The Effect Of Ly6E On Biological Behavior Of Esophageal Squamous Cell Carcinoma Cells And The Mechanism Study

BackgroundEsophageal carcinoma(EC)is one of the most common malignant tumors in the world,the incidence and mortality rank the sixth and the fifth position respectively showed by statistics.It is also the second most common malignant tumor in gastrointestinal tract.The incidence distribution of EC varies from different regions.China is one of the hot regions.The health of Chinese people is threatened by EC seriously.Squamous cell carcinoma is the major histological subtype,accounting for over 90% of the total cases.Esophageal squamous cell carcinoma(ESCC)is a highly invasive disease,with rapid progressing,easily resistant and prone to recurrence.The prognosis factors include primary site,histological differentiation,infiltrating depth,number and range of lymph node metastasized,and clinical TNM staging.Despite comprehensively adjuvant therapies such as surgical intervention,radiotherapy and chemotherapy used,few significant improvement have been achieved and the five-year overall survival rate is still hovering around 5%-45%.Studies have demonstrated that the activation of oncogenes and the inactivation of tumor suppressor genes are key factors in the development and progression of tumors.So,analyzing the functions of the key genes could help to find new therapeutic targets.At present,the targeted gene therapy is more and more popular due to its small toxic and side effects,good tolerance and significant curative prognosis,and has been a new orientation in fighting with tumors.For example,gefitinib,an epidermal growth factor receptor(EGFR)inhibitor,has been used effectively in treating patients suffered from non-small cell lung cancer with EGFR gene mutations,and it could extend the survival,decrease the toxicity and produce tolerance.Trastuzumab(herceptin)is effective in the treatment of metastatic breast cancer patients with human epidermal growth factor receptor-2(HER2)gene overexpression.There are only a few reports about targeted gene therapies of EC.Studies with small samples and clinical trials have showed that erlotinib,hydrochloride and nidotuzumab were effective for few patients.So,it is of great significance to looking for more effective therapeutic target genes of ESCC.Lymphocyte antigen 6 complex(Ly6)is a small cysteine-rich and surface protein of cells.It contains a group of allogenic antigens encoded by multiple genes,which are mainly and differentially expressed in hematopoietic cells,especially in T lymphocytes,attributing to an important role in signal transduction,complement membrane attack complex and T cell activation et al.Researches have shown that members of Ly6 superfamily took part in the tumorigenesis and progression of malignant tumors.Lymphocyte antigen 6 complex,locus E(Ly6E),known as stem cell antigen 2(SCA2)and thymic shared antigen-1(TSA-1),is a member of the Ly6 superfamily.Studies have showed that the expression of Ly6 E was up-regulated in many malignant tumors,such as lung cancer,gastric cancer and breast cancer et al,and was correlated with the histological differentiations and /or prognosis closely.It suggested that Ly6 E might participate in the tumorigenesis and progressions.Furthermore,Ly6 E siRNA could suppress the migrating ability of gastric cancer cells and up-regulate the expressions of PTEN and E-cadherin protein;As a key regulatory factor,Ly6 E could also inhibit breast cancer invasion and metastasis significantly by down-regulating its expression.All studies above suggested that Ly6 E might play an important role in the invasion and metastasis of tumors.In addition,the increasing expression of Ly6 E was related to the decreasing of the tumor suppressor gene PTEN,leading to the activation of the PI3K/Akt signaling pathway and increasing the expression and activity of hypoxia inducible factor-1(HIF-1α),which produced the effects of promoting tumor cell growth,inhibiting the apoptosis,inducing angiogenesis,and promoting cell invasion and metastasis.Through transcriptome analysis,Khammanivong A et al.found that Ly6 E expression was obviously up-regulated in EC induced by N-Nitrosonornicotine in rats in 