| BackgroundEsophageal cancer(EC)is one of the most common malignancies in the world.Although we have achieved some advances in the diagnosis and treatment of EC,the overall prognosis of these patients is still very dismal.Lymph node metastasis has been proved to be an independent prognostic factor for EC patients.However,the mechanism of lymphangiogenesis in EC is largely unclear.PGRN(progranulin)is a secretory glycoprotein,which plays important roles in regulating neurotrophic function and inflammatory reaction.PGRN has been shown to be highly expressed in various cancer types including the colorectal and epithelial ovarian cancers.Besides,studies have also proved that PGRN could promote tumor growth,apoptosis resistance and epithelial mesenchymal transition(EMT)in these cancers through PKB/AKT or ERK signal pathways.However,expression status of PGRN in EC and effects of PGRN on EC lymphangiogenesis is still unknown.VEGF-C is an important factor regulating tumor lymphangiogenesis,promoting the formation and growth of lymph tube through activation of VEGFR-3.The expression of VEGF-C has been shown to be positively correlated with LMVD and lymph node metastasis in a variety of cancers.Interestingly,phosphorylated ERK,and AKT are believed to be the upstream transducers of VEGF-C.The relationship between PGRN expression and VEGF-C expression in esophageal cancer and its possible mechanisms also need to be researched.AimTo determine the expression levels of PGRN in EC;to explore the roles of PGRN in EC lymphangiogenesis and the molecular mechanisms;to elucidate the effects of PGRN expression on the prognosis of EC patients;to establish a clicinal useful nomogram.Methods1.The expression of PGRN and lymphangiogenesis factor VEGF-C was detected using RT-PCR and Western blot in EC cell lines including TE-1,Ecal 09,K410,K450.2.The expression of PGRN in EC tissue was detected using immunohistochemistry(IHC);the correlation of PGRN with clinical parameters was further analyzed.Proliferation marker Ki67 and LECs marker D2-40 in EC tissue was detected using IHC.The correlation of PGRN with Ki67 and lymph micro vessel density(LMVD)and lymph vessel space invasion(LVSI)was further analyzed.3.p-GPU6/GFP/Neo/PGRN plasmid and empty vector were used to transfect EC cell lines TE-1 to get EC cell lines with silenced PGRN expression(TE-1-PGRN-sh),as well as control cell lines(TE-1-NC-sh);pEZ-M61/GFP/PGRN plasmid and empty vector were used to transfect EC cell lines TE-1 to get EC cell lines with PGRN overexpression(TE-1-PGRN-vector),as well as control cell lines(TE-1-NC-vector).RT-PCR and Western blot were used to confirm transfection efficiency.After upregulation or downregulation of PGRN expression in TE-1 cells,RT-PCR and Western blot were performed to detect changes of VEGF-C.4.Phosphorylated ERK,AKT and JNK are believed to be the upstream transducers of VEGF-C.The effect of PGRN on activation of extracellular signal regulated kinases(ERK),protein kinase B(PKB or AKT)and(c-JunN-terminalkinase)JNK was detected using Western blot.After the downstream signal tergets were selected,adding the signals inhibitors and then the changes of the VEGF-C expression induced by PGRN were detected to confirm the signal pathways.5.The expression of VEGF-C,p-ERK,AKT,p-AKT in EC tissue were detected using IHC;and the correlation of PGRN with VEGF-C,p-ERK,AKT,p-AKT was further analyzed.6.Kaplan-Meier curve was applicated to analyze the overall survival(OS)and the progression-free survival(PFS)of the EC patients with high and low PGRN expression,and the log-rank method was applied for the statistical analysis.7.Cox regression model was used to analyze the independent risk factors for overall survival of EC patients,and a nomogram was established to predict the prognosis of EC patients.Results1.Expression of PGRN in EC cell lines and its correlation with VEGF-CPGRN was expressed in all EC cell lines including TE-1,Eca109,k410 and k450 by RT-PCR analysis.Then,the PGRN protein was further confirmed in these cell lines except for k410 cells lines assayed