Clinical Significance,Function And Mechanism Of Receptor Expressed In Lymphoid Tissues In Esophageal Squamous Cell Carcinoma

ObjectivesTo investigate the expression level of receptor expressed in lymphoid tissues(RELT)in esophageal squamous cell carcinoma(ESCC)and its relationship with clinicopathological parameters and prognosis,and further to investigate the specific molecular mechanisms by which RELT promotes ESCC proliferation.MethodsqRT-PCR,Western blot and immunohistochemistry were used to detect 83 cases of ESCC tissues,adjacent tissues and esophageal cancer cell lines ECA-109,KYSE140,TE-1,TE-11,TE-14 and human esophageal epithelial cells(HEEC)RELT expression level,analyze the relationship between RELT expression and ESCC clinicopathological parameters and patient prognosis.We constructed RELT low expression TE-1 and TE-14 stable strains,and examined the efficiency of RELT overexpression and knockdown strains using qRT-PCR and Western blot,respectively;the proliferation ability of ESCC stable strains was examined by CCK-8 assay,the cycle distribution and apoptosis of ESCC stable strains were examined by flow cytometry.The expression levels of cycle and apoptosis marker proteins such as P27,Cleaved Casapase3 and Survivin in the stable strain were detected by Western blot.The expression of NF-κB(plasma)and NF-κB(nuclear)was detected by Western blot.ResultsThe results of qRT-PCR indicated that the expression level of RELT m RNA in ESCC tissue was evidently higher than that in the corresponding adjacent tissues(P <0.05).Western blot showed that RELT protein expression levels were remarkably higher in ESCC tissues than in the corresponding paraneoplastic tissues(P<0.05).Immunohistochemical results showed that RELT protein expression level was higher in ESCC tissues than in paraneoplastic tissues,and RELT was mainly distributed in the nucleus and cytoplasm;The positive expression rate of RELT protein was higher in ESCC tissues(78.31%)than in paraneoplastic tissues(16.87%)(P < 0.001).The expression level of RELT was evidently correlated with the depth of tumour infiltration,lymph node metastasis,vascular infiltration and TNM stage in ESCC patients.The survival analysis by Kaplan ～ Meier method sindicated that the median disease-free survival of ESCC patients in the high expression.group was lower than that in the low expression group(P<0.05);Cox univariate regression analysis indicated that age,degree of tumour infiltration,lymph node metastasis,vascular invasion,high RELT expression and TNM stage were risk factors affecting the prognosis of patients with ESCC(P < 0.05).Cox multi-factor regression analysis indicated that the high expression of RELT and TNM stage were independent risk factors affecting the prognosis of ESCC patients(P < 0.05).The results of qRT-PCR and Western blot indicated that the expression level of RELT in ECA-109,KYSE140,TE-1,TE-11 and TE-14 esophageal cancer cell lines was higher than that of HEEC in human normal esophageal epithelial cells.The qRT-PCR and Western blot results indicated that RELT expression levels were significantly decreased in the knockdown TE-1 stable strain compared to the control,and significantly up-regulated in the overexpression TE-14 stable strain compared to the control.The CCK-8 results indicated that down-regulation of the expression of TE-1 significantly inhibited the proliferation of cells.The cell proliferation ability of TE-14 stable strain was evidently increased after overexpression of RELT.Flow cytometry results showed that after down-regulation of RELT expression,the proportion of cells in G1 phase was evidently increased and the proportion of cells in S and G2 phases was evidently decreased in the experimental group(knockdown TE-1 group)compared to the control group;The proportion of cells in G1 phase was evidently decreased and the number of cells in S and G2 phases was evidently increased in the RELT overexpression(overexpression TE-14 group)group.The apoptosis rate was evidently increased in the experimental group compared to the control group after knockdown of RELT expression,and evidently decreased in the overexpressed RELT group compared to the control group.Silencing of RELT expression resulted in increased expression of the cycle marker protein p27,increased expression of the apoptosis-related protein caspase 3 and decreased expression of survivin.Overexpression of RELT resulted in the opposite trend of these marker proteins.GSEA analysis showed that high level of RELT was significantly enriched in the NF-κB pathway.Western blot results showed that the level of NF-κB protein in the cytoplasm of the experimental group was significantly increased and the level of NF-κB protein in the nucleus was significantly decreased after knocking down the expression of RELT.On the contrary,after overexpression of RELT,the level of NF-κB protein decreased significantly in the cytoplasm of the experimental group and increased significantly in the nucleus.Conclusion1.RELT is highly expressed in ESCC tissues and cells;2.High RELT expression in ESCC tissues was closely related to the depth of tumor infiltration,the presence or absence of metastasis in lymph nodes,whether the vasculature was invaded,and the TNM stage;ESCC patients with high RELT expression had poor prognosis,and elevated RELT expression level was an independent risk factor affecting the poor prognosis of ESCC patients.3.Knockdown of RELT expression inhibited the proliferative capacity of ESCC cells and promoted their apoptosis,and upregulation of RELT expression promoted the proliferative capacity of ESCC cells and inhibited ESCC apoptosis.4.RELT can promote ESCC cell proliferation through the NF-κB signaling pathway.