2016,suggesting that Ly6 E might play an important role in the development and progression of EC and might be an excellent target gene for ESCC therapy.To date,there has been no research about the expression and effect of Ly6 E on human ESCC.ObjectiveIn order to clarify the role of Ly6 E in the tumorigenesis,progression,invasion and metastasis of ESCC and to find a new effective targeted gene for the treatment,our study was mainly divided into the following 3 aspects: Firstly,we analyzed the expression of Ly6 E protein in ESCC tissues and the corresponding normal parts by Immunohistochemistry(IHC)EnVision and studied its relationship with the clinicopathological parameters to elucidate the role of Ly6 E in the occurrence and development of ESCC preliminarily.In addition,four fresh ESCC tissues and the corresponding normal parts were selected randomly,then compared the compliance of the expression of Ly6 E protein and mRNA between the two different kind of tissues by using Western blot and real-time fluorescence polymerase chain reaction(RT-PCR)respectively.Secondly,we down-regulated the expression of Ly6 E in ESCC cell lines by RNA interference(RNAi)and analyzed the effect on the proliferation,cell cycle,apoptosis,invasion and metastasis of ESCC cells by CCK-8 kit,flow cytometry and Transwell chamber et al.we also analyzed the expressions of E-cadherin,N-cadherin,Vimentin and ZEB1,key molecules in the epithelial to mesenchymal transition(EMT)signal pathway,to explore the possible mechanism of the decreased migration and invasion ability of ESCC cells.Most importantly,we analyzed the relationship between down-regulated expression of Ly6 E and the PTEN/Akt signaling pathway,and tried to illuminant the role and the mechanism of Ly6 E in the occurrence,development,invasion and metastasis of ESCC.Finally,a xenograft model of ESCC transfected with Ly6 E siRNA was constructed.We analyzed the effect of Ly6 E siRNA on the growth of ESCC cells and the regulatory effect on E-cadherin,N-cadherin,Vimentin and ZEB1 expressions in vivo.That might provide more theoretical and experimental proofs for Ly6 E as a targeted gene in treating human ESCC.Part I: Expression and clinical significance of Ly6 E in esophageal squamous cell carcinoma Methods1.Immunohistochemical EnVision staining was used to detect the expression of Ly6 E protein in 89 cases of ESCC and the corresponding normal esophageal mucosa tissues,then,analyzing the correlation between the expressions with the clinicopathological features.2.Real-time quantitative PCR and Western blot were used to analyze the expression of Ly6 E mRNA and protein in 4 fresh ESCC tissues and the corresponding fresh normal esophageal mucosa tissues.3.Statistical methods: All statistical data were performed by SPSS 17.0 software.Chi-square test was used to compare the data in two or more groups.P<0.05 was considered as statistically significant.Results1 The expression of Ly6 E protein in ESCC tissues was significantly increased compared with that in normal esophageal mucosa tissues.The differences were statistically significant(P<0.01).The positive signals were mainly detected in the cytoplasm and/or the membrane,with brown and yellow granular staining.2.The expression of Ly6 E protein in ESCC tissues were closely correlated with clinical TNM staging,invasion depth,histological grading and lymph node metastasis,the differences between groups were statistically significant(P<0.01),but were not correlated with age,sex,gross classification,and the diameter,there were no significant difference between groups(P>0.05).3.The expression levels of Ly6 E mRNA and protein in fresh ESCC tissues were higher than those in the corresponding normal tissues,the differences between groups were statistically significant(P<0.05).Part II: Down-regulated expression of Ly6 E plays a role in conquering ESCC by suppressing the PTEN/Akt signaling pathway Methods1.Real-time quantitative PCR and Western blot were used to analyze the different expressions of Ly6 E mRNA and protein respectively in ESCC cell lines(Eca109,EC9706,EC1,TE1 and KYSE70)and normal esophageal squamous cell line Het-1A.2.Ly6 E siRNA and control siRNA were transfected into the ESCC EC9706 and EC1 