过表达铜锌超氧化物歧化酶促进食管癌细胞增殖和Wnt信号通路活化

Overexpression of copper/zinc superoxide dismutase promotes cell proliferation and Wnt signaling pathway activation in esophageal cancer

目的:探讨食管癌中铜锌超氧化物歧化酶(SOD1)的表达及其对细胞增殖的影响和机制。方法:选取食管癌组织和癌旁组织各32例,利用实时定量反转录聚合酶链反应(RT-qPCR)、蛋白质印迹法(Western blot)和免疫组织化学法检测SOD1表达。将食管癌细胞系KYSE510随机分为3组:对照组、腺相关病毒(AAV)-绿色荧光蛋白(GFP)组和AAV-SOD1组。AAV-GFP组和AAV-SOD1组细胞分别转染AAV-GFP和AAV-SOD1重组病毒载体,对照组则加磷酸盐缓冲液(PBS)处理。利用噻唑蓝(MTT)法检测细胞活性、二氢乙锭(DHE)探针检测活性氧簇(ROS)产生、Western blot检测Wnt5a和β-连环蛋白(β-catenin)蛋白表达。两组间比较采用t检验,3组间比较采用单因素方差分析并采用Tukey法进行两两比较。结果:与癌旁组织(1.00±0.33)比较,食管癌组织中SOD1 mRNA表达升高(3.76±0.51,t=-25.595,P<0.01)。食管癌中SOD1蛋白表达水平(1.93±0.35)也高于癌旁组织(1.00±0.28,t=-11.734,P<0.01)。转染24、48、72 h后,AAV-SOD1组细胞活性(0.165±0.016、0.283±0.023、0.391±0.036)明显高于对照组(0.133±0.017、0.213±0.028、0.326±0.038)和AAV-GFP组(0.129±0.015、0.209±0.024、0.316±0.032,P<0.01)。转染48 h后,AAV-SOD1组细胞ROS水平(0.757±0.062)明显低于对照组(1.000±0.079)和AAV-GFP组(1.048±0.108,P<0.01),而SOD1、Wnt5a和β-catenin蛋白表达水平则明显高于对照组和AAV-GFP组(P<0.01)。结论:SOD1的表达在食管癌中升高,过表达SOD1通过抑制ROS和调节Wnt信号通路活化诱导细胞增殖。

Objective To investigate the expression of copper/zinc superoxide dismutase(SOD1)in esophageal cancer and its effect and mechanism on cell proliferation.Methods Thirty-two esophageal cancer tissues and adjacent tissues were selected.SOD1 expression was analyzed using real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR),Western blotting and immunohistochemistry methods.Esophageal cancer cell lines were randomly allocated into three groups:control,adeno-associated virus(AAV)-green fluorescent protein(GFP)group and AAV-SOD1 group.Cells in AAV-GFP group and AAV-SOD1 group were transfected with AAV-GFP and AAV-SOD1 recombinant viral vectors,while control was treated with phosphate buffered saline(PBS).Cell viabilities were evaluated with methyl thiazolyl tetrazolium(MTT)assay,reactive oxygen species(ROS)production was detected with dihydroethidium(DHE)probe,and protein expression of Wnt5a andβ-catenin was analyzed with Western blotting.Results Compared with adjacent tissues(1.00±0.33),expression of SOD1 mRNA(3.76±0.51)was enhanced in esophageal cancer tissues(t=-25.595,P<0.01).SOD1 protein expression was also higher in cancer tissues(1.93±0.35)than in adjacent tissues(1.00±0.28,t=-11.734,P<0.01).At 24,48 and 72 h after transfection,cell viabilities in AAV-SOD1 group(0.165±0.016,0.283±0.023,0.391±0.036)were markedly higher than those in control(0.133±0.017,0.213±0.028,0.326±0.038)and AAV-GFP group(0.129±0.015,0.209±0.024,0.316±0.032,P<0.01).At 48 h after transfection,ROS levels in AAV-SOD1(0.757±0.062)were lower than control(1.000±0.079)and AAV-GFP group(1.048±0.108,P<0.01),while protein expression of SOD1,Wnt5a andβ-catenin was higher(P<0.01).Conclusion SOD1 expression was increased in esophageal cancer,and SOD1 overexpression seduced cell proliferation via inhibition of ROS and modulation of Wnt signaling pathway.