ABSTRACT
Background@#It is well-established that serum testosterone in men decreases with age, yet the underlying mechanism of this change remains elusive. @*Methods@#The expression patterns of Fancd2 opposite-strand (Fancd2os) in BALB/c male mice and testicular tissue derived cell lines (GC-1, GC-2, TM3, and TM4) were assessed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence. The Fancd2os-overexpressing or knockdown TM3 cells were constructed by infecting them with lentivirus particles and were used to evaluated the function of Fancd2os. The testosterone production was measured using enzyme linked immunosorbent assay (ELISA) and the steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) were analysed using RT-PCR. The apoptosis of TM3 cells induced by ultraviolet light or testicular tissues was detected using flow cytometry, Western blot or dUTP-biotin nick end labeling (TUNEL) assays. Pearson correlation analysis was used to assess the correlation between the Fancd2os expression and TUNEL-positive staining in mouse testicular Leydig cells. @*Results@#The Fancd2os protein was predominantly expressed in mouse testicular Leydig cells and its expression increased with age. Fancd2os overexpression inhibited testosterone levels in TM3 Leydig cells, whereas knockdown of Fancd2os elevated testosterone production. Fancd2os overexpression downregulated the levels of StAR, P450scc and 3β-HSD, while Fancd2os knockdown reversed this effect. Fancd2os overexpression promoted ultraviolet light-induced apoptosis of TM3 cells. In contrast, Fancd2os knockdown restrained apoptosis in TM3 cells. In vivo assays revealed that higher Fancd2os levels and mouse age were associated with increased apoptosis in Leydig cells and decreased serum testosterone levels. Pearson correlation analysis exhibited a strong positive correlation between the expression of Fancd2os and TUNEL-positive staining in mouse testicular Leydig cells. @*Conclusion@#Our findings suggest that Fancd2os regulates testosterone synthesis via both steroidogenic enzymes and the apoptotic pathway.
ABSTRACT
Objective: To explore the effect of 5-Aza-2′-deoxycytidine (5-Aza-CdR) on the molecular biological behaviors of SW480 cells and the expression of 15-hydroxyprostaglandin dehydrogenase (PGDH). Methods: Colorectal carcinoma SW480 cells were treated with 5-Aza-CdR. Cell proliferation was determined by MTT assay and plate colony formation test. The methylation status of the tumor suppressor gene PGDH was detected by methylation-specific PCR. The expression of PGDH protein was measured by Western blotting and the cell cycle was analyzed by flow cytometry. Results: It was shown that 5-Aza-CdR decreased the proliferation rate of SW480 cells compared with control group. The clony formation rate of SW480 cells decreased significantly after treatment with 5-Aza-CdR in 5 and 10μmol/L compared with the control group (35.4% and 26.5% vs 59.35%, P <0.01). The methylation degree in the promoter of PGDH gene was decreased and the expression of PGDH protein was detected after 5-Aza-CdR treatment. 5-Aza-CdR treatment induced G1 phase arrest of the SW480 cells. Conclusion: The hypermethylation of CpG islands in the promoter of PGDH gene results in the loss of PGDH protein expression in human colorectal carcinoma cell line. The demethylating agent 5-Aza-CdR could restore PGDH gene expression. These findings provide theoretic evidence for clinical treatment of human colorectal carcinoma with demethylation agent 5-Aza-CdR.