SLC1A5协同TM4SF1通过mTOR信号通路调控食管鳞癌细胞迁移

SLC1A5 combined with TM4SF1 regulates the migration of esophageal squamous cell through mTORsignaling pathway

目的探讨氨基酸转运载体溶质载体家族1成员5(SLC1A5)在食管鳞癌(ESCC)中的表达及其与临床病理特征的相关性,以及SLC1A5对ESCC细胞迁移的影响及潜在分子机制。方法采用TNM plot在线数据库分析SLC1A5基因在ESCC组织和正常组织中的表达情况。通过实时定量逆转录聚合酶链反应(qRT-PCR)法检测ESCC组织和癌旁组织中SLC1A5 mRNA表达情况;采用免疫组化(IHC)法检测SLC1A5蛋白表达情况,并分析其与患者临床病理资料的相关性。分别构建SLC1A5过表达(SLC1A5-OE组)、四跨膜蛋白超家族成员1过表达(TM4SF1-OE组)、SLC1A5和TM4SF1共同过表达的Eca109细胞。采用细胞增殖试验、Transwell实验检测Eca109细胞的增殖、迁移和侵袭能力。通过免疫共沉淀(IP)实验证实SLC1A5和TM4SF1的相互作用;采用蛋白质免疫印迹(WB)法检测哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白的表达水平。结果TNM plot数据库分析显示,ESCC组织中SLC1A5基因表达水平高于正常组织,差异有统计学意义(P<0.05)。ESCC组织中SLC1A5 mRNA表达高于成对的癌旁组织,差异有统计学意义(P<0.01)。IHC结果显示,SLC1A5蛋白在癌组织中高表达,并且与临床分期(P=0.036)、淋巴结转移(P=0.029)显著相关。细胞增殖实验结果显示,SLC1A5-OE组和对照细胞(Con组)增殖能力比较,差异无统计学意义(P>0.05)。Transwell实验结果显示,与Con组比较,SLC1A5-OE组细胞迁移及侵袭能力增强,差异有统计学意义(P<0.01)。IP结果表明,SLC1A5与TM4SF1存在相互作用。WB结果显示,相对于SLC1A5-OE组或TM4SF1-OE组,p-mTOR、p-S6蛋白表达水平在SLC1A5和TM4SF1共同过表达的细胞中最高。Transwell实验证实,共同过表达SLC1A5和TM4SF1的ESCC细胞迁移能力最强。结论ESCC中SLC1A5呈高表达,且与患者临床分期、淋巴结转移显著相关,SLC1A5可能通过与TM4SF1相互作用,激活mTOR信号通路,进而促进ESCC细胞迁移。

Objective To explore the expression of amino acid transporters solute carrier family-1 member-5(SLC1A5)in esophageal squamous cell carcinoma(ESCC)and its correlation with clinicopathological features,and investigate the potential molecular mechanisms of SLC1A5 in ESCC metastasis.Methods The expression of SLC1A5 gene in ESCC and normal tissues was analyzed by TNM plot online database.The SLC1A5 mRNA expression in ESCC and paracancer tissues was examined by quantitative real time polymerase chain reaction(qRT-PCR);the protein expression of SLC1A5 was detected by immunohistochemistry(IHC)and the correlations between SLC1A5 and clinic pathological characteristics in patients were analyzed;SLC1A5 overexpression(SLC1A5-OE group),transmembrance-4 L-six family member-1 overexpression(TM4SF1-OE group),SLC1A5 and TM4SF1 co-overexpression Eca109 cells were constructed respectively.Cell proliferation test and Transwell test were used to detect the proliferation,migration and invasion ability of Eca109 cells.The interaction between SLC1A5 and TM4SF1 was confirmed by immunoprecipitation(IP)assays;the expression level of mammalian target of rapamycin(mTOR)signaling pathway-related protein was detected by Western blot(WB).Results TNM plot database analysis showed that the expression level of SLC1A5 gene in the ESCC tissues was significantly higher than that in the normal tissues(P<0.05).The expression of SLC1A5 mRNA in ESCC tissues was significantly higher than that in paired para-cancerous tissues(P<0.01).IHC results showed that SLC1A5 protein was highly expressed in cancer tissues,and was significantly correlated with clinical stage(P=0.036)and lymph node metastasis(P=0.029).The results of cell proliferation experiment showed that there was no significant difference in proliferation ability between the SLC1A5-OE group and control group(Con group)(P>0.05).The results of Transwell experiment showed that compared with the Con group,the cell migration and invasion ability of the SLC1A5-OE group were significantly enhanced(P<0.01).The IP results showed that SLC1A5 interacted with TM4SF1.WB results showed that the expression levels of p-mTORand p-S6 proteins were the highest in the cells co-overexpressing SLC1A5 and TM4SF1,relative to the SLC1A5-OE group or TM4SF1-OE group.Transwell experiment confirmed that ESCC cells overexpressing SLC1A5 and TM4SF1 had the strongest migration ability.Conclusion SLC1A5 is highly expressed in ESCC,and is significantly correlated with clinical stage and lymph node metastasis of patients.SLC1A5 may activate mTORsignaling pathway through interaction with TM4SF1,thus promoting ESCC cell migration.