| Background Nasopharyngeal carcinoma(NPC)is an epithelial malignant tumor of nasopharynx.The incidence rate of NPC is relatively high in southern China.Currently,the standard treatment for nasopharyngeal cancer is radiotherapy or radiotherapy combined chemotherapy.Although the 5-year survival rate of primary NPC is high after radiotherapy or radiotherapy combined chemotherapy,locally advanced and metastatic nasopharyngeal carcinoma caused treatment failure.Increasing evidence shows that tumor cells which have the characteristics of self-renewal and stem cell-like cells are a subset of tumor stem cells,which plays an important role in tumor initiation and resistance to radiotherapy or chemotherapy.The presence of tumor stem cells may be one of the causes of tumor recurrence after standard treatment.Therefore,targeted therapy targeting tumor stem cells,which are the special cell population,are a new strategy to overcome tumor recurrence after nasopharyngeal cancer treatment.In the past few years,many studies have described the EBV-associated nasopharyngeal carcinoma cell lines and tumor stem cell subsets in primary tumors.Recent studies have shown that EBV-encoded proteins can induce stem cell-like properties in epithelial cells.All EBV-infected NPC cells exhibited type II latency,which expressed Epstein-Barr virus nuclear antigen 1(EBNA1),LMP1,LMP2 A,Epstein-Barr virus-encoded small RNA(EBER),BART,and many EBV-encoded micro RNAs(mi RNAs).Among these potential genes,LMP1 encodes a viral oncoprotein that is responsible for altering multiple cellular mechanisms and signaling pathways in host cells.There is increasing evidence that LMP1 plays a role in acquiring stem-like or progenitor-like cells in nasopharyngeal carcinoma.Studies have shown that LMP1 can induce the expression of factors related to cell invasion and metastasis and epithelial-mesenchymal transition(EMT)through Twist and Snail.LMP1 is the only latent protein involved in nasopharyngeal carcinoma cell differentiation,transformation,and malignant tumor regulation.The study also found that LMP1 induces nasopharyngeal carcinoma epithelial cells to transform into tumor stem cells,possesses the phenotypic characteristics(CD44high and CD24low),and epithelial-mesenchymal transition.It also indicates that LMP1-positive nasopharyngeal carcinoma tumor stem cells are important factor that are resistant to radiotherapy and chemotherapy,and relapse or metastasis.Therefore,the antigen LMP1 may be an ideal target for inhibiting nasopharyngeal carcinoma tumor stem cells,providing a basis for further studying the molecular mechanism of LMP1 epithelial-mesenchymal transition in nasopharyngeal carcinoma cells and obtaining stem cell characteristics.Our group firstly prepared the human-derived anti-LMP1 TES1 antibody(htes Fab)for the first time by screening the fully human antibody library method.Gene sequencing showed that the Fab antibody gene sequence was obtained by assembly and secretion.It was identified by ELISA,flow cytometry,and immunofluorescence technology that htes Fab can specifically bind to LMP1 intracellular functional regions of LMP1-positive nasopharyngeal carcinoma cell.MTT showed that htes Fab has the potential to inhibit nasopharyngeal carcinoma cell proliferation.Aim It was investigated that the effect of LMP1 on tumor stem cells in nasopharyngeal carcinoma and the role of LMP1 in nasopharyngeal carcinoma tumor formation.The anti-LMP1-specific antibody htes Fab was prepared,and the inhibitory effect of the antibody on the growth and metastasis of nasopharyngeal cancer stem cells and their transplanted tumors was analyzed at molecular mechanism of cell growth in vivo and vitro.This study will help elucidate the molecular mechanism of Fab targeting LMP1 to inhibit the biological activity of nasopharyngeal cancer stem cells,and it will be of great significance for the development of new drugs for nasopharyngeal cancer.Method 1.LMP1-p CMV eukaryotic expression plasmid were constructed,and transferred it into nasopharyngeal carcinoma tumor stem