The Study On The Role And Related Mechanisms Of Chemine Receptor-4 In Metastasis Of Nasopharyngeal Carcinoma

ObjectiveMetastasis of nasopharyngeal carcinoma(NPC) is the major factor influencing prognosis and survival rates of NPC patients.It was reported that more than 60%diagnosed NPC patients had metastasis of neck lymph node or distant organs.About 1/3 patients at their first time seeing doctor had metastasis symptoms.Furthermore,the middle survival was less than one year if metastasis was detected.Therefore,it is very important to study the metastasis mechanisms of NPC and search for targets for improving curative effects of NPC and prolonging overall survivals of patients.Chemine receptor-4(CXCR4),which was secreted by inflammatory cells and tumor cells,was proved by many reachers.It was reported that CXCR4 played an important role in the process of progression and metastasis in many tumors,but rarely studied in NPC.Our study will observe the expression of CXCR4 in NPC tissues and cell lines with different metastatic potential;investigate the role of CXCR4 in metastasis of NPC and its relationships with clinical stage and cell differentiation in NPC;identify the relationships between CXCR4 and LMP-1 in NPC;explore the role of CXCR4/SDF-1αin organ-specific metastasis of NPC. Methods1.Clinical samples collecting and groupingThirty patients with NPC(16 cases with cervical lymph nody metastasis,14 cases without cervical lymph nody metastasis) and fifteen normal cases(volunteers as controll group) were recruited in this study from October 2006 to April 2007.Normal neck lymph node,bone marrow,lung,liver where NPC easily metastasizes(5 cases/each group) and kidney,colon tissues where NPC not easily metastasizes(5 cases/each group) were also collected.Normal neck lymph node were from NPC patients,other specimen were gotten from patients who suffered tumors and needed to be operated and drawn far away from(more than 3cm)safe cutting edge of resected tumor.All the specimen were confirmed as normal condition by HE staining method.2.Culture of NPC cell linesThe follwing cell lines were maintained in our department:poorly differentiated NPC cell line with EB virus C666-1,NPC cell line 5-8F(with ability to metastasize) and 6-10B(without ability to metastasize),well-differentiated NPC cell lines CNE-1 and Hone-1,poorly differentiated NPC cell line CNE-2,B95-8 cell inverted by EB virus.The cells were digested by 0.25% parenzyme(containing 0.02%EDTA) and maintained in RMPI-1640 medium which was supplemented with 15%heated inactivated fetal calf serum,penicillin(100ug/ml) and streptomycin(100ug/ml) at 37℃with 5%CO₂.3.RT-PCRTotal RNA were extracted from NPC tissues and cell lines according to routine methods, then reversely transcripted into cDNA and PCR amplified according to the sequences of primers.All the production of PCR reaction were underwent ionophoresis in 1.5%agarose gel with EB,then image analysis software Bandscan5.0 was used to scan the densities of straps.The log numerus of gray scale about CXCR4 and LMP-1 compared with those of GAPDH were used to represent the relative expression levels of CXCR4 and LMP-1 mRNA.4.IHC detection Paraffin sections of tissues prepared for detection were stained by SP method,and the results of IHC were observed by light microscope and judged by the modified formula used by Bresalier: Sections were observed in 10X objective lens and the staining intensities of all cells in 5 fields of vision were counted randomly,then scores were counted according to intensity stained on cell(four grades):negative,no colour on cell(0 score);weakly positive,pale yellow stained on cell(1 score);midrange positive,palm yellow stained on cell(2 scores);weakly positive,dark brown stained on cello scores),finally the average staining intensities of