lines by RNA interference technology(RNAi).We analyzed the differences of the expressions of Ly6 E mRNA and protein in Ly6 E siRNA groups,control siRNA groups and untreated groups(normally cultured ESCC EC9706 and EC1 cell)by real-time quantitative PCR and Western blot respectively.3.CCK-8 kit assay was used to analyze the effect of Ly6 E siRNA on the proliferation ability of EC9706 and EC1 cells.4.Flow cytometry and Annexin V-FITC/PI double staining were used to examin the effects of Ly6 E siRNA on the cell cycle and apoptosis ability of EC9706 and EC1 cells.5.Tranwell chamber was used to examin the impact of Ly6 E siRNA on the invasion and metastasis ability of EC9706 and EC1 cells.6.Real-time quantitative PCR was used to analyze the impacts of Ly6 E siRNA on the expression of E-cadherin,N-cadherin,Vimentin and ZEB1 mRNA related in epithelial-mesenchymal transition(EMT)pathway of ESCC cells.7.Western blot was used to detect the effect of Ly6 E siRNA on the expression of PTEN、p-Akt and total Akt protein of the ESCC cells,and to analyze the relationship between Ly6 E and PTEN/Akt signaling pathway of ESCC.8.Statistical methods: All data were analyzed by GraphPad Prism 6.0 software.Data were expressed as (?)±s.Comparison between two groups was expressed by t-test,three or more groups by one-way ANOVA.P<0.05 was defined as the difference was significant.Results1.Compared to normal esophageal epithelial cells Het-1A,the expression levels of Ly6 E mRNA and protein in Eca109,EC9706,EC1,TE1 and KYSE70 cells were all significantly increased,the differences were statistically significant(P<0.001).The EC9706 and EC1 cell lines were the two highest expression levels of Ly6 E,which were selected for the subsequent experiments.2.After transfected for 48 h,EC9706 and EC1 cells showed significantly lower Ly6 E mRNA and protein expression levels in Ly6 E siRNA groups than those in untreated groups and control siRNA groups,the differences were statistically significant(P<0.05).No significant differenceswere examined between untreated groups and control siRNA groups(P>0.05).3.After transfected for 24 h,48h,72 h and 96 h,the proliferation abilities of EC9706 and EC1 cells detected in Ly6 E siRNA groups were all significantly lower than those in untreated groups and control siRNA groups,the differences were statistically significant(P<0.05).No significant differences were examined between untreated groups and control siRNA groups(P>0.05).4.After transfected for 48 h,the ratios of EC9706 and EC1 cells in G0/G1 phases were significantly increased in Ly6 E siRNA groups compared with those in untreated groups and control siRNA groups,the differences were statistically significant(EC9706 cells: F=176.260,P=0.000;EC1 cells: F=104.834,P=0.000);No significant differences were examined between untreated groups and control siRNA groups(EC9706 cells: P=0.939;EC1 cells:P=0.25).The ratios of EC9706 and EC1 cells in S phase were significantly lower in Ly6 E siRNA groups than those in untreated groups and control siRNA groups,the differences were statistically significant(EC9706 cells: F=56.798,P=0.000;EC1 cells: F= 53.610,P=0.000).No significant differences were examined between untreated groups and control siRNA groups(EC9706 cells: P= 0.25;EC1 cells: P=0.320).5.After transfected for 48 h,the ratios of EC9706 and EC1 cells apoptosis in Ly6 E siRNA groups were significantly higher than those in untreated groups and control siRNA groups,the differences were statistically significant(EC9706 cells: P<0.0001;EC1 cells: P<0.0001);No significant differences were examined between untreated groups and control siRNA groups(P>0.05).6.After transfected for 48 h,the numbers of migration EC9706 and EC1 cells in Ly6 E siRNA groups were significantly decreased compared with those in untreated groups and control siRNA groups,the differences were statistically significant(EC9706 cells: P<0.001;EC1 cells: P<0.01).No significant differences were examined between untreated groups and control siRNA groups(P>0.05).7.After transfected for 48 h,the numbers of invasived EC9706 and EC1 cells in Ly6 E siRNA groups were significantly decreased compared with those in untreated groups and control siRNA groups,mthe differences