cell HNE2.Nasopharyngeal carcinoma tumor stem cell HNE2-LMP1 were confirmed to m RNA and protein expression of LMP1 by real-time quantitative PCR and Western blot.The positive expression of LMP1 on the surface of nasopharyngeal carcinoma tumor stem cells were confirmed by flow cytometry.2.The proliferation of HNE2 and HNE2-LMP1 nasopharyngeal cancer stem cell subpopulations were compared by cell proliferation assay and cell colony formation test.The invasion and transfer effect of HNE2 and HNE2-LMP1 nasopharyngeal cancer stem cell subsets compare by Transwell test and cell scratch test in vitro.Real-time quantitative PCR and Western blot were used to analyze the m RNA and protein expression of tumor stem cell markers Nanog,Oct4,CD44,CD24,Sox2,and ABCG2 target genes,and the effects of LMP1 on nasopharyngeal carcinoma stem cells were observed.3.Anti-LMP1 antibody Fab were prepare.The anti-LMP1 TES1 antibody Fab(htes Fab)were observed to inhibit the proliferation of nasopharyngeal cancer stem cells through cell proliferation assay and cell colony experiments.The induction of nasopharyngeal cancer stem cells was analyzed by flow cytometry apoptosis.Real-time quantitative PCR and Western blot were used to analyze the effect of htes Fab on the expression of cyclin molecules CCNA2 and CCND1 in nasopharyngeal carcinoma stem cell proliferation-related downstream genes.4.After htes Fab was applied to nasopharyngeal carcinoma stem cells,Western blot detection was used to detect changes in PI3 K,phosphorylated PI3 K,Akt and phosphorylated Akt protein levels.The effect of htes Fab on the proliferation of nasopharyngeal carcinoma stem cells through PI3 K / Akt pathway were investigated.5.Nasopharyngeal carcinoma tumor-bearing nude mouse animal model were constructed.Tumor growth or size of the tumor-bearing nude mice were observed after the action of htes Fab.The tumor tissue necrosis by HE staining and apoptosis by TUNEL were observed after the nude mice were sacrificed.The changes in protein levels of phosphorylated PI3 K and phosphorylated Akt were analyzed by immunohistochemistry.6.The effect of htes Fab on the invasion and metastasis of nasopharyngeal cancer stem cells were observed by Transwell test and cell scratch test.Real-time quantitative PCR and Western blot were used to analyze the relative m RNA expression levels and protein expression of EMT-related genes E-cadherin,N-cadherin,vimentin,?-catenin and transcription factor slug in nasopharyngeal carcinoma stem cells after anti-LMP1 antibodies treated.Western blot was used to detect the expression of β-catenin protein,and to investigate the effect of htes Fab on Wnt / β-catenin signalling pathway.7.The effect of antibody on tumor metastasis of nasopharyngeal carcinoma was observed by animal fluorescence imaging on a tumor-bearing nude mouse model after htes Fab injected.The effect of antibodies on the expression of E-cadherin,N-cadherin,vimentin,?-catenin and transcription factor slug in tumor tissues was observed by immunohistochemistry.Result 1.HNE2 cells line of nasopharyngeal carcinoma were seeded in serum-free culture medium and aggregated to grow in a spherical shape.It was confirmed by q RT-PCR and Western blot analysis that stem cell related genes CD44,Sox2,ABCG2,Nanog and Oct4 in nasopharyngeal carcinoma cell line HNE2 spheres were highly expressed,and CD24 has a low level of expression,while the parental HNE2 cell line were the opposite result.2.The results of double enzyme digestion analysis showed that the LMP1-p CMV eukaryotic expression plasmid has the LMP1 gene.After transfection of nasopharyngeal cancer stem cells HNE2,nasopharyngeal cancer stem cells HNE2-LMP1 expressed LMP1 protein.Western blot confirmed that the m RNA and protein levels of LMP1 in nasopharyngeal carcinoma tumor stem cells HNE2-LMP1 were increased,and the expression of LMP1 on the surface of nasopharyngeal carcinoma tumor stem cells was detected by flow cytometry.3.The results of cell proliferation assay showed that HNE2-LMP1 microsphere cells had significantly higher