Roles Of MUC1 In CCR7/CCL21 Induced Lymphatic Metastasis Of Tongue Squamous Cell Carcinoma

Objectives:By investigating the epidemiological trend and the possible relationship between cervical lymph nodes(CLN)metastasis and clinicopathologic characteristics of patients of tongue squamous cell carcinoma(TSCC),and observing the expression of MUC1 and CCR7 in TSCC tissues,and studying the possible role of MUC1 in CCR7/CCL21 signal transduction pathway as it induces the lymphatic metastasis of TSCC,to find out the possible effect and molecular regulation mechanism of CCR7/CCL21 signal transduction pathway on lymphatic metastasis of TSCC,and to introduce new molecular targets to the biotherapy of TSCC.Methods:1.The medical records of 78 patients of TSCC who were treated in Tianjin stomatological hospital were collected to analyse the epidemiological trends of TSCC.The relationship between CLN metastasis and clinicopathologic characteristics of the patients was analysed by Chi-square test.The influence factors of CLN metastasis of TSCC were analysed by Logistic regressive method.2.The recent 60 out of 78 of the patients were selected to inplement the Immunohistochemistry(IHC)stain to observe the expression of MUC1 and CCR7 in TSCC tissues.The relationships between the expression of both molecular and the clinicopathologic characteristics were analysed by Chi-square test.The relationship between the expression of MUC1 and CCR7 was analysed by Kappa’s consistency check.3.SCC15 and CAL27 TSCC cell lines were cultured in vitro.A method of q RT-PCR and Western blot was used to detect the expression variation of MUC1 while the cells were induced by recombinant human CCL21(r CCL21)and specific antibody of CCR7.The Wound healing assay and Transwell invasion assay were used to analyse the influences of migration and invasion capacity of the TSCC cells while up or down regulating the expression of MUC1.Results:1.The 78 TSCC patients concluded 48 males and 30 females,M/F=1.6/1.The age of the patients ranged from 26 to 73,and the middle age was 59,and the high morbidity age group was considered from 50 to 69.33 patients out of 78 were diagnosed to be CLN metastasis(42.3%)by biopsy.Chi-square test showed that CLN metastasis was correlated with T staging(P=0.026),clinical staging(P=0.024),invasion depth(P=0.001),but not correlated with gender(P=0.425),age(P=0.896),area of tumor site(P=0.302).Logistic regressive analysis showed that the invasion depth was the most important influence factor of CLN metastasis of TSCC(P=0.033,OR=10.919).2.In IHC stain experiment,43 patients out of 60 were considered as positive expression of MUC1(71.7%),and 41 patients out of 60 were considered as positive expression of CCR7(68.3%).Chi-square test showed that MUC1 and CCR7 expressions in TSCC tissues were only correlated with CLN metastasis(P=0.026,0.041,respectively)among all the clinicopathologic characteristics.Kappa’s consistency check showed that the consistency of the positive expression between MUC1 and CCR7 in TSCC tissues was fairly good(Kappa=0.683,P=0.000).3.In cell culture experiment,r CCL21 could up-regulate the expression of MUC1 in SCC15 cell line(P<0.05),and the up-regulation behaved in a dose-dependent manner to some extent whlie increasing the concentration of r CCL21.The up-regulation effect was inhibited when CCR7 was blocked by specific antibody of CCR7(P<0.05).But the CAL27 cell line didn’t show up the sensitivity of r CCL21(P>0.05).Wound healing assay showed that cell migration capacity was different between blank group and MUC1-up-regulate group,and between MUC1-up-regulate group and MUC1-down-regulate group in both cell lines in both 24 h and 48 h of culture time(P<0.05).Transwell invasion assay showed that cell invasion capacity was different between blank group and MUC1-up-regulate group,and between MUC1-up-regulate group and MUC1-down-regulate group in SCC15 cell lines in both 24 h and 48 h of culture time(P<0.05).But CAL27 cells were not transmembraned under the same condition.Conclusions:1.CLN metastasis of TSCC patients was correlated with T staging,clinical staging and invasion depth,and the invasion depth was the most important influence factor of CLN metastasis of TSCC,which means the staging and invasion depth of TSCC patients were crucial risk factors for lymphatic metastasis.2.The positive expression of MUC1 and CCR7 were both correlated with CLN metastasis in TSCC patients,which means high expression of MUC1 and CCR7 could both remind of lymphatic metastasis of TSCC.The correlation between the expression of MUC1 and CCR7 indicated that MUC1 might have some relationships with CCR7/CCL21 signal transduction pathway.3.r CCL21 could up-regulate the expression of MUC1 in SCC15 cell line,but not in CAL27 cell line.Blocking CCR7 could inhibit the effect of up-regulation.MUC1 might be a downstream molecular in CCR7/CCL21 signal transduction pathway as it induced the lymphatic metastasis of TSCC.The up-regulation of MUC1 could enhance cell migration and invasion capacities of TSCC.4.CCR7/CCL21 signal transduction pathway probably promotes the CLN metastasis of TSCC by up-regulating MUC1,and MUC1 and CCR7 could be the new molecular targets for the biotherapy of TSCC.