The Role Of MiRNA200c-vimentin Signaling Pathway In Steroidogenesis Stimulated By Mono-butyl Phthalate

Di-butyl phthalate (DBP) can be found in the environment ubiquitously. They can end up in human body via ingestion, inhalation or dermal absorption. Studies on rodents have identified that DBP as one of the endocrine-disrupting chemicals (EDCs) is a potent testicular toxicant. DBP as a kind of widely used phthalic acid esters (PAEs) is present in many consumer products.Data reported that DBP can inhibit steroidogenesis and damage the male reproductive function. DBP play effect anti-androgenic through its active metabolite mono-butyl phthalate (MBP). Cholesterol is the precursor of the steroid hormones, providing the backbone of the steroid molecule. The involvement of the cytoskeleton in this process of the transfer of cholesterol from the cytoplasm into mitochondria has long been under investigation. Recent studies have shown that intermediate filament (IF) proteins, which constitute part of the cytoskeleton, could be involved in steroidogenesis. Vimentin is an IF protein and few studies are available to explore the role of vimentin in the process of steroidogenesis induced by DBP and/or MBP.In this study, we used mouse Leydig tumor cells (MLTC-1) and murine adrenocortical tumor cells (Y1) as the vitro models to investigate the expression of vimentin and steroidogenesis-related proteins when the cells exposed to low doses of MBP. Subsequently, we discussed the role of miRNA200c-vimentin signaling pathway in steroidogenesis stimulated by low levels of MBP.Part I Role of miRNA200c-vimentin signaling pathway in steroidogenesis stimulated by mono-butyl phthalate in MLTC-1 cellsObjectiveTo explore the role of miRNA200c-vimentin signaling pathway in steroidogenesis stimulated by low doses of MBP in MLTC-1 cells.Methods1. The MLTC-1 cells were used as the experimental model. The function of cells are similar to the Leydig cells isolated from normal testicular tissue. Steroidogenesis is elevated after the cells stimulated by hCG or CT and the main of steroidogenesis is progesterone.2. The cell viability of MLTC-1 cells was analyzed using the method of MTT after exposed to MBP for 24h or 48h and the final doses of MBP were determined by these results.3. MLTC-1 cells were treated with different concentrations of MBP for 24h. Then hCG(0.1U/L)was added into the medium for 4h. Levels of progesterone in the medium were measured by RIA.4. mRNA expression of vimentin and miRNA200c in MLTC-1 cells was determined by Real-time PCR.5. Protein expression of StAR, P450scc,3β-HSD, vimentin and tubulin in MLTC-1 cells was assessed by western blot.6. MLTC-1 cells were treated by vim-siRNA for 6h, then cells were stimulated by MBP (10-7 M). Protein expression and progesterone levels were detected by western blot and RIA, respectively.7. miRNA200c was overexpression or lowexpression in MLTC-1 cells, which were stimulated by MBP (10"7 M), vimentin expression and progesterone levels were detected by western blot and RIA, respectively.Result1. MBP treatment did not affect cell viability at doses ranging from 10-7 to 10-4 M, while MBP caused cell viability decreased at 10-3M.2. Compared with the control group, progesterone levels were increased at 10-7 M and 10-6 M of MBP.3. Results of western blot showed that protein expression levels of vimentin and StAR corrected by corresponding GAPDH were increased in MLTC-1 cells treated by MBP at 10-7 M and 10-6 M. The expression levels of other proteins related to steroid hormone synthesis including tubulin, b-actin, P450scc, and 3β-HSD remained unchanged compared with control group.4. Vimentin protein expression and progesterone levels were inhibited after silencing of the vimentin gene as compared to cells treated with control siRNA. In contrast, vimentin protein expression and progesterone levels were promoted after the cells were stimulated with MBP at 10-7 M compared to cells treated with control siRNA.5. In MLTC-1 cells, expression of miRNA200c was downregulated at 10-7,10-6, and 10-5 M of MBP treatment compared with the control group.6. Levels of vimentin mRNA and protein were increased when miRNA200c expression was artificial inhibited in MLTC-1 cells. At the same time, progesterone synthesis was increased about 30%.7. Progesterone and expression levels of vimentin corrected by corresponding GAPDH were decreased after miRNA200c overexpression, but were increased after MBP at 10-7M treatment with or without miRNA200c overexpression.Conclusion1. MBP (10-7 and 10-6 M) increase progesterone production in MLTC-1 cells.2. miRNA200c-vimentin signaling pathway play important roles in steroidogenesis stimulated by MBP in MLTC-1 cells.Part II Role of miRNA200c-vimentin signaling pathway in steroidogenesis simulated by mono-butyl phthalate in Yl cellsObjectiveTo explore the role of miRNA200c-vimentin signaling pathway in steroidogenesis simulated by low levels of MBP in Y1 cells,Methods1. The Y1 cells were used as the experimental model. The function of cell is similar to the original generation of adrenal cortex cells. Steroidogenesis is elevated after the cells stimulated by ACTH or forskolin and the main of steroidogenesis is progesterone.2. The cell viability of Y1 cells were analyzed using the method of MTT after exposed to MBP for 24h or 48h and the final doses of MBP were determined by these results.3.Y1 cells were treated with different concentrations of MBP for 24h. Then forskolin was added into the medium for 12h. Levels of progesterone in the medium were measured by RIA.4. mRNA expression of vimentin and miRNA200c in Y1 cells was determined by Real-time PCR.5. Protein expression of StAR, P450scc,3β-HSD, vimentin and tubulin in Y1 cells was assessed by western blot.6. Y1 cells were treated by vim-siRNA for 6h, then cells were stimulated by MBP (10-7 M). Protein expression and progesterone levels were detected by western blot and RIA, respectively.7. miRNA200c was overexpression or lowexpression in Yl cells, then cells were stimulated by MBP (10-7 M), vimentin expression and progesterone levels were detected by western blot and RIA, respectively.Result1. MBP treatment did not affect cell viability at doses ranging from 10-7 to 10-4 M, while MBP caused cell viability decreased at 10-3 M.2. Compared with the control group, progesterone levels were increased at 10-7 M of MBP.3. Results of western blot showed that protein expression levels of vimentin and StAR corrected by corresponding GAPDH were increased in Y1 cells treated by MBP at 10-7 M and 10-6M. The expression levels of other proteins related to steroid hormone synthesis including tubulin, b-actin, P450scc, and 3β-HSD remained unchanged compared with control group.4. Vimentin protein expression and progesterone levels were inhibited after silencing of the vimentin gene as compared to cells treated with control siRNA. In contrast, vimentin protein expression and progesterone levels were promoted after the cells were stimulated with MBP at 10-7 M compared to cells treated with control siRNA.5. In Y1 cells, expression of miRNA200c was downregulated at 10-7,10-6, and 10-5 M of MBP treatment compared with the control group.6. Levels of vimentin mRNA and protein were increased when miRNA200c expression was artificial inhibited in Y1 cells. At the same time, progesterone synthesis was increased.7. Progesterone and expression levels of vimentin corrected by corresponding GAPDH were decreased after miRNA200c overexpression, but were increased after MBP at 10’7M treatment with or without miRNA200c overexpression.Conclusion1. MBP (10-7 M) increases progesterone production in Yl cells.2. miRNA200c-vimentin signaling pathway play important roles in steroidogenesis stimulated by MBP in Y1 cells.