by western blot.VEGF-C was found to be co-expressed with PGRN in EC cell lines including TE-1,Eca109 and k450.Thus,we speculate that the causal relationship of PGRN and VEGF-C may exist in esophageal cancers.2.The effects of PGRN upregulation/downregulation on the expression of VEGF-C in TE-1 cells.The expression of VEGF-C was changed accordingly,when PGRN-vector or siRNA was used to upregulate or downregulate the PGRN expression.Thus,PGRN may induce the expression of VEGF-C in TE-1 cells.3.PGRN induced the expression of VEGF-C through p-ERK and p-AKT signal pathways in TE-1 cells.Western blot found that PGRN-vector could induce the expression of p-ERK and p-AKT and VEGF-C,but no effects on the expression of ERK.AKT,JNK and p-JNK.Furthermore,p-ERK inhibitor U0126 could indeed abrogate the inducing effects of PGRN-vector on the p-ERK and VEGF-C levels,whereas it had nothing to do with the ERK expression;p-AKT inhibitor LY294002 could alleviate the effects of PGRN-vector on the expression of p-AKT and VEGF-C,whereas it had no effects on AKT levels.Thus,PGRN may induce the expression of VEGF-C through p-ERK and p-AKT signal pathways in EC cells.4.Expression of PGRN in the EC samples and its association with lymph node metastasis,LMVD and LVSI.We collected 96 tumor samples of EC patients as well as the clinicopathologic information of these patients.The patients expressing high levels of PGRN were more likely having lymph node metastasis(69%vs 46%,P=0.03)compared to those with low levels of PGRN.Yet,the expression of PGRN was not associated with age,gender,TNM stage,tumor diameter,depth of invasion,histologic types and tumor grades.Patients expressing high levels of PGRN had high levels of D2-40(marker of LMVD)(P=0.016)and positive LVSI(67%vs 46%,P=0.04).The expression of PGRN was significantly correlated with LMVD(r=0.30,P=0.003),and LVSI(r=0.21,P=0.039).5.The expresion of PGRN was significantly correlated with the expression of VEGF-C,p-ERK and p-AKT in EC tissues.3.The patients expressing high levels of PGRN were more likely having high levels of VEGF-C(P<0.001),p-ERK(P<0.001),and p-AKT(P=0.035).And the expression of PGRN was linearly correlated with VEGF-C(r=0.39,P=0.001),p-ERK(r=0.33,P=0.001)and p-AKT(r=0.30,P=0.03).Thus,the reseach in vivo confirm that PGRN induces the expression of VEGF-C through the p-ERK and p-AKT signaling pathways to promote the lymphangiogenesis and lymph node metastasis of EC.6.Survival analysis of EC patients expressing different levels of PGRN.Kaplan-Meier curves found that patients expressing high levels of PGRN had less favorable OS and PFS than those expressing low levels of PGRN.Cox proportional hazard model found that patients with high levels of PGRN had 1.99 increased fold of mortality(95%confidence interval:1.05-3.78;P=0.035),and 2.88 increased fold of morbidity(95%confidence interval:1.55-5.35;P=0.001),compared to those with low levels of PGRN.7.Nomogram was established to predict the mortality for individual EC patient.The Cox regression model found that TNM stages(hazard ratio:2.64,95%confidence interval:1.07?6.50;P=0.035),lymph node metastasis(hazard ratio:1.31,95%confidence interval:2.26?7.56;P<0.001),PGRN status(hazard ratio:1.36,95%confidence interval:1.05?1.77;P=0.022),and p-ERK(hazard ratio:1.42,95%confidence interval;1.07?1.89;P=0.016)were independent prognostic factors for EC mortality.A nomogram was established using these four independent risk factors to predict the mortality for individual EC patient.Conclusion1.PGRN was highly expressed and closely correlated with lymph node metastasis and lymphangiogenesis in EC.2.PGRN may promote VEGF-C expression via p-ERK and p-AKT signaling pathways to promote lymph node metastasis and lymphangiogenesis in EC.3.The high expressin of PGRN was closely correlated with poor prognosis,and was the indepengdent risk factor for EC patient survival.4.A nomogram was established using TNM stages,lymph node metastasis,PGRN status and p-ERK to predict the mortality for EC patients. |
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