proliferation ability than HNE2 microsphere cells,and the difference was statistically significant.The results of cell colony formation test showed that HNE2-LMP1 nasopharyngeal cancer stem cell subsets were formed to more cell colonies in the same time than compared with HNE2.The results of Transwell experiments showed that the average number of transmembrane cells in the HNE2-LMP1 microsphere cell group was significantly higher than that in the HNE2 microsphere cell group.Cell scratch test in vitro test showed that HNE2 microsphere cells recovered nearly 43% and HNE2-LMP1 microsphere cells recovered 94% in the wound after 48 h of scratch.The real-time quantitative PCR and Western blot were used to analyze the m RNA and protein expression of the tumor stem cell markers Nanog,Oct4,CD44,Sox2,and ABCG2 but CD24 was low expressed.The results showed that the expression of tumor stem cells of HNE2-LMP1 nasopharyngeal cancer stem cell subsets were higher than HNE2.4.The purified htes Fab were successfully prepared using the original mature technology of the experiment.Cell proliferation assays results showed that tumor microsphere cells proliferated fastest in the PBS group over time,followed by the htes Fab group and LY294002(PI3K inhibitor)group,while the Fab + LY294002 group had the slowest cell proliferation after 72 hours.Cell colony experiments showed that the number of colonies in the PBS group were significantly higher than that in the LY294002(PI3K inhibitor)group,the cells in the htes Fab group,and the Fab + LY294002 group.The results of flow cytometry analysis showed that the early apoptosis rates of the PBS group,LY294002(PI3K inhibitor)group,htes Fab group,and htes Fab+LY294002 group were 5.89%,9.32%,14.6%,and 21.8%.Real-time Quantitative PCR and Western blot analysis showed that the m RNA and protein expression levels of CCNA2 and CCND1 in the htes Fab group,LY294002 group,and htes Fab+LY294002 group were decreased compared with the negative control PBS group.5.After htes Fab acted on nasopharyngeal cancer stem cells HNE2-LMP1,Western blot results showed that the protein levels of PI3 K,Akt,phosphorylated PI3K(p-PI3K)and phosphorylated Akt(p-Akt)in PBS group were no significant change,while the phosphorylated PI3K(p-PI3K)and phosphorylated Akt(p-Akt)in the other three groups were down-regulated,and the htes Fab+ LY294002 group was significantly down-regulated.However,there was no significant change in the protein levels of the four groups of PI3 K and Akt.6.Different drugs were injected into the peritoneum of nude mice on day 1,6,11,16,and 21 respectively.The results were shown in the growth and size of the tumor.After 10 days of intratumoral injection,the tumor volume of the htes Fab antibody + LY294002 group was smaller than that of the PBS group;at 15,20,25,and 30 days,the tumor volume of the htes Fab + LY294002 group,htes Fab group,and LY294002 group was significantly less than the PBS group.The tumor suppression rates of htes Fab + LY294002 group,htes Fab group and LY294002 group were 45%,19%,and 27.4%,respectively.The htes Fab antibody and LY294002 caused necrosis of tumor tissue in nude mice by HE staining.Tumor cells in the htes Fab + LY294002 group,the htes Fab group,and the LY294002 group were significantly increased in staining with TUNEL,but no obvious staining was observed in the PBS group.The same results were observed by immunohistochemical analysis.7.Through Transwell experiments,it was observed that the average number of transmembrane cells per field in the PBS group was significantly higher than that of the htes Fab group,XAV-939 group and the htes Fab+ XAV-939 group after 48 hours of cell migration.Cell scratch test observed that 48 hours after the scratch,the cells in the PBS group recovered nearly 87%,while the wound recovery in the htes Fab group,XAV-939 group,and htes Fab+XAV-939 group was 42%,38%,and 17%,respectively.Real-time quantitative PCR and Western blot analysis showed that htes Fab down-regulated the relative m RNA and protein expression levels of N-cadherin,vimentin,?