选择性自噬接头蛋白、微管相关蛋白轻链3和信号转导与转录激活因子3在食管鳞癌中的表达及临床意义

Expression of sequestosome-1,microtubule-associated protein light chain 3 and signal transduction and activators of transcription factor 3 in esophageal squamous cell carcinoma and their clinical significance

目的探讨选择性自噬接头蛋白(P62)、微管相关蛋白轻链3(LC3)和信号转导与转录激活因子3(STAT3)在食管鳞癌(ESCC)中的表达及意义。方法收集在川北医学院附属医院于2018年7月至2019年4月收治的48例ESCC患者行根治术后的癌组织和癌旁组织,采用实时定量反转录聚合酶链反应(RT-qPCR)法和蛋白质印迹法(Western blot)检测ESCC组织及癌旁组织中P62、LC3及STAT3的mRNA和蛋白表达,分析其与临床病理特征的关系及其三者之间的相关性。敲低食管癌EC109细胞中的STAT3,Western blot检测LC3、P62、STAT3、非受体型酪氨酸蛋白激酶2(JAK2)和磷酸化信号转导与转录激活因子3(p-STAT3)的表达。组间比较采用χ^(2)检验、t检验和Spearman相关性分析法进行统计分析。结果P62、STAT3在ESCC组织中的阳性表达率高于癌旁组织(62.5%比37.5%,66.7%比33.3%,χ^(2)=13.520、23.120,P<0.05),LC3在ESCC组织中的阳性表达率低于癌旁组织(35.4%比64.6%,χ^(2)=18.000,P<0.05);P62、STAT3在ESCC组织中的表达高于癌旁组织(1.520±1.172比0.980±2.539,1.320±1.163比0.860±2.560,t=7.453、7.337,P<0.05),LC3的表达低于癌旁组织(0.957±1.074比1.587±2.630,t=8.525,P<0.05);STAT3与P62的表达呈正相关(rs=0.438,P<0.05),与LC3的表达呈负相关(rs=-0.400,P<0.05),P62与LC3的表达呈负相关(rs=-0.585,P<0.01)。P62、LC3、STAT3与肿瘤T分期、临床分期以及淋巴结转移密切相关(χ_(P62-T分期)^(2)=5.581,χ_(P62-临床分期)^(2)=6.817,χ_(P62-淋巴转移)^(2)=5.689;χLC3-T分期2=6.947,χ_(LC3-临床分期)^(2)=6.919,χ_(LC3-淋巴转移)^(2)=9.108;χ_(STAT3-T分期)^(2)=5.270,χ_(STAT3-临床分期)^(2)=7.054,χ_(STAT3-淋巴转移)^(2)=6.000;均P<0.05)。敲低食管癌EC109细胞中的STAT3后,敲低组(ShSTAT3-1和ShSTAT3-2)中JAK2、p-STAT3、STAT3、P62的表达低于空载组(0.300±0.190、0.430±0.190比1.273±0.114,0.520±0.120、0.410±0.070比1.070±0.200,0.430±0.060、0.500±0.080比1.000±0.170,1.185±0.125、0.847±0.067比1.793±0.130),LC3的表达高于空载组(1.527±0.197、1.410±0.170比0.221±0.040),差异有统计学意义(tJAK2/ShSTAT3-1=7.613、tJAK2/ShSTAT3-2=6.597,tp-STAT3/ShSTAT3-1=4.084、tp-STAT3/ShSTAT3-2=5.395,tSTAT3/ShSTAT3-1=11.640、tSTAT3/ShSTAT3-2=8.660,tP62/ShSTAT3-1=5.844、tP62/ShSTAT3-2=11.220,tLC3/ShSTAT3-1=11.260、tLC3/ShSTAT3-2=11.790,均P<0.05)。结论STAT3能通过调控P62和LC3,参与ESCC的进展。

Objective To explore the expression and significance of sequestosome-1(P62),microtubule-associated protein light chain 3(LC3)and signal transduction and activators of transcription factor(STAT)3 in esophageal squamous cell carcinoma(ESCC).Methods The 48 cases of ESCC tissues and adjacent tissues after radical resection were collected in the Affiliated Hospital of North Sichuan Medical College from July 2018 to April 2019.The real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the mRNA and protein expression of P62,LC3 and STAT3 in ESCC tissues and adjacent tissues.The relationship between P62,LC3,STAT3 and clinicopathological characteristics and the correlation among P62,LC3 and STAT3 were analyzed.STAT3 was knocked down in EC109 cells by transfection.Western blotting was used to detect the expression of LC3,P62,non-receptor tyrosine protein kinase 2(JAK2)and phosphorylated signal transducer and activator of transcription 3(p-STAT3).The data were analyzed byχ^(2)test,t test and Spearman correlation analysis method.Results The positive expression rate of P62 and STAT3 in ESCC tissues was higher than that in adjacent tissues(62.5%vs.37.5%,66.7%vs.33.3%,χ^(2)=13.520,23.120,all P<0.05).The positive expression rate of LC3 in ESCC tissues was lower than that in adjacent tissues(35.4%vs.64.6%,χ^(2)=18.000,P<0.05).The expression levels of P62 and STAT3 in ESCC tissues were higher than those in adjacent tissues(1.520±1.172 vs.0.980±2.539,1.320±1.163 vs.0.860±2.560,t=7.453,7.337,all P<0.05).The expression level of LC3 in ESCC tissues was lower than that in adjacent tissues(0.957±1.074 vs.1.587±2.630,t=8.525,P<0.05).The expression of STAT3 was positively correlated with the expression of P62(rs=0.438,P<0.05)and negatively with the expression of LC3(rs=-0.400,P<0.05).The expression of P62 was negatively correlated with the expression of LC3(rs=-0.585,P<0.01).P62,LC3 and STAT3 were closely related to tumor T staging,clinical staging and lymph node metastasis(χP62-T stage2=5.581,χ_(P62-clinical stage)^(2)=6.817,χP62-lymph node metastasis2=5.689;χ_(LC3-T stage)^(2)=6.947,χLC3-clinical stage2=6.919,χ_(LC3-lymph node metastasis)^(2)=9.108;χSTAT3-T stage2=5.270,χ_(STAT3-clinical stage)^(2)=7.054,χ_(STAT3-lymph node metastasis)^(2)=6.000;allP<0.05).After knocking down STAT3 in EC109 cells,the expression of JAK2,p-STAT3,STAT3 and P62 in the knockdown group(ShSTAT3-1 and ShSTAT3-2)was lower than that in control group(0.300±0.190,0.430±0.190 vs.1.273±0.114;0.520±0.120,0.410±0.070 vs.1.070±0.200;0.430±0.060,0.500±0.080 vs.1.000±0.170;1.185±0.125,0.847±0.067 vs.1.793±0.130).The expression of LC3 in the knockdown group(ShSTAT3-1 and ShSTAT3-2)was higher than that in control group(1.527±0.197,1.410±0.170 vs.0.221±0.040).The difference was statistically significant(tJAK2/ShSTAT3-1=7.613,tJAK2/ShSTAT3-2=6.597;tp-STAT3/ShSTAT3-1=4.084,tp-STAT3/ShSTAT3-2=5.395;tSTAT3/ShSTAT3-1=11.640,tSTAT3/ShSTAT3-2=8.660;tP62/ShSTAT3-1=5.844,tP62/ShSTAT3-2=11.220;tLC3/ShSTAT3-1=11.260,tLC3/ShSTAT3-2=11.790,all P<0.05).Conclusion STAT3 probably participated in the progression of ESCC by regulating P62 and LC3.