The Effects Of AGR2 Downregulation On Cell Proliferation,Cell Cycle And Invasion Ability In Esophageal Squamous Cell Carcinoma

Esophageal cancer(EC)is the eighth most common cancer worldwide,with450.000 new cases and 400.000 estimated deaths per year.The two main types of esophageal cancer,squamous cell carcinoma(SCC)and adenocarcinoma(AC),differ in pathogenesis,epidemiology,tumor biology,prognosis and treatment strategies.At present,Multimodality treatment for the patients with EC,combining esophagectomy with perioperative chemotherapy or neoadjuvant chemoradiotherapy,has been shown to improve patients' survival and is therefore the standard treatment for curable esophageal cancer.However,due to the aggressive character of the tumor and the lack of effective individualized treatment,the survival remains poor with 5-year survival rates of merely 36-47%.Therefore,it is imperative to develop the novel therapeutic strategies and seek for new molecular target for improving the prognosis of the patients with EC.Anterior gradient 2(AGR2)is a human homolog of Xenopus laevis cement gland protein XAG-2.The gene for human AGR2 lies at chromosomal position7p21.3,consisting of eight exons and nine transcripts in total have been described for this gene.AGR2 belongs to one of the members of the protein disulfide isomerase(PDI),localizing in endoplasmic reticulum(ER).At present,its overexpression has been widely reported in human many tumors,as well as its promotion of tumor progression and malignant transformation.Most importantly,the potential of AGR2 as a promising diagnostic cancer biomarker has been extensively demonstrated,and may be a novel prognostic factor.These findings suggest that AGR2 may play a pivotal role in occurrence,development,progression and metastasis of a large amount of tumors,and thus may be a potential molecular target and prognostic factor of many tumors.However,the role of AGR2 in occurrence and development of esophageal squamous cell carcinoma(ESCC)remains unclear,to further explore its role in occurrence and development of ESCC,in the current study,immunohistochemistry,real-time quantitative PCR and Western blot were employed to investigate the expressions of AGR2 mRNA and protein in ESCC tissues and cells,and the correlations between its expression and clinicopathological features as well as prognosis were investigated.Furthermore,AGR2 siRNA and siRNA control were utilized to transfect ESCC EC1 cells,and the effects of its downregulation on cell proliferation,cell cycle and invasion were analyzed,which will provide theoretical foundation of targeted therapy of the patients with ESCC using AGR2 as a novel target.Methods(1)Immunohistochemistry was used to detect the expression of AGR2 protein in97 cases of ESCC tissues and corresponding normal esophageal epithelial tissues.(2)SPSS 17.0 software was utilized to investigate the associations of AGR2 expression with clinicopathological features as well as prognosis of the patients with ESCC.(3)Real-time quantitative PCR was employed to investigate the expression of AGR2 mRNA in randomly selected 8 cases of ESCC tissues and corresponding normal esophageal epithelial tissues.(4)Real-time quantitative PCR and Western blot were used to detect the expressions of AGR2 m RNA and protein in different ESCC cell lines and normal esophageal epithelial cells.(5)AGR2 siRNA and siRNA control were transfected to ESCC EC1 cells by Lipofectamine 2000,which was divided into three groups,including untreated group,siRNA control group and AGR2 siRNA group.(6)CCK-8 kit was used to detect the changes of cell proliferation ability in different treatment ESCC EC1 cells.(7)Flow cytometry was employed to the changes of cell cycle distribution in different treatment ESCC EC1 cells.(8)Transwell chamber was utilized to examine the changes of cell invasion ability in different treatment ESCC EC1 cells.(9)Statistical treatment: All data were analyzed using SPSS17.0 software.The results of immunohistochemistry were investigated using Chi-square test.The correlation of AGR2 protein with the prognosis of the patients with ESCC was investigated using Log-rank(Mantel-Cox).The comparison of two groups were investigated using t test,and the comparison of three groups or above was analyzed using One way ANOVA.P<0.05 was considered to be statistical significance.Results(1)Positive expression ratio of AGR2 in ESCC tissues(82/97,84.5%)was significantly higher than that in normal esophageal epithelial tissues(11/97,11.3%),and significant differences were found between two groups(c2=104.115,P<0.05).(2)Relative level of AGR2 mRNA expression in randomly selected 8 cases of ESCC tissues was markedly higher than that in paired normal esophageal epithelial tissues,and there was statistical difference(P<0.05).(3)Expression of AGR2 protein wasn't related to the gender and age of the patients with ESCC(both P>0.05),but closely correlated with histological differentiation,clinical staging and lymph node metastasis(all P<0.05).(4)The results of Log-rank(Mantel-Cox)test revealed that AGR2 expression was obviously associated with the prognosis of the patients with ESCC,and the survival time of the patients with high AGR2 expression was significantly lower than that with low AGR2 expression,and there was statistical difference(P<0.05).(5)The relative levels of AGR2 mRNA and protein in 3 ESCC cell lines(EC1,Eca109 and EC9706)were significantly higher than those in normal esophageal epithelial cell Het-1A,and there was statistical difference(P<0.05).Most importantly,the relative level of AGR2 mRNA and protein in EC1 cell was markedly higher than in other cells investigated above,and there was statistical difference(P<0.05).(6)Compared with untreated group and siRNA control group,the relative level of AGR2 protein was significantly downregulated in AGR2 siRNA group,and there was statistical difference(P<0.05).(7)Compared with untreated group and siRNA control group,cell proliferation ability was significantly inhibited in AGR2 siRNA group,and there was statistical difference(P<0.05).(8)The percentage of cell number of EC1 cells in G0/G1 phase in AGR2 siRNA group was 64.77±1.12%,which was significantly higher than that in untreated group(52.48±1.26%)and siRNA control group(52.29±1.24%),and there was statistical difference(F=105.850,P<0.05).Further investigation revealed that the percentage of cell number of EC1 cells in S phase in AGR2 siRNA group was 21.71±0.87%,which was significantly lower than that in untreated group(29.09±0.71%)and siRNA control group(29.40±1.91%),and there was statistical difference(F=34.859,P<0.05).Besides,the percentage of cell number of EC1 cells in G2/M phase in AGR2 siRNA group was 13.52±1.89%,which was significantly lower than that in untreated group(18.43±1.63%)and siRNA control group(18.31±1.11%),and there was statistical difference(F=9.437,P<0.05).(9)The invading cell numbers of EC1 cells in AGR2 si RNA group were52.67±15.01,which was significantly lower than those in untreated group(132.67±16.50)and siRNA control group(125.33±14.57),and there was statistical difference(F=24.791,P<0.05).Conclusion(1)AGR2 plays an essential role in the occurrence and development of ESCC,and may be a novel prognostic factor for the patients with ESCC.(2)AGR2 downregulation mediates the inhibition of cell proliferation,the changes of cell cycle distribution and the decreases of cell invasion ability,and thus inhibition of AGR2 expression may be a novel therapeutic strategy for the patients with ESCC.