-catenin,and the transcription factor slug in EMT-related genes of nasopharyngeal cancer stem cells while E-cadherin were up-regulated.Western blot showed that both htes Fab and XAV-939 can inhibit the expression of β-catenin protein.It suggested that htes Fab may affect the invasion and metastasis of nasopharyngeal carcinoma stem cells through the Wnt / β-catenin signaling pathway.8.No significant metastasis was observed in tumor cells of htes Fab group,XAV-939 group,htes Fab+ XAV-939 group,but the control group PBS control was the opposite result which had tumor metastasis.Immunohistochemical results showed that the levels of EMT-related genes N-cadherin,vimentin,?-catenin and the transcription factor slug decreased and E-cadherin increased in tumor tissues of the htes Fab + XAV-939 group,the htes Fab group,and the XAV-939 group compared with the control PBS group. Conclusion 1.The nasopharyngeal carcinoma cell line HNE2 cells were inoculated in serum-free culture medium and grown in a spherical shape.The stem cell-related genes CD44,Sox2,ABCG2,Nanog and Oct4 in the nasopharyngeal carcinoma cell line HNE2 cells were highly expressed,and CD24 had a relatively low expression.Strong tumor stem cell characteristics can be used as a good model of nasopharyngeal cancer stem cells.2.LMP1-p CMV eukaryotic expression plasmid were successfully constructed and transfected nasopharyngeal carcinoma tumor stem cells HNE2,prepared nasopharyngeal carcinoma tumor stem cells HNE2-LMP1,which express high LMP1.HNE2-LMP1 nasopharyngeal carcinoma stem cell subsets have stronger proliferation ability and invasion and metastasis ability than HNE2.HNE2-LMP1 tumor stem cell markers were highly expressed in nasopharyngeal carcinoma stem cell subsets.It has been confirmed that LMP1 plays an important role in maintaining the tumor stemness of nasopharyngeal carcinoma.3.Through cell proliferation assay and cell colony experiments,htes Fab and LY294002(PI3K pathway inhibitor)synergistically inhibit the proliferation of nasopharyngeal cancer stem cells,and induce apoptosis.In addition,htes Fab inhibited the expression of cyclin molecules CCNA2 and CCND1(nasopharyngeal carcinoma stem cell proliferation-related downstream genes),and had similar inhibitory effects as LY294002.After htes Fab and LY294002acted on nasopharyngeal cancer stem cells,the levels of phosphorylated PI3 K and phosphorylated Akt protein decreased significantly,while the protein levels of PI3 K and Akt remained unchanged.It is suggested that htes Fab can inhibit the proliferation of nasopharyngeal cancer stem cells through PI3 K / Akt pathway.4.The htes Fab and LY294002 can inhibit tumor growth of tumor-bearing nude mice,leading to necrosis of tumor tissue in nude mice,and significantly increasing apoptosis of tumor tissue cells.The experiments confirmed that the htes Fab has the same effect as LY294002 in inhibiting the phosphorylation of PI3 K and Akt in vivo.5.The htes Fab can inhibit the invasion and metastasis of nasopharyngeal cancer stem cells,and has a similar effect to XAV-939(Wnt / β-catenin inhibitor).The htes Fab down-regulates the relative m RNA and protein expression levels of N-cadherin,vimentin,?-catenin,transcription factor slug and E-cadherin up-regulates in EMT-related genes of nasopharyngeal carcinoma stem cells.At the same time,both htes Fab and XAV-939 can inhibit the expression of β-catenin protein,suggesting that htes Fab may affect the invasion and metastasis of nasopharyngeal cancer stem cells through the Wnt / β-catenin signaling pathway.6.The htes Fab inhibited nasopharyngeal carcinoma tumor metastasis through fluorescence imaging of animals.The experiments in vivo showed that the htes Fab down-regulated the expression levels of EMT-related genes N-cadherin,vimentin,?-catenin,and the transcription factor slug and up-regulated E-cadherin in tumor tissues.htes Fab provides new ideas in the treatment of nasopharyngeal carcinoma,especially recurrent nasopharyngeal carcinoma. |
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