were statistically significant(EC9706 cells: P<0.01;EC1 cells: P<0.05).No significant differences were examined between untreated groups and control siRNA groups(P>0.05).8.After transfected for 48 h,the E-cadherin mRNA expression levels of EC9706 and EC1 cells in Ly6 E siRNA groups were significantly higher than those in untreated groups and control siRNA groups;the N-cadherin,Vimentin and ZEB1 mRNA expression levels in Ly6 E siRNA groups were significantly lower,the differences were all statistically significant(EC9706 cells: P< 0.001;EC1 cells: P< 0.0001).9.After transfected for 48 h,the PTEN protein expression levels of EC9706 and EC1 cells in Ly6 E siRNA groups were significantly up-regulated compared with those in untreated groups and control siRNA groups(P<0.0001),The p-Akt protein expression levels were significantly down-regulated(P<0.0001).No significant differences of total Akt protein expression were examined among the three groups(P>0.05).Part III: The effect of Ly6 E gene on the growth and the EMT signal pathway of xenograft tumors of esophageal squamous cell carcinoma in nude mice Methods1.The ESCC EC9706 cells stable transfected with Ly6 E siRNA and control siRNA were subcutaneously injected into the right armpit of nude mice,then compared the sizes and speeds of the xenograft tumor in Ly6 E siRNA group,control siRNA group and untreated group.2.Real-time quantitative PCR and Western blot were used to detect mRNA and protein expression levels of Ly6 E in xenograft tumors in each group.3.Real-time fluorescence quantitative PCR was used to detect the expression levels of E-cadherin,N-cadherin,Vimentin and ZEB1 mRNA in fresh tissue of xenograft tumors in each group.4.Immunochemistry EnVision was used to detect the expression of E-cadherin protein in xenograft tumors in each group.5.All data were statistically processed using SPSS 17.0 statistical package.The comparison of count data was performed by χ2 test,the difference of quantitative data was examined by t test between two groups and by analysis of variance among multiple groups.Quantitative data were expressed as mean ± standard deviations((?)±s).P<0.05 was considered statistically significant.Results1.Established the xenograft tumor model of esophageal squamous cell carcinoma transfected with Ly6 E siRNA successfully.The volumes of xenograft tumors in Ly6 E siRNA group were significantly lower than those in untreated group and control siRNA group.The difference was statistically significant(P<0.001).2 The Ly6 E mRNA and protein expression of xenograft tumors in Ly6 E siRNA group were significantly lower than those in untreated group and control siRNA group.The differences were statistically significant(P<0.01).The results detected by Immunohistochemistry and Western Blot were consistent.3.The E-cadherin mRNA and protein expression levels of xenograft tumors in Ly6 E siRNA group were significantly higher than those in unteated group and control siRNA group,the differences were statistically significant(P<0.01).The N-cadherin,Vimentin and ZEB1 mRNA expression levels were significantly lower than those in untreated group and control siRNA group.The differences were statistically significant(P<0.001).Conclusions1.Ly6 E gene was overexpressed in both ESCC tissues and cells,which was closely correlated with the clinical TNM stage,infiltration depth,histopathological grading and the state of lymph node metastasis.It suggested that Ly6 E might play an important role in occurrence,development,invasion and metastasis of ESCC.2.In vitro,the decreased expression of Ly6 E could inhibite the growth of the cells,and the EMT signal pathway attributing to decrease the invasion and migration ability,which suggested that Ly6 E could be an oncogene in ESCC and be related to the invasion and metastasis.The decreased expression of Ly6 E could play an anti-effect on ESCC by affecting the PTEN/Akt signaling pathway.3.Established the xenograft tumor model of ESCC transfected with Ly6 E siRNA successfully.In vivo,the decreased expression of Ly6 E could prevent the growth、invasion and metastasis of xenograft tumors of ESCC in nude mice,which suggested that Ly6 E could be an effective therapeutic target gene for ESCC.