every section were counted according to the following formula:the average staining intensities of every section =∑{(0×F0)+(1×F1)+(2×F2)+(3×F3)},Fi=%10×fields of vision(i=0,1,2,3).5.StatisticsThe values given were means±SEM.The significance of difference about CXCR4 and LMP-1 mRNA among different groups were assessed by one-way ANOVA and LSD(if multiple comparison was needed).The expression levels of CXCR4 protein and LMP-1 protein between the primary tumors and corresponding metastasis of lymph node of neck were assessed by non-parameter test of two related samples(Wilcoxo method),and Kruskal-Wallis test were used to compare the other results of IHC.Pearson linear correlation and Sperman rank correlation were used to analyze the relationships of mRNA expression and protein expression, respectively.The difference was significant if the P value was＜0.05.All the data were statistically treated by adopting statistical package of SPSS 11.5.Results1.The expressions of CXCR4 and LMP-1 in normal nasopharynx and NPC tissuesThe expression levels of CXCR4mRNA detected by RT-PCR in normal nasopharynx tissues,nasopharyngeal carcinoma tissues without metastasis of neck lymph node,nasopharyngeal carcinoma tissues with metastasis of neck lymph node were 0.185±0.126,0.631±0.287, 0.856±0.299 respectively,and the differences among them were significant(F=28.449,P=0.000) by One-way ANOVA and LSD test.The expression levels of CXCR4mRNA were highest in nasopharyngeal carcinoma tissues with metastasis of lymph node of neck and lowest in normal nasopharynx tissues.The expression levels of CXCR4 protein detected by IHC in normal nasopharynx tissues,nasopharyngeal carcinoma tissues without metastasis of neck lymph node, nasopharyngeal carcinoma tissues with metastasis of neck lymph node were 9.47,21.79,36.75,and the differences among them were also significant(X²=34.521,P=0.000) by Kruskal-Wallis test.The expression levels of CXCR4 protein were highest in nasopharyngeal carcinoma tissues with metastasis of neck lymph node and lowest in normal nasopharynx tissues.Furthermore,the expression levels of CXCR4mRNA and protein in NPC were positively correlated with clinical stage and metastasis of neck lymph node,negatively correlated with extent of cell differentiation (P＜0.01).Finally,the expression levels of CXCR4 protein were higher in metastasis than those of corresponding primary turnouts in the patients(15 cases) who were made biopsy in nasopharynx and neck lymph node,and the differrences of them were significant(Z=-2.724,P= 0.006) by Wilcoxon test.The expression levels of LMP-lmRNA detected by RT-PCR in normal nasopharynx tissues,nasopharyngeal carcinoma tissues without metastasis of neck lymph node,nasopharyngeal carcinoma tissues with metastasis of neck lymph node were 0.541±0.284,0.637±0.288,0.966±0.198,and the differences among them were significant(F=28.449,P=0.000) by One-way ANOVA and LSD test but except the difference between normal nasopharynx tissues and nasopharyngeal carcinoma tissues without metastasis of lymph node of neck(P＞0.05).The expression levels of LMP-1mRNA were highest in nasopharyngeal carcinoma tissues with metastasis of neck lymph node and lowest in normal nasopharynx tissues.The expression levels of LMP-1 protein detected by IHC in different groups of nasopharynx tissues were 11.83,19.46,36.56,and the differences among them were also significant(X²=30.082,P=0.000) by Kruskal-Wallis test.The expression levels of LMP-1 protein were highest in nasopharyngeal carcinoma tissues with metastasis of neck lymph node and lowest in normal nasopharynx tissues.Furthermore,the expression levels of LMP-1mRNA and protein in NPC were positively correlated with clinical stage and metastasis of neck lymph node(P＜0.01),but not significantly correlated with extent of cell