were assessed by Real time PCR and Western blot.(4) HK-2were cultured and divided into5groups:NG, HG, E-cad group, E-cad+High glucose group, blank plasmid+High glucose group. To further study the effect of over-expression about E-cad on EMT the induced by high glucose. The level of mRNA and protein expression in48h about E-cad and a-SMA were assessed by Real time PCR and Western blot. ELISA were used to detect the protein expression of COL-Ⅰ, COL-Ⅲand FN.Results:(1) The recombinant plasmids were verified by sequencing analysis, identifying with the sequence in genebank.(2) HK-2stimulated by high glucose became elongated and the number of cells decreased, especially remarkable in48h,72h. The cell in MG didn’t show any morphological change. After treating with high glucose, compared with NG and MG, the levels of E-cad mRNA and protein were significantly decrease in HG(p<0.05); The levels of a-SMA mRNA and protein were significantly increased with the treatment of high glucose (p<0.05). ELISA results showed upregulations of COL-Ⅰ COL-Ⅲ and FN after48h treated with high glucose, there was significant difference compared with the NG and MG (p<0.05)(3) Fluorescence microscopy showed that constructing vector can be transfected effectingly and expressed successfully. Real time PCR and Western blotshowed that, the mRNA and protein levels of E-cad and a-SMA compared with0time point were inecreased in a time-dependent manner (24h,48h,72h, P<0.05).(4)The results showed that upon the stimulation of high glucose, the expression of E-cad significantly decreased(p<0.05), while the level significantly decreased by pretransfecting with E-cad plasmid; Compared with NG, the levels of a-SMA mRNA and protein were significantly increased in the group HG and group blank plasmid+high glucose (p<0.05), but significantly decreased in group E-cad+high glucose (p<0.05); ELISA showed that pretransfecting with E-cad plasmid, the up-regulations of COL-Ⅰ, COL-Ⅲ and FN can be significantly suppressed(p<0.05).Conclusions:(1) High glucose promote EMT in HK-2.(2) Over-expression of E-cad can restrain epithelial-mesenchymal transition induced by high glucose in HK-2, and down-regulation the expression of.