Figure 3. Dex downregulates DOT1L and its target gene via GR in B lymphoma cells. (A) Raji and THP-1 cells were incubated with incremental concentrations of Dex, and then the CCK-8 assay was taken to evaluate the cell viability. (B) Raji cells were incubated with 0.4 μM Dex, following the mRNA levels of DOT1L and its target gene MEIS1 were detected by RT-qPCR. (C) Raji and THP-1 cells were conducted with 0.4 μM Dex, then Western blot was applied to detect the protein levels of the DOT1L effector H3K79me2, H3 served as the loading control. (D) After pretreatment with RU486 for 30 min, Raji and THP-1 cells were incubated with Dex, and then the CCK-8 assay was taken to evaluate the cell viability. (E) After pretreatment with 4 μM of RU486 for 30 min, Raji cells were conducted with Dex, afterwards the mRNA levels of DOT1L and its target gene MEIS1 were detected by RT-qPCR. (F) After pretreatment with 4 μM of RU486 for 30 min, Raji and THP-1 cells were conducted with 0.4 μM Dex, then the DOT1L effector H3K79me2 was measured by Western blot.
3.4 Dex reduces the mRNA level of DOT1L through decreasing its mRNA stability
Since GR generally functions as a transcriptional factor [21], we next investigated whether DOT1L was a novel target gene of GR. By bioinformatics, we found several potential GR binding sites in the -1,800 bp to +200 bp of the DOT1L gene promoter region (-2800 bp to +200 bp) (Figure 4A). The fragment containing the -1,800 bp to +200 bp region was fused with the pGL3-Basic vector to generate pGL3-DOT1L. The reporter assay revealed a significantly higher luciferase activity of pGL3-DOT1L compared to that of pGL3-Basic (Figure 4B). However, Dex did not decrease the luciferase activity of pGL3-DOT1L, suggesting that Dex had no impact on the transcriptional activity of DOT1L gene promoter. In addition, RU486 did not influence the luciferase activity. Next, we explored whether Dex weakened the mRNA stability of DOT1L. As illustrated in Figure 4C-4D, treatment with actinomycin D (Act D), the transcriptional inhibitor, significantly decreased the mRNA level ofDOT1L , which could be further reduced by Dex. These results suggested that Dex downregulated DOT1L through decreasing its mRNA stability.