differentiation (P＞0.05).Finally,the expression levels of LMP-1 protein were higher in metastasis than those of corresponding primary tumours in the patients(15 cases)who were made biopsy in nasopharynx and neck lymph node,and the differrences were significant(Z=-2.233,P=0.026) by Wilcoxon test.2.The expressions of CXCR4 and LMP-1 in nasopharyngeal carcinoma cell linesThe expression levels of CXCR4mRNA detected by RT-PCR in C666-1,5-8F,6-10B,CNE-1,CNE-2 were 0.974±0.015,0.836±0.023,0.648±0.013,0.642±0.026,0.628±0.019, but CXCR4 mRNA was not expressed in Hone-1.Results of One-way ANOVA and LSD test indicated that the differences among cells were significant(P=0.000) except 6-10B and CNE-1(P=0.642),6-10B and CNE-2(P=0.131),CNE-1 and CNE-2(P=0.284),the expression levels of CXCR4mRNA were highest in C666-1 and lowest in CNE-2.None of 5-8F,6-10B,CNE-1,CNE-2,Hone-1 had the expression of LMP-1 mRNA except C666-1(1.0140±0.0421)by nest PCR,and the expressions of CXCR4 and LMP-1 mRNA in C666-1 were obviously correlated.3.Relationships between CXCR4 and LMP-1 in NPCThe expressions of CXCR4mRNA(0.751±0.310) and LMP-1mRNA(0.813±0.292) in NPC were significantly correlated(r=0.491,P=0.006) by Pearson linear correlation analysis,but the expressions of CXCR4mRNA(0.185±0.126) and LMP-1mRNA(0.541±0.284)in normal nasopharynx tissues were not significantly correlated(r=0.067,P=0.812).The expressions of CXCR4 protein(1.176±0.911) and LMP-1 protein(0.687±0.645) in NPC were significantly correlated(r=0.724,P=0.000) by Sperman rank correlation analysis,but the expressions of CXCR4 protein(0.303±0.195) and LMP-1 protein(0.170±0.186)in normal nasopharynx tissues were not significantly correlated(r=0.001,P=0.997).Compared with C666-1 transfected with rAAV-shRNA-EGFP, expression of CXCR4mRNA was significantly downregulated after LMP-1 was silenced in C666-1 transfected with rAAV-shRNA-LMP-1.4.The expressions of SDF-1αin distant target organs of NPCThe expression levies of SDF-1αin normal lymph node of neck,lung,bone marrow,liver (distant organs where NPC easily metastasizes) were 20.80,22.40,20.60,17.20 by Kruskal-Wallis test,nevertheless they were 6.40,5.60 in kidney and colon tissues(distant organs where NPC not easily metastasizes ),and the expression levels of SDF-1αprotein in former group were significantly higher than those of the latter group(X²=19.759,P=0.001).Conclusion1.The expression levels of CXCR4 mRNA and protein in normal nasopharynx tissues, nasopharyngeal carcinoma tissues without metastasis of neck lymph node,nasopharyngeal carcinoma tissues with metastasis of neck lymph node,were steped up significantly in order.2.The expression levels of CXCR4 protein were higher in metastasis site than those of corresponding primary tumours in the same patients.3.The expression levels of CXCR4mRNA and protein in NPC were positively correlated with clinical stage and metastasis of neck lymph node,negatively correlated with extent of cell differentiation.4.The expressions of CXCR4,LMP-1 mRNA and protein in NPC were sinificantly correlated.5.CXCR4mRNA was observed to express in NPC cell lines C666-1,5-8F,6-10B,CNE-1 and CNE-2 to some extent,and it was highest in C666-1 and lowest in CNE-2;but LMP-1mRNA was only expressed in C666-1.The expression levels of CXCR4mRNA and LMP-1mRNA were significantly corelated in C666-1.6.Compared with C666-1 transfected with rAAV-EGFP,the expression of CXCR4mRNA in C666-1 transfected with rAAV-shRNA-LMP-1 was significantly downregulated after LMP-1 was silenced.It had altered the expression of CXCR4 on transcriptional level.7.CXCR4 was observed to express highly in NPC cells and tissues.SDF-1αwas proved to express highly in distant organs where NPC easily metastasizes.It showed that CXCR4/SDF-1αaxis may participate in organ-specific metastasis of NPC.