| Part1 The expression and clinical significance of MIIP in nasopharyngeal carcinomaObjective:To detect the expression of Migration and Invasion Inhibitory protein(MIIP)expression in nasopharyngeal carcinoma tissue,and to analysis the relationship between the MIIP levels and clinicopathological characteristics and prognosis.Methods:1.Using immunohistochemistry to detect MIIP expression of NPC(n=96)paraffin specimens,and analyzes its relationship with clinical pathological factors.2.The 3 years progress-free survival differences between the positive MIIP protein expression and negative MIIP protein expression group were analyzed by Kaplan-Meier survival curve.3.The prognostic value of MIIP was estimated by the Cox regression model.Results:1.MIIP positive expression is found in 29 cases(positive rate:30.21%).MIIP protein expression in NPC positively correlated with T stage,N stage,TNM stage,3 years related recurrence and metastasis(p<0.05).2.The 3 year progress-free survival rate in all NPC was 79.8%(95%CI:0.708-0.863),3 year progress-free survival rate of positive and negative MIIP protein expression group was 89.7%(95%CI:0.816-0.947)and 70.1%(95%CI:0.515-0.765)respectively,the difference was statistically significant(p<0.05).Univariate analysis showed that T stage,N stage,TNM staging and MIIP were predictors of poor prognosis.Multivariate analysis showed that only N stage(HR:7.892,95%CI:2.731-22.807,p<0.001),TNM stage(HR:4.065,95%CI:1.878-6.239,p=0.034)and MIIP(HR:3.583,95%CI:2.185-4.077,p=0.043)were independent predictors of poor prognosis.Conclusion:These results demonstrated that MIIP,an independent prognostic factor,may relate to NPC occurrence and progression,and MIIP may be considered a potential therapeutic target in NPC.Part2 The expression of MIIP suppresses nasopharyngeal carcinoma cells malignant biological behaviorsObjective:To investigate the effect of MIIP in regulating proliferation,migration and invasion in NPC in vitro.Methods:1.The expression of MIIP mRNA and protein were estimated by qRT-PCR and Western blot in human immortalized nasopharyngeal epithelium NP-69 cell line and human nasopharyngeal carcinoma cell line CNE-1,CNE-2,5-8F,6-10B,SUNE1 and HNE-1.2.Si-RNA Was used to knockdown MIIP in CNE1 cell.Adenovims-MIIP was used to overexpress MIIP in 5-8F cell.The viability and metastatic ability were analyzed by colony formation assay,and transwell assay.The changes of cell cycle were evaluated by flow cytometry.Results:1.MIIP mRNA expression in each NPC cell lines was significantly lower than that of NP-69(p<0.01).Also,MIIP protein high expression in NP69,low expression in NPC cells,the results were statistically significant(p<0.05).MIIP expression in low differentiated and high capacity of Invasion and metastasis cell lines was decreased obviously compare with high differentiated cell line.2.Clone formation rate was increased by 40%(p<0.001)in CNE-1 si-MIIP group and decreased by 50%(p<0.001)in 5-8F MIIP group.3.Knockdown of MIIP expression with siRNA in CNEI cells significantly increased cell migration and invasion when compared with control siRNA treated cells(migration:44.45 ± 8.45 vs.150.45 ± 10.43,p<0.001;invasion:34.03 ±6.46 vs.100.66 ± 8.18,p<0.001).Cell invasion migration and invasion was significantly decreased in MIIP overexpressed cell 5-8F compared with control cells(migration:201.32 ± 1.67 vs.60.88 ± 12.66,p<0.001;invasion:144.16 ±10.31 vs.43.03 ± 10.77,p<0,001).4.By flow cytometry,G1 phase cells decreased(p<0.01),and G2/M phase cells proportion increased(p<0.01)in CNE1 si-MIIP group.Consistently,G1 phase cells increased(p<0.01),and G2/M phase cells proportion reduced(p<0.01)in 5-8F MIIP group.Conclusion:MIIP might take part in some malignant biological behaviors including proliferation,tumor growth,migration,invasion and cell cycle.Part3 The mechanism of MIIP suppresses nasopharyngeal carcinoma cells malignant biological behaviorsObjective:To assess the relationship between the expression of MIIP and EGFR and study the mechanism of MIIP suppresses malignant biological behaviors in NPC.Methods:1.The expression of EGFR in 96 cases of NPC paraffin sample were evaluated by immunohistochemistry and analyze the relationship between EGFR expression and prognosis.The relationship between MIIP and EGFR were evaluated by Spearman rank correlation analysis.2.The expression of EGFR,pEGFR and HDAC6 were investigated by Western blot in MIIP knockdown or overexpression cells.3.Following the knockdown or overexpression of MIIP,the key proteins expression of PI3K/AKT pathway and EMT were analyzed by Western blot.After the application of exogenous EGF stimulation in MIIP-overexpression cells,the changes of cell migration ability and AKT protein was observed.Results:1.MIIP expression(showed in Part 1),EGFR positive particles mainly located in cell membrane.EGFR protein was positive expression in 66 cases of NPC(positive rate:68.8%).EGFR negatively correlated to the expression of MIIP(r =-0.780,p<0.01).2.The 3 year progress-free survival rate in all NPC was 79.8%(95%CI:0.708-0.863),3 year progress-free survival rate of positive and negative EGFR protein expression group was 71.2%(95%CI:0.659-0,772)and 86.7%(95%CI:0.798-0.921)respectively,the difference was not statistically significant(p =0.109).3.By Western blot,in MIIP knockdown cells,EGFR and pEGFR is relatively higher than the control.Consistently,in MIIP overexpression cells,EGFR and pEGFR is relatively lower than the control.4.In MIIP knockdown cells,pAKT and Vimentin expression is relatively higher than the control,E-cadherin expression is relatively lower than the control.Consistently,in MIIP overexpression cells,pAKT and Vimentin expression is relatively lower than the control.E-cadherin expression is relatively higher than the control.After the application of exogenous EGF stimulation in MIIP-overexpression cells,the cell migration ability increased obviously(p<0.01),and pAKT protein expression up-regulated in the control group.Nevertheless,the increase of cell migration ability and pAKT protein expression was not found in MIIP-overexpression group.Conclusion:MIIP expression and EGFR negatively correlated.MIIP may suppresses NPC EMT process through EGFR/AKT signaling pathways which affect the proliferation of NPC,metastasis and other malignant biological behavior. |
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