摘要
目的探讨钙激活的氯离子通道A4(CLCA4)对食管癌细胞增殖、迁移、侵袭及JAK激酶2/信号转导及转录激活蛋白3(JAK2/STAT3)信号通路的影响。方法实时荧光定量逆转录聚合酶链反应(qRT-PCR)与蛋白质印迹法(Western blotting)分别检测正常人食管鳞状上皮细胞与人食管癌细胞中CLCA4的表达;将合成的CLCA4过表达载体及其对照分别转染至食管癌细胞Eca109,分别记作CLCA4过表达(pcDNA-CLCA4)组、CLCA4阴性对照(pcDNA-NC)组,并将未转染的细胞作为阴性对照(NC)组。四甲基偶氮唑盐微量酶反应比色法(MTT法)检测细胞增殖能力;细胞迁移实验(Transwell)检测细胞迁移及侵袭能力。JAK2/STAT3信号通路激活剂p-JAK2多肽对细胞增殖、迁移及侵袭的影响。Western blotting检测细胞周期蛋白D1(Cy⁃clinD1)、依赖性激酶抑制因子(P21)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、JAK2、STAT3、磷酸化JAK激酶2(p-JAK2)、磷酸化信号转导及转录激活蛋白3(p-STAT3)的表达水平。结果与Het-1A相比,人食管癌细胞KYSE170、Eca109、TE10中CLCA4 mRNA及蛋白表达水平降低(P<0.05);与pcDNA-NC组比较,pcDNA-CLCA4组细胞存活率显著降低[(52.16±11.41)%比(99.57±13.49)%,P<0.05],迁移细胞数[(56.47±10.03)%比(112.49±13.52)%]与侵袭细胞数[(63.43±9.87)%比(123.47±16.58)%]减少(P<0.05),CyclinD1、MMP-2、MMP-9、p-JAK2、p-STAT3的表达水平降低(P<0.05),P21的表达水平升高(P<0.05);激活JAK2/STAT3信号通路可逆转CLCA4过表达对Eca109细胞增殖、迁移及侵袭的抑制作用。结论CL⁃CA4过表达可抑制食管癌细胞增殖、迁移及侵袭,其作用机制可能与抑制JAK2/STAT3信号通路活化有关。
Objective To explore the effects of calcium-activated chloride channel A4(CLCA4)on the proliferation,migration,inva⁃sion and non-receptor tyrosine protein kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway of esophageal cancer cells.Methods qRT-PCR and Western blotting were used to detect the expression of CLCA4 in normal human esophageal squamous cells and human esophageal cancer cells;the synthetic CLCA4 overexpression vector and its control were trans⁃fected into esophageal cancer cells(Eca109),denoted as CLCA4 overexpression(pcDNA-CLCA4)group,CLCA4 negative control(pcDNA-NC)group,and untransfected cells as negative control(NC)group.Thiazolan(MTT)detected cell proliferation ability;cell mi⁃gration test(Transwell)detected cell migration and invasion ability.The effect of JAK2/STAT3 signaling pathway activator p-JAK2 polypeptide on cell proliferation,migration and invasion.Western blotting detected Cyclin D1(CyclinD1),dependent kinase inhibitor(P21),matrix metalloproteinase 2(MMP-2),matrix metalloproteinase 9(MMP-9),non-receptor tyrosine protein kinase 2(JAK2),signal transducer and activator of transcription 3(STAT3),phosphorylated tyrosine kinase 2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3).Results Compared with Het-1A,the expression levels of CLCA4 mRNA and protein in human esophageal cancer cells KYSE170,Eca109 and TE10 were significantly lower(P<0.05).Compared with the pcDNA-NC group,the cell viability of the pcDNA-CLCA4 group was significantly decreased[(52.16±11.41)%vs.(99.57±13.49)%,P<0.05],and the number of mi⁃grated cells[(56.47±10.03)%vs.(112.49±13.52)%]and the number of invasive cells[(63.43±9.87)%vs.(123.47±16.58)%]were signifi⁃cantly decreased(P<0.05),the expression levels of CyclinD1,MMP-2,MMP-9,p-JAK2,p-STAT3 were significantly decreased(P<0.05),and the expression level of P21 was significantly increased(P<0.05).Activation of the JAK2/STAT3 signaling pathway reversed the inhibitory effect of CLCA4 overexp
作者
蒋可心
李宁
张旭
JIANG Kexin;LI Ning;ZHANG Xu(1Department of Digestive and Lymphatic Radiotherapy,Cancer Hospital of China Medical University,Shenyang,Liaoning 110042,China;Department of Digestive and Oncology,245 Hospital of Shenyang,Shenyang,Liaoning 110042,China)
出处
《安徽医药》
CAS
2021年第3期474-478,共5页
Anhui Medical and Pharmaceutical Journal
