Methods: HuR expression levels were estimated in surgically removed oral carcinomas by both Western blot and immunohistochemistry methods. Normal oral keratinocytes and oral cancer UM74B cells were used to study the HuR alterations during hypoxia. Western blot, Immunocytochemistry and RT-qPCR confirmed the over expression and cleavage of HuR in oral cancer tissues and cells. To assess the HuR role in mRNA stability, we knockdown HuR by siRNA and studied their associated mRNA stability by performing RT-qPCR.
Results: Our results indicate that during hypoxic stress, HuR was significantly overexpressed and undergoes caspase-dependent cleavage in OSCC cells. Unexpectedly, the HuR-cleavage product 1 (HuR-CP1) was found to strongly associate with the 3'-untranslated region (UTR) of c-myc mRNA and block mRNA translation. The binding efficiency of HuR to the 3'-UTR of c-myc mRNA was confirmed using ribonucleoprotein immunoprecipitation and site-directed mutagenesis at the AU-rich element sequences of the c-myc mRNA. Overexpression of a non-cleavable isoform, HuR-D226A, revealed a potent dominant-negative effect, repressing cleavage of endogenous HuR and promoting cell viability and proliferation. Surprisingly, under hypoxia, siRNA knockdown of HuR elevated c-Myc protein expression suggest that HuR act as a translational repressor.
Conclusions: These findings suggest an important role for HuR in hypoxia, and we may have revealed a novel post-transcriptional mechanism that controls c-Myc expression in oral cancer progression.
Keywords: Apoptosis, Epithelium/epithelial, Gene expression and post-transcriptional regulation