Dicer1e depletion mediates miR-548 biogenesis and induces G2/M-DNA-damage checkpoint control

Objectives: To determine the significance of Dicer1e expression in relation to miRNA biogenesis, the DNA damage response (DDR), and oral cancer pathogenesis.
Methods: Dicer1e silencing experiments were performed in oral cancer cells followed by RNA-sequencing and qPCR to determine its effects on miRNA biogenesis levels. Furthermore, qPCR was performed using total RNA extracted from oral cancer patient tumor samples, as well as oral cancer cell lines, to determine the levels of a specific miRNA in relation to Dicer1e protein levels. Lastly, Western blotting, comet assays, and fluorescence microscopy analyses were used to assess the effects of Dicer1e depletion on DNA damage and the mediators of the DDR.
Results: RNA-sequencing demonstrated that Dicer1e silencing in oral cancer cell lines overexpressing Dicer1e protein altered the expression level of a subset of miRNAs from the miR-548 gene family. Subsequent qPCR experiments further corroborated that Dicer1e protein levels appeared to correlate specifically with the expression levels of miR-548k. Moreover, qPCR analyses demonstrated that 6 of 8 (75%) oral cancer patient tumor samples that were previously found to have high-Dicer1e protein expression levels, also showed an increase in miR-548k levels compared to low-Dicer1e-expressing normal adjacent tissues and that high-Dicer1e-expressing oral cancer cell lines also appeared to have higher miR-548k levels, compared to low-Dicer1e-expressing normal human oral keratinocytes. Lastly, Dicer1e silencing was also found to induce DNA damage, as well as DDR foci formation of ATM and increased expression/activation of several additional proteins involved in the G2/M-DNA-damage checkpoint, including ATR and Wee1.
Conclusions: Together, our data appear to demonstrate a correlation between miR-548k levels and oral cancer pathogenesis, with a potential connection between Dicer1e and miR-548k biogenesis. Moreover, Dicer1e also appears to play a role in regulating genome integrity and stability, with its depletion promoting DNA damage and a G2/M-DNA-damage checkpoint response in oral cancer cells.