EBV LMP1/NF-κB\PINX1 Signaling Pathway Stimulates Telomerase Activation During Nasopharyngeal Carcinoma Development

ObjectivesTo clarify the expression of PIN2/TRF1-interacting telomerase inhibitor l(PINX1)in nasopharyngeal carcinoma(NPC),and further to investigate the potential mechanism of aberrant PINX1 expression in NPC from the aspects of mutation,DNA methylation and transcriptional regulation.MethodsImmunohistochemistry,real-time PCR,and Western blotting were used to explore the expression of PINX1.PCR sequencing was conducted to analyze deletion,insertion and mutation of PINX1 gene.Bisulfite sequencing PCR(BSP)and methylation-sensitive high-resolution melting(MS-HRM)were performed to determine the methylation level of the PINX1 promoter region.Chromatin immunoprecipitation(ChIP)and dual luciferase reporter gene assay were exploited to elucidate the regulatory mechanism between NF-ΚB and PINX1.TRAP-SYBR Green and Southern Blotting assays were utilized to detect telomerase activity and telomere length.CCK8 and EdU tests were conducted to measure proliferation ability of NPC cens.ResultsHere,we demonstrated that the mRNA and protein level of PINX1 is down-regulated in NPC.Mechanistically,we found that the LMP1 could inhibit the transcriptional activity of PINX1 promoter by promoting the binding of NF-κB p65 to three specific sites in the PINX1 promoter region,significantly,two(-1698/-1689,tgcaatttcc;-206/-197,cgggctttac)of which have not been reported.However,neither promoter methylation nor somatic mutations or deletions was detected in the PINX1 gene.In addition,we also observed that overexpression of LMP1 in NPC cells resulted in increased telomerase activity and proliferation ability and prolonged telomere length.ConclusionsIn summary,we first discovered EBV LMP1 led to reduced PINX1 expression through LMP1-NF-κB-PINX1 axis,which up-regulates telomerase activity in NPC.And hence,the tumor cells acquire the ability to proliferate indefinitely.This signaling pathway enriches the study on the molecular mechanism of EBV carcinogenesis,and further provides new thinking for early diagnosis and treatment in NPC.