| [Background and Objective]Nasopharyngeal carcinoma (NPC) is one of common malignant tumors in South China and Southeast Asia. NPC lesion position is covert and uneasily discovered in the early stage, and furthermore NPC may trigger lymph gland and blood metastasis in the early stage, had high rate of recurrence even accepting therapy. So the five-year survival rate always below50%. In addition to genetic predisposition, EBV infection, environmental factors such chemical carcinogens were thought to be important in the aetiology of NPC. EBV infection play an important role in occurrence and development of NPC, the virus proteins work in the procession of tumorigene, immunosuppression, metastasis and inhibiting tumor cells apoptosis, which can promote tumongenesis.BCRF-1that is located in EBV gene can produce viral IL-10, which shares83%protein sequences with human IL-10(hIL-10) and displays immune suppressive function. There are several ways by which vIL-10inhibit the immunity and even help tumor cells to escape the immune surveillance. During tumor immune response process, the major histocompatibility complex (MHC) class I molecules(mainly HLA-I) play a crucial role in the elimination of oncogenesis and tumor progression by cytotoxic T cells (CTLs). The HLA-I antigen processing and presentation pathway starts with tumor antigen being degradation by the proteasome, which consists of multiple catalytic subunits (majorly LMP-2, LMP-7). The peptides are then translocated across the endoplasmic reticulum (ER) membrane via antigenprocessing subunits TAP-1, TAP-2(the transporter associated with antigen processing, TAP).In the ER, HLA-I interact with TAP associated peptides, which are then translated to the surface. The location of MHC-I genes, TAP genes and LMP genes are adjacent, and the transcriptional activity and expression of those genes are induced by interferon simultaneously, so they are called as MHC-I antigen processing "the operon" in Eukaryotes. In the antigen processing procession, the abnormal expression of whatever protein will influence the antigen processing and inhibit anti-tumor immunological response.So we ask whether expression of MHC-I antigen processing "the operon" are influenced by vIL-10in NPC and further explore the possible mechanism.The objects include:To determine the expression of "MHC-I antigen processing the operon"(i.e. TAP-1, TAP-2, LMP-2, LMP-7and HLA-I)in NPC paraffin section and to explore the relationship between the expressions and the clinical factors; To investigate the expression of BCRF-1mRNA and protein vIL-10, and to explicit the influence of vIL-10on immune cells of the patients with NPC; To explore the different expression of "MHC-I antigen processing the operon" in NPC cells by vIL-10; To explore the influence of vIL-10on associated proteins in NF-κB signal pathway and further modulation on the expression of "MHC-I antigen processing the operon[Methods]1. The research on expression of "MHC-I antigen processing the operon" inNasopharyngeal carcinoma tissueTo collect the biopsy of NPC patients who remitted to the department Head and Neck Surgery, The Third affiliated Hospital of Kunming Medical University, and confirmed squamous cell carcinoma by pathologic examination. For control group, healthy people were recruited who remitted to the hospital and were excluded to be NPC cases by pathological examination. The expression of TAP-1, TAP-2, LMP-2, LMP-7and HLA-I were analyzed by Immunohistochemistry and the percentage of CD4+T cells and CD8+T cells in peripheral blood (PB) of patients with NPC or non-carcinoma disease by Flow Cytometry and the content of IL-10in PB by enzyme linked immunosorbent assay.2. The expression of BCRF-1gene and the influence on immunity in Nasopharyngeal carcinomaThe mRNA of BCRF-1in NPC tissue was detected by RT-PCR and protein of vIL-10was detected by Western Blot; the frequency of CD4+T cells and CD8+T cells were calculated by FCM.3. The influence of vIL-10on "MHC-I antigen processing the operon" in Nasopharyngeal carcinoma cell linesCollected nasopharyngeal carcinoma cell treated by vIL-10in different time, detected the changes of "MHC-I antigen processing the operon" by RT-PCR and Western Bolt.4. The mechanism of vIL-10modulate "MHC-I antigen processing the operon" through NF-κB signal pathway in nasopharyngeal carcinoma cells(1) NPC cells were transfected by pEGFP-N1-TNF-a and the TNF-a level secreted by transfected cells was detected;(2) After different concentrations of TNF-α were added to NPC cell lines, the influence on the activity of NF-κB at different times were detected by Western Blot;(3) The nuclear translocation of NF-κB p65influenced by vIL-10was observed by immunofluorescence;(4) The binding DNA affinity of NF-κB in cell nuclei by EMS A;(5) The influence of the genes of "MHC-I antigen processing the operon" as the NF-κB targeted genes by vIL-10was detected with CHIP.