Gulo Regulates The Proliferation,Apoptosis And Mesenchymal-to-epithelial Transformation Of Metanephric Mesenchyme Cells Via Inhibiting Six2

Objective:To study the expression pattern of L-gulono-?-lactone oxidase(Gulo)in embryonic kidney of C57BL/6 mice,and to explore its effects on proliferation,apoptosis and mesenchymal-to-epithelial transformation(MET)in metanephric mesenchyme(MM)cells,so as to illustrate the potential biological function of Gulo in kidney development.Methods:1.Embryonic kidneys of C57BL/6 mouse at different development stages were isolated and the Gulo expression was detected by RT-qPCR;The expressions of Gulo in MM cells at different development stages,mK3 cells and mK4 cells,were detected by RT-qPCR.2.Gulo was overexpressed in mK3 cells and knocked down in mK4 cells,and then the markers in MET process and Sine oculis related homeobox 2(Six2)were detected by Western Blot.Cell scratch assay was used to detect the change of cell migration ability.5-Ethynyl-2'-deoxyuridine(Edu)assay was used to detect cell proliferation.Cell apoptosis was detected by flow cytometry.3.Six2 was over-expressed in mK3 cells with Gulo over-expression,and Six2 was silenced in mK4 cells with Gulo knocked-down.Western Blot was performed to detect the related markers of MET process.Cell scratch assay was conducted to detect migration ability.The proliferation ability of cells was detected by Edu assay.Cell apoptosis was detected by flow cytometry.Results:1.Gulo expression was significantly increased at embryonic 12.5 days(E12.5)in C57BL/6 mouse embryonic kidney,and lower in undifferentiated MM(mK3)cells than in differentiated MM(mK4)cells.2.Over-expression of Gulo can promote the MET and apoptosis of mK3 cells and inhibit proliferation.Knock-down of Gulo in mK4 cells made its epithelial character cells unstabilized,promoted proliferation and inhibited apoptosis.3.Six2 is negatively regulated by Gulo.Over-expressing Six2 in mK3 cells with over-expression of Gulo and silencing Six2 in mK4 cells with knock-down of Gulo could partially rescue the MET,proliferation and apoptosis of MM cells induced by Gulo's increase or deficiency.Conclusion:In summary,these findings reveal that Gulo expression was significantly increased in mouse embryonic kidney E12.5,and Gulo expression in undifferentiated MM(mK3)cells was significantly lower than that in differentiated MM(mK4)cells.In addition,Gulo promotes the MET and apoptosis of MM cells and inhibites their proliferation by negatively regulating Six2.