Effects Of The Variant Of Epstein-Barr Virus-encoded RNA2 To Inhibit Apoptosis Of Nasopharyngeal Carcinoma Cell Lines Through PKR

Background and objective: It has been confirmed that EBV-encoded RNAs(EBERs, including EBER1 and EBER2) contribute to carcinogenesis in multiple cell lines. Our previous researches about the polymorphism of EBERs demonstrated that EB-8m variant with 6 common mutants in the EBER2 gene area may be associated with nasopharyngeal carcinoma(NPC), especially endemic NPC. Previous studies have indicated that EBERs confer resistance to interferon-α(IFN-α)-induced apoptosis by binding RNA-activated protein kinase(PKR) and blocking its phosphorylation. This study aims to determine the cell viability and the resistance of EBER2 with EB-8m variant to IFN-α-induced apoptosis in EBV-negative NPC cell lines, and to explore its effects on the binding and the phosphorylation of PKR as well as the phosphorylation and expression of PKR-related proteins.Methods: The cell viablity analysis and anti-apoptosis observation induced by IFN-α was conducted in EBV-negative NPC cell lines(CNE1 and HONE1) with stable expression of EBER2 prototype gene and EB-8m variant gene under the control of lentivirus vector transfection group. The expression of basal PKR, Phospho-PKR, basal e IF2α, Phospho-e IF2α, basal c-Jun, Phospho-c-Jun and Bcl-2 were determined by Western blot. RNA-binding protein immunoprecipitation against anti-FLAG was done in EBER2-expressing cells after pc DNA3.1-PKR-FLAG transfection. The level of EBER2 in immunoprecipitated RNA and PKR in immunoprecipitate were detected by Real-time PCR and Western blot, respectively.Results:(1) Expression of variant EBER2 in CNE1 or HONE1 cell lines increased cell proliferation and colony formation ability compared prototype EBER2-expressed or control cells(P<0.05).(2) Expression of prototype and EB-8m variant EBER2 in NPC cell lines enhanced their anti-apoptosis ability compared with the control cells(P<0.05). Furthermore, the cells expressed EB-8m variant increased the anti-apoptosis ability compared with those expressed prototype EBER2(P<0.05).(3) Phosphorylation of PKR, e IF2α and c-Jun were inhibited and the expression of Bcl-2 was upregulated in EBER2-expressing cells. The variant EBER2 showed stronger inhibition of phosphorylation to PKR compared to prototype EBER2.(4) Both prototype and variant EBER2 were co-precipited along with PKR. The fold enrichment of EB-8m variant EBER2 in immunoprecipitated RNA was significantly higher than that of prototype EBER2(P<0.05).Conclusion:(1) The EB-8m variant EBER2 enhances the cell viability and anti-apoptosis ability in NPC cell lines.(2) EBER2 confers resistance to IFN-α induced apoptosis in NPC cell lines through binding to PKR and inhibiting its phosphorylation, as well as inhibiting the phosphorylation of e IF2α and c-Jun and upregulating Bcl-2 expression. The EBER2 with EB-8m variant may enhance its abilities to bind PKR and inhibit phosphorylation of PKR.