[Results]1.The low expression of TAP-1, TAP-2, LMP-2, LMP-7and HLA-I in NPC and the association with clinical factors:①The expression of TAP-1, TAP-2, LMP-2, LMP-7 and HLA-I in NPC were43.1%,46.55%,41.38%,44.83%,50.00%respectively (25/58,27/58,24/58,26/58,29/58),which were significantly lower than that in control group90.00%,95.00%,95.00%,95.00%,95.00%(18/20,19/20,19/20,19/20,19/20)(P<0.05) respectively;②CD4+T cells in NPC group are less to different extents, obviously lower than those of control group (33.41±10.04%vs40.15±3.56%, P<0.05), while CD8+T cells increase slightly without any statistical difference from that of control group (25.32±8.29%vs22.89±2.24%, P>0.05). IL-10expression in NPC was obviously higher than those of control group (13.12±1.23vs3.69±1.03ng/ml, P<0.05);③TAP-1, TAP-2, LMP-2, LMP-7and HLA-I expression in NPC were irrelevant with age and gender (P>0.05), while they were closely associated with TNM stage, lymph gland metastasis or not and distance metastasis or not (P<0.05);④There was a positive correlation between CD8+T cells and TAP-1, TAP-2, LMP-2, LMP-7and HLA-I expression, a negative correlation between IL-10and TAP-1, TAP-2, LMP-2, LMP-7and HLA-I expression, while there was no correlation between CD4+T cells and TAP-1, TAP-2, LMP-2, LMP-7and HLA-I expression;(5)Clinical stage, lymph node metastasis, distant metastasis, pathological type, TAP-1expression, TAP-2expression,LMP-2expression, LMP-7expression and HLA-I expression were prognostic factors by Kaplan-Meier analysis.Multivariate analysis by a Cox regression model indicated that distant metastasis and down-expression of HLA-I were independent, unfavorable prognostic factors.2. The high expression of vIL-10and the relationship with immune cells:①The mRNA of BCRF-1in85%NPC samples were positive (34/40) and that in80%normal nasopharynx samples were positive (16/20), there was no significant difference of BCRF-1mRNA expression between two kinds of tissue(P>0.05);②The protein of vIL-10in75%NPC samples were positive(30/40) and that in50%normal nsopharynx samples were positive (10/20), there was significant difference between two kinds of tissue (P<0.05);③40NPC patients were divided into vIL-10positive group and vIL-10negative group. The frequency of CD4+T cells and ratio of CD4+T/CD8+T in vIL-10positive group were lower than that in vIL-10negative group (24.99±3.88% VS35.92±6.31%,0.86VS1.99, P<0.05);The frequency of CD8+T cells in vIL-10positive group were higher than that in vIL-10negative group (30.61±3.34%VS20.10±7.68%,P<0.05).3. vIL-10inhibit the expression of "MHC-I antigen processing the operon" in Nasopharyngeal carcinoma cell lines:(1)mRNA expression:①there was no difference among times in expression of TAP-1in CNE-land CNE-2cells;②The expression of TAP-2in CNE-1and CNE-2did not show any change at1h,4h,6h,12h, but downregulation even disappear at24h;③The expression of LMP-2in CNE-1was lower at24h, and that in CNE-2disappear at same time;④The expression of LMP-7in CNE-1showed decrease after adding vIL-101h, and that in CNE-2showed decrease at6h;⑤The expression of HLA-I in CNE-1and CNE-2showed significant decrease at24h.(2) protein expression:①The expression of TAP-1in CNE-1showed subtle decrease at6h, but increased at12h, pronouncedly decreased at24h(P<0.05), The expression of TAP-1in CNE-2were higher at6h and12h than that at1h, and became declined at24h(P<0.05);②The expression of TAP-2in CNE-1and CNE-2were gradually downregulated at different periods(P<0.05);③The expression of LMP-2in CNE-1was significantly decreased at12h, and that in CNE-2was gradually downregulated at different periods(P<0.05);④The expression of LMP-7in CNE-1was significantly decreased at12h, and that in CNE-2was gradually downregulated at different periods(P<0.05);⑤The expression of HLA-I in CNE-land CNE-2were gradually downregulated, but there were not any significantly differences at every period in CNE-1(P>0.05), while the expression of HLA-I in CNE-2at24h was significantly downregulated(P<0.05).4. vIL-10inhibit the expression of "MHC-I antigen processing the operon" through NF-κB signal pathway in nasopharyngeal carcinoma cells:(1) vIL-10inhibit the NF-κB signal pathway:①The TNF-a secretion from NPC cells transfected with TNF-a gene was increased, but which did not reach the needed concentration.;②The expression of NF-κBp-p65was up-regulated when CNE-land CNE-2cells were treated by TNF-α, and the concentration of TNF-a were positive proportional to the expression;③The expression of IKK a、IKKβ in CNE-2were increased when treated by TNF-α, but there was no change in CNE-1, and the p-IKK a/β in two cell lines was up-regulated;④The expression of IKB a in CNE-1and CNE-2was decreased when treated by TNF-a, and there was negative correlation. On the contrary, the expression of p-IKB a was increased when induced by TNF-α;⑤The expression of p-IKK a/β、NF-icBp-p65in CNE-1and CNE-2were negatively correlated to vIL-10, but the expression of IKB was positively correlated to vIL-10;⑥The activity of nuclear translocation of NF-κBp65in cytoplasm was decreased when CNE-1and CNE-2cells were treated by vIL-10;⑦In NPC cells, vIL-10could inhibit the binding DNA affinity of NF-κB, which was dependent on the dosage of vIL-10in CNE-2especially.(2) vIL-10could inhibit the gene expression of TAP-1, TAP-2, LMP-2, LMP-7and HLA-I in CNE-2detected by ChIP.[Conclusion]1. The low expression of TAP-1, TAP-2, LMP-2, LMP-7and HLA-I in NPC influence the immune function for the patients with NPC, lower expression of HLA-I in NPC patients was associated with a poor prognosis.2. The gene of BCRF-1and protein of vIL-10were significantly higher expression in tissue of NPC, which would cause immunosuppression.3. The vIL-10could inhibit the expression of "MHC-I antigen processing the operon" in NPC, and the inhibition in TAP-1,TAP-2,LMP-2,LMP-7and HLA-I were different time-dependent.4. The vIL-10could inhibit the activation, nuclear translocation and the binding DNA affinity of NF-κBp65, and then inhibit the transcription of NF-κB targeted geneTAP-1, TAP-2, LMP-2, LMP-7and HLA-I, down-regulated the antigen presentation. |
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