脑和肌肉芳香烃受体核转运样蛋白1在食管鳞状细胞癌组织中表达变化及对KYSE150细胞增殖和迁移的影响

Expression of BMAL1 in esophageal squamous cell carcinoma and its influence on proliferation and migration of KYSE150 cells

目的观察食管鳞状细胞癌(鳞癌)组织脑和肌肉芳香烃受体核转运样蛋白1(brain and muscle ARNT-like protein 1,BMAL1)表达变化,探讨BMAL1对食管鳞癌KYSE150细胞增殖和迁移的影响。方法食管鳞癌患者82例,取手术切除癌组织及癌旁组织,采用实时荧光定量PCR法检测BMAL1 mRNA相对表达量,采用Western blot法检测BMAL1蛋白相对表达量,并进行比较。以癌组织BMAL1 mRNA相对表达量中位数为界,将82例患者分为BMAL1高表达组(BMAL1 mRNA相对表达量≥6.94)37例和BMAL1低表达组(BMAL1 mRNA相对表达量<6.94)45例,比较BMAL1高、低表达组临床病理特征。取对数生长期食管鳞癌KYSE150细胞,分为对照组(转染空载慢病毒)、BMAL1过表达组(转染BMAL1过表达慢病毒)、回复实验组(转染BMAL1过表达慢病毒及BMAL1 shRNA慢病毒),转染72 h,采用实时荧光定量PCR法检测3组细胞BMAL1 mRNA相对表达量,采用Western blot法检测BMAL1蛋白相对表达量,采用CCK-8实验检测细胞增殖的吸光度(optical density,OD)值,采用细胞克隆形成实验检测细胞克隆形成数目,采用细胞划痕实验检测细胞迁移率。结果食管鳞癌组织BMAL1 mRNA及蛋白(6.81±0.62、0.65±0.15)相对表达量均低于癌旁组织(7.74±0.79、0.97±0.13)(P<0.05)。BMAL1低表达组年龄<60岁(80.0%)、T分期T;~T;期(86.7%)、临床分期Ⅱb~Ⅲ期(86.7%)、脉管侵犯(66.7%)、神经侵犯(71.1%)及淋巴结转移(80.0%)比率均高于BMAL1高表达组(48.6%、54.1%、54.1%、43.2%、32.4%、29.7%)(P<0.05),性别、肿瘤直径、病理分级与BMAL1高表达组比较差异均无统计学意义(P>0.05)。BMAL1过表达组细胞BMAL1 mRNA及蛋白相对表达量(11.03±0.56、0.83±0.09)均高于对照组(9.27±0.48、0.35±0.09)和回复实验组(10.04±0.45、0.44±0.08)(P<0.05);回复实验组细胞BMAL1 mRNA及蛋白相对表达量与对照组比较差异均无统计学意义(P>0.05)。3组细胞转染后培养0 h时细胞增殖OD值比较差异无统计学意义(P>0.05);BMAL1过表达组转染后培养24 h时细胞增殖OD值(0.44±0.03)低于对照组(0.55±0.04)(P<0.05),BMAL1过表达组与回复实验组(0.48±0.12)、回复实验组与对照组比较差异均无统计学意义(P>0.05);BMAL1过表达组转染后培养48、72 h时细胞增殖OD值(0.73±0.04、0.90±0.03)均低于对照组(0.94±0.04、1.23±0.03)和回复实验组(0.86±0.03、1.15±0.05)(P<0.05),回复实验组与对照组比较差异均无统计学意义(P>0.05)。BMAL1过表达组细胞克隆形成数目[(322.88±31.37)个]少于对照组[(517.63±10.51)个]和回复实验组[(494.13±9.46)个](P<0.05),细胞迁移率[(11.67±0.88)%]低于对照组[(32.26±1.41)%]和回复实验组[(29.93±0.14)%](P<0.05);回复实验组细胞克隆形成数目、细胞迁移率与对照组比较差异均无统计学意义(P>0.05)。结论食管鳞癌组织BMAL1呈低表达,提示肿瘤恶性程度高,上调BMAL1表达可抑制食管鳞癌KYSE150细胞增殖和迁移。

Objective To observe the changes of brain and muscle ARNT-like protein 1(BMAL1 protein)in esophageal squamous cell carcinoma(ESCC)tissues,and to explore the influence of BMAL1 on the proliferation and migration of KYSE150 cells.Methods Eighty-two patients with ESCC were obtained the resected ESCC tissues and paracancerous tissues.The relative expressions of BMAL1 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot,and were compared.Taking the median relative expression of BMAL1 mRNA in ESCC tissues as the boundary,82patients were divided into 37patients with BMAL1mRNA≥6.94(highly-expressed BMAL1group)and 45patients with BMAL1 mRNA<6.94(lowly-expressed BMAL1 group),and the clinicopathological features were compared between two groups.The KYSE150cells in logarithmic growth phase were divided into control group(transfected with negative control lentivirus),BMAL1overexpression group(transfected with BMAL1overexpressed lentivirus),and recovery experimental group(transfected with shRNA-BMAL1overexpressed lentivirus).After 72-h transfection,the relative expressions of BMAL1 mRNA and protein in three groups were detected by real-time fluorescence quantitative PCR and Western blot,the CCK-8assay was used to detect the optical density(OD),the cell clone formation assay was used to detect the number of cell clones,and the cell scratch assay was used to detect the cell migration rate.Results The relative expressions of BMAL1 mRNA and protein were lower in ESCC tissues(6.81±0.62,0.65±0.15)than those in paracancerous tissues(7.74±0.79,0.97±0.13)(P<0.05).The percentages of patients aged<60years,in staging T;-T;,in clinical stageⅡb-Ⅲ,and with vascular invasion,nerve invasion and lymph node metastasis were higher in lowly-expressed BMAL1group(80.0%,86.7%,86.7%,66.7%,71.1%,80.0%)than those in highly-expressed BMAL1 group(48.6%,54.1%,54.1%,43.2%,32.4%,29.7%)(P<0.05),and there were no significant differences in gender,cancer diameter,and degree of differentiation between two groups(P>0.05).The relative expressions of BMAL1mRNA and protein were higher in BMAL1overexpression group(11.03±0.56,0.83±0.09)than those in control group(9.27±0.48,0.35±0.09)and recovery experimental group(10.04±0.45,0.44±0.08)(P<0.05),and showed no significant differences between recovery experimental group and control group(P>0.05).The OD value showed no significant difference at 0 hamong three groups(P>0.05).The OD value was lower in BMAL1overexpression group(0.44±0.03)than that in control group(0.55±0.04)(P<0.05),and showed no significant difference between BMAL1overexpression group and recovery experimental group(0.48±0.12)and between recovery experimental group and control group(P>0.05).The OD values at 48and72hwere lower in BMAL1overexpression group(0.73±0.04,0.90±0.03)than those in control group(0.94±0.04,1.23±0.03)and recovery experimental group(0.86±0.03,1.15±0.05)(P<0.05),and showed no significant differences between recovery experimental group and control group(P>0.05).The number of cell clones was less in BMAL1overexpression group(322.88±31.37)than that in control group(517.63±10.51)and recovery experimental group(494.13±9.46)(P<0.05),and the cell migration rate was lower in BMAL1overexpression group[(11.67±0.88)%]than that in control group[(32.26±1.41)%]and recovery experimental group[(29.93±0.14)%](P<0.05),which showed no significant differences between recovery experimental group and control group(P>0.05).Conclusion The BMAL1is lowly expressed in ESCC tissues,which indicates a high degree of malignancy,and the up-regulation of BMAL1expression can inhibit the proliferation and migration of KYSE150cells.