Effect and mechanism of lentiviral vector-mediated FoxO1 expression on proliferation of rat mesangial cells cultured under high glucose conditions

Abstract

Objective To study the effect and mechanism of forkhead transcriptionfactor O1(FoxO1) on proliferation of rat mesangial cells(MCs) cultured under high glucose conditions. Methods Constructing lentiviral vectors of LV-CA-FoxO1 and LV-siRNA-FoxO1 were used to upregulate or downregulate FoxO1. Moreover, negative control LV-NC-FoxO1 was also constructed. Rat MCs were separately cultured in normal glucose(5.6 mmol/L, NG group), only high glucose(30 mmol/L, HG0 group), LV-NC-FoxO1 with HG(HG1 group), LV-CA-FoxO1 with HG(HG2 group), and LV-siRNA-FoxO1 with HG(HG3 group) for 72 h. MTT assay and flow cytometrywas were used to analyze cell proliferation and cell cycle distribution. The expression of FoxO1, cyclin-dependent kinase inhibitor(p27), cyclinD1, and cyclin-dependent kinase 4(CDK4) were detected by QRT-PCR and Western blot. Results The MCs proliferation rate in HG0 group was faster than that in NG group. Besides, there were no statistical differences in FoxO1 expression and proliferation rate of MCs between HG0 group and HG1 group.Nevertheless, LV-CA-FoxO1 promoted cell cycle arrest at the G1 phase and attenuated proliferation rate, along with upregulation of FoxO1 and p27 and downregulation of cyclin D1 and CDK4 in HG2 group(all P<0.05). Moreover, degradation of FoxO1 by LV-siRNA-FoxO1 stimulated hyperproliferation of MCs, associating with decline of p27 and increase of cyclin D1 and CDK4 in HG3 group(all P<0.05). Conclusion The proliferation of MCs induced by high glucose is regulated by utilizing transgenic technology targeted and regulated FoxO1 expression and consequently through FoxO1/p27 signaling pathway. These findings indicate that FoxO1 seems to be a new therapeutic target for early diabetic nephropathy. (Chin J Endocrinol Metab, 2015, 31: 162-168) Key words: Forkhead transcription factor O1; High glucose; Lentiviral vectors; Mesangial cells; Proliferation