The Effects And Mechanism Of Transcriptional Inactivation Of GLI3 Gene In Nasopharyngeal Carcinoma

Objective: To study the expression of the major members(PTCH1,GLI1,GLI2,and GLI3)of the Hedgehog(HH)signaling pathway and the latent membrane protein 1 and epigenetic alterations of GLI3 in nasopharyngeal carcinoma(NPC)tissues and NPC cell lines,analyzing the possible causes of HH pathway activation and its mechanism.Method:Part Ⅰ: Immunohistochemistry(IHC)was used to determine the expression of HH pathway members(PTCH1,GLI1,GLI2,GLI3)in 4 cases of pathologically confirmed NPC tissues,according to the Immuno Reactive Score(IRS).Quantitative Real-time PCR was used to detect the expression of key members(PTCH1,GLI1,GLI2,and GLI3)mRNA in NPC C666-1,CNE1,CNE2 cells and normal nasopharyngeal epithelial NP69 cell lines.Part Ⅱ: The methylation status of GLI3 was assessed by Bisulfite Sequencing PCR,and selecting two hypermethylated NPC cells for subsequent experiments.CCK8,scratch test,transwell,QRT-PCR and WB were used to detect the cell viability,l migration and invasion ability and the expression of GLI3 mRNA and protein after treated by DNA methylation inhibitor 5-Aza-2’deoxycytidine(5-Aza-dC),respectively.Part Ⅲ: The protein expression of LMP1 in 4 cases of NPC tissues was assessed by IHC.The morphological changes of above cell lines were observed after treated with ATO.QRT-PCR and WB were used to detect the mRNA and protein expression of LMP1.Meanwhile,the DNMTs(DNMT1,DNMT3 a and DNMT3b)expressons were tested in these cells after treatment with ATO.Result:Part Ⅰ: HH signaling pathway is activated in different degrees in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cell lines.Part Ⅱ:1.The methylation level of GLI3 gene promoter in 4 cases of nasopharyngeal carcinoma was 16.5%,16.5%,31.8%,58.8%,which was the middle to low level of methylation.2.All of three nasopharyngeal carcinoma cell lines were methylated,and the methylation ratio was 44.7%(CNE1),43.1%(CNE2),70.6%(C666-1).These C666-1 and CNE1 NPC cells were subjected to subsequent experiments;3.The results of CCK8,scratch test and transwell experiment showed that the proliferation,migration and invasion ablitiy of C666-1 and CNE1 cells were decreased with the increase of 5-Aza-dC concentration(P<0.01),respectively.4.The expression of GLI3 mRNA and protein in C666-1 and CNE1 cell lines was up-regulation with the increase of 5-Aza-dC concentration(P<0.01).Part Ⅲ:1.It was showed the different expression levels of LMP1 mRNA and protein in nasopharyngeal carcinoma tissues and cell lines.2.The morphology of CNE1,CNE2 and NP69 cells were changes profoundly,and the viability was decreased after 48 hours of treatment with ATO.But it was not obvious in morphologic change of C666-1 cells after ATO treatment.3.After treated with the ATO,the expression levels of DNMTs mRNA and proteins in CNE2 and C666-1 cells were decreased.The expression of DNMT1 and DNMT3 a mRNA and proteins in NP69 cells were up-regulated,as the expression of DNMT3 was slided.The expression of DNMT3 a in CNE1 cells was increased slightly,while the expression of DNMT1,DNMT3 mRNA and proteins were down-regulated.Conclusion:1.It is evidenced that HH signaling pathway is abnormally activated in nasopharyngeal carcinoma.2.It is demonstrated that methylation and demethylation of GLI3 in nasopharyngeal carcinoma affect the proliferation,migration and invasion of nasopharyngeal carcinoma cells.Then,the GLI3 can be used as a new target for the treatment of nasopharyngeal carcinoma.3.It is suggested that ATO can be used for target therapy ofnasopharyngeal carcinoma.