Study On The Association And Mechanism Of NFX1-123 With HPV16 E6 On Cervical Cancer

ObjectiveFollowing breast cancer,cervical cancer has become the second leading cause of cancer death in women aged 20 to 39 years and is closely associated with persistent infection with high-risk human papillomavirus(HR-HPV).NFX1(Nuclear transcription factor,X-box binding 1)is a transcriptional repressor of MHC class Ⅱgenes.Human NFX1-123,one of the isoforms of NFX1,is highly expressed in HPV-positive cells and primary cervical cancer,so it is hypothesized that NFX1-123 may play an important role in the mechanism of cervical cancer,which may be associated with cellular telomerase activity and cellular senescence.The aim of our study was1.To construct the eukaryotic expression recombinant plasmid of NFX1-123 gene and identify its interaction and co-localization with HPV16 E6 in HEK 293T cells.2.To explore the cellular localization relationship and possible mechanism of NFX1-123 with HPV16 E6.3.To elucidate whether the effects of NFX1-123 on telomerase activity and cellular senescence in cells overexpressing HPV E6 are influenced by highor low-risk human papillomavirus types.4.To explore whether there is a synergistic or competitive relationship between the E6-NFX1-123 pathway and the E6-E6AP-p53/p21 pathway.Methods1.NFX1-123 gene fragment was obtained by PCR and inserted into the nHA/pRK5 vector.Restriction enzyme digestion and sequencing were used to identified the NFX1-123 recombinant plasmid.2.Co-immunoprecipitation was used to certify its interaction with HPV16 E6 and immunofluorescence was chosen to study the cellular localization relationship between them.3.Senescence-associated beta-galactosidase(SA-β-gal)activity assay was commonly used to evaluate the beta-galactosidase(β-gal)activity in senescent cells.4.Western Blot was used to confirm the protein expression of NFX1-123,p53,p21 and others.Results1.The result of enzyme digestion and sequencing showed that NFX1-123 was successfully cloned into nHA/pRK5.Western blot showed that HA-NFX1-123 protein could be normally expressed in HEK 293T cells.The immunofluorescence revealed that NFX1-123 was mainly localized in the cytoplasm.2.The interaction between NFX1-123 and HPV16 E6 was demonstrated by co-immunoprecipitation.When HPV16 E6 was co-expressed,NFX1-123 underwent a change in localization from the cytoplasm to the nucleus and co-localizes with HPV16 E6 in the nucleus of HEK 293T cells.3.Co-expression of HPV16 E6 and NFX1-123 significantly increased telomerase activity in HEK 293T cells,while overexpression of low-risk HPV6 E6 and NFX1-123 had no effect on it;4.NFX1-123 significantly enhanced the inhibitory effect of HPV16 E6 on the senescence of HEK 293T cells,but not on cells overexpressing HPV6 E6;5.NFX1-123 had no effect on the protein expression of p53 and p21.However,when HPV16 E6 and NFX1-123 were co-expressed,the degradation of p53 and p21 by HPV16 E6 in HEK 293T cells was markedly enhanced,thus the E6-NFX1-123 pathway synergistically enhanced the E6-E6AP-p53 pathway.ConclusionsNFX1-123 was induced by high-risk HPV16 E6 to change its cellular localization and bind to E6,which contributed to the altered biological functions of NFX1-123:NFX1-123 significantly increased telomerase activity and markedly inhibited cellular senescence in HEK 293T cells overexpressing HPV16 E6 which is mediated by the E6-E6AP-p53 pathway.However,low-risk HPV6 E6 was unable to induce these changes in the host protein NFX1-123,which may be an important reason for the different outcome of cervical epithelial tissue infected with high-risk HPV versus low-risk HPV.