| ObjectiveNasopharyngeal carcinoma is a malignant tumor with high incidence in southern China,with hidden sites,atypical early clinical symptoms,high rates of distant metastasis,and poor prognosis.The pathogenesis of nasopharyngeal carcinoma is not completely clear,and Epstein-Barr virus infection is closely related to the occurrence and development of nasopharyngeal carcinoma.Epstein-Barr virus oncogenic protein LMP1 can activate NF-κB signaling pathway.OTUD7B is involved in the regulation of multiple signal transduction pathways such as cell proliferation and tumor angiogenesis,such as NF-κB and EGFR signaling pathway.Therefore,in this study,the oncogenic protein LMP1 of Epstein-Barr virus was used as the starting point to preliminarily investigate whether the oncogenic protein LMP1 of Epstein-Barr virus regulated the expression of OTUD7B through NF-κB signaling pathway.Materials and Methods1.Construction and extraction of LMP1 recombinant plasmidThe LMP1 insert and linearized vector was obtained first,and then the LMP1 insert was cloned into vector with the catalysis of recombinase.After the bonding product was transformed and cultured,a number of monoclones were selected for colony PCR identification,and the colonies identified as positive clones were sent for sequencing and plasmid was extracted for subsequent experiments.2.Study on the relationship between LMP1 and OTUD7B,NF-κB signaling pathwayThe expression of OTUD7B and NF-κB signaling pathway related proteins(IκBα,p-IκBα,p100/p52,p-p100/p52)in human nasopharyngeal carcinoma cell line CNE1 and stable cell line CNE1-LMP1 was detected by qPCR and WB assay,and the relationship between LMP1 and OTUD7B,NF-κB signaling pathway was investigated.3.Study on whether LMP1 regulated the expression of OTUD7B through the NF-κB signaling pathwayStable cell line CNE1-LMP1 were treated with NF-κB signaling pathway inhibitor MG-132,and the protein expression levels of OTUD7B and NF-κB signaling pathway related proteins(IκBα,p-IκBα,p100/p52,p-p100/p52)before and after treatment were detected by WB to further investigate whether LMP1 regulated OTUD7B expression through NF-κB signaling pathway.Results1.Identification of LMP1 recombinant plasmidColony PCR identification results of the LMP1 recombinant plasmid showed that there was a band with a length slightly larger than the size of the inserted fragment of LMP1 in the selected monoclones.Colonies identified as positive by PCR were sent for sequencing,and the sequencing results showed that the sequence was correct,indicating that the construction of the LMP1 recombinant plasmid was successful.2.Relationship between LMP1 and OTUD7B,NF-κB signaling pathwayIdentification of stable cell line CNE1-LMP1 cells:qPCR showed that the expression level of LMP1 mRNA in stable cell line CNE1-LMP1 cells was higher than that in CNE1 cells(P=0.022<0.05),the difference was statistically significant.WB showed overexpression of LMP1 protein in stable cell line CNE1-LMP1.It suggested that the stable cell line CNE1-LMP1 was successfully constructed.Study on the relationship between LMP1 and OTUD7B:mRNA and protein expression levels of OTUD7B in stable cell line CNE1-LMP1 were higher than those in CNE1 cells(P=0.021,P=0.006,respectively),and the differences were statistically significant,suggesting that LMP1 was related to the expression of OTUD7B.Study on the relationship between LMP1 and NF-κB signaling pathway:Comparing the stable cell line CNE1-LMP1 with the nasopharyngeal carcinoma cell line CNE1 in the expression level of NF-κB signaling pathway related proteins IκBα,p-IκBα,p100/p52 and p-p100/p52,P values were 0.130,0.034,0.149,0.015,respectively.The expression levels of IκBα and p100/p52 in CNE1 and CNE1-LMP1 cells were not statistically significant,while the protein expression levels of p-IκBαand p-p100/p52 in CNE1-LMP1 cells were higher than that in CNE1 cells,and the differences were statistically significant,suggesting that LMP1 might activate NF-κB signaling pathway.3.Further investigating that LMP1 regulated the expression of OTUD7B through the NF-κB signaling pathwayAfter the treatment of stable cell line CNE1-LMP1 with NF-κB signaling pathway inhibitor MG-132,the expression level of OTUD7B protein was decreased(P=0.004),suggesting that the expression of OTUD7B was regulated by NF-κB signaling pathway.In addition,the differential expression of NF-κB signaling pathway related proteins IκBα,p-IκBa,p100/p52,and p-p100/p52 before and after inhibitor treatment,P values were 0.107,0.010,0.134,0.003,respectively.There was no significant difference in the protein expression of IκBα and p100/p52 before and after treatment with inhibitor,while the expression of corresponding phosphorylated proteins p-IκBα and p-p100/p52 in CNE1-LMP1 cells after treatment with inhibitor was lower than that before treatment,and the difference was statistically significant,suggesting that the inhibitor MG-132 could effectively inhibit the activation of NF-κB signaling pathway.Conclusion1.Compared with CNE1 cells before transfection,both mRNA and protein expression levels of OTUD7B were increased in stable cell line CNE1-LMP1,suggesting that LMP1 could promote the expression of OTUD7B.2.The expression level of NF-κB pathway related phosphorylated proteins in stable cell line CNE1-LMP1 was higher than that in CNE1 cell,suggesting that LMP1 could activate NF-κB signaling pathway.3.After inhibiting the NF-κB pathway with MG-132,the expression level of OTUD7B protein in the stable cell line CNE1-LMP1 was lower than that before inhibition,suggesting that LMP1 might regulate the expression of OTUD7B through NF-κB signaling pathway. |
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