Oral Cancer is a 6th leading cause of death worldwide. Oral cancer cells undergo post-transcriptional modifications for their growth and proliferation. The RNA-binding protein CUG-Binding protein-1 (CUGBP1) regulates the expression of genes essential for programmed cell death (apoptosis) via post-transcriptional processes. Hence, we hypothesize that expression of CUGBP1 in oral cancer cells destabilize the apoptosis-related mRNAs and controls cell proliferation.
CUGBP1 expression was estimated in oral cancer and adjacent normal tissues from SCC patients by using the methods of Western blot, RT-qPCR and immunohistochemistry (IHC). The expression and localization of CUGBP1 in oral cancer cells were confirmed through immunocytochemistry. Role of CUGBP1 on mRNA stability was confirmed through siRNA knockdown and actinomycin pulse-chase analysis. Binding of CUGBP1 with pro-apoptosis mRNAs BAX, BAD and JunD was analyzed using RNA immunoprecipitation (RNP-IP) approach.
Our data revealed overexpression of CUGBP1 in oral cancer tissues and cell lines suggest that CUGBP1 may play a key role in mRNA destability. Further, siRNA-mediated inhibition of CUGBP1 impairs cell proliferation and enhances apoptosis. CUGBP1 deficiency resulted in overexpression of BAX, BAD and JunD creating a cellular environment conducive to apoptosis. Further, serum starvation disrupts this binding and stabilizes the BAX, BAD and JunD mRNAs through signaling kinases leading to protein kinase-C (PKC) activation. Interestingly, Bis (indolyl) maleimide-1 (BIM-1) a PKC inhibitor disrupted the RNA binding activity of CUGBP1 and increase apoptosis through stabilization of BAX and BAD mRNAs.
These results suggest that phosphorylation of CUGBP1 by PKC plays a critical role in RNA binding activity and degradation. In addition, ectopic expression of CUGBP1 in oral cancer cells destabilizes the apoptosis coded mRNAs and enhances cell proliferation. Thus, CUGBP1 is an important RNA binding protein in cancer and a potential therapeutic target.
Keywords: Apoptosis, Carcinogenesis, Cell culture, Gene expression and Proteins
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