摘要
目的:探讨人表皮生长因子受体4(HER4)在非小细胞肺癌(NSCLC)中的表达意义及其对NSCLC细胞生物学行为的影响。方法:选取2015年1月至2017年1月长治市人民医院呼吸与危重症医学科收治的经手术切除并经病理学证实的84例NSCLC患者的肿瘤组织及癌旁正常组织,男55例,女29例,年龄(54.58±9.67)岁,年龄范围为33~75岁。采用实时荧光定量聚合酶链式反应(RT-qPCR)检测肺癌组织和癌旁正常肺组织中HER4的表达,分析HER4与患者临床病理特征的相关性,通过Kaplan-Meier生存曲线分析HER4与NSCLC预后的关系。HER4-siRNA转染H522细胞系沉默HER4的表达,采用四甲基偶氮唑蓝(MTT)法、侵袭小室(Transwell)法,流式细胞术检测细胞增殖、迁移和侵袭及凋亡情况。蛋白免疫印迹(Western blot)检测HER4和间质上皮转化(EMT)相关蛋白的表达。结果:NSCLC组织中HER4 mRNA的表达水平(3.80±0.64)显著高于癌旁正常肺组织(2.07±0.57),NSCLC细胞系H522中HER4 mRNA的表达水平(1.45±0.12)也显著高于人正常肺上皮细胞系BEAS-2B(0.78±0.06),差异有统计学意义(P<0.05)。将NSCLC患者按照HER4表达水平的中位值分为高表达组和低表达组,两组肿瘤淋巴结转移(TNM)分期、淋巴结转移比较,差异有统计学意义(P<0.05)。进一步采用Kaplan-Maier法分析两组生存函数,结果显示低表达组总体生存期为(40.14±2.58)个月,优于高表达组的(30.45±2.58)个月,差异有统计学意义(P<0.05)。低表达组5年生存率[38.1%(16/42)]高于高表达组[23.8%(10/42)],差异有统计学意义(P<0.05)。HER4-siRNA及阴性载体转染细胞后,RT-qPCR检测发现,HER4-siRNA组中HER4 mRNA的相对水平(0.52±0.04)低于空白组(1.00±0.01)和阴性组(0.96±0.06),Western blot检测显示,HER4-siRNA组中HER4蛋白表达水平(0.64±0.04)低于空白组(1.16±0.09)和阴性组(1.12±0.08),差异有统计学意义(P<0.05)。MTT试验结果显示,转染后,HER4-siRNA组H522细胞的增殖活性低于阴性组和空白组,差异有统计学意义(P<0.05)。Transwell法检测结果显示,与空白组和阴性组相比,HER4-siRNA组中迁移和侵袭的细胞数量明显降低,差异有统计学意义(P<0.05)。Western blot检测结果显示,HER4-siRNA组上皮标志蛋白E-cadherin的表达水平显著高于空白组和阴性组,N-cadherin和Vimentin的表达水平则显著低于空白组和阴性组,差异有统计学意义(P<0.05)。流式细胞术结果显示,HER4-siRNA组中细胞凋亡率为(12.48±1.02)%,高于空白组(2.25±0.28)%和阴性组(2.69±0.35)%,差异有统计学意义(P<0.05)。结论:HER4可能是潜在的NSCLC预后和治疗的新的靶点。但关于HER4在NSCLC细胞的增殖、转移和凋亡中具体的作用机制,尚有待深入研究。
Objective To investigate the expression significance of human epidermal growth factor receptor 4(HER4)in non-small cell lung cancer(NSCLC)and its influence on the biological behavior of NSCLC cells.Methods The tumor tissues and adjacent normal tissues of 84 patients with NSCLC admitted to the department of Respiratory and Critical Care Medicine,Changzhi People′s Hospital from January 2015 to January 2017 who had been surgically removed and pathologically confirmed,including 55 males and 29 females,aged(54.58±9.67)years old,ranged from 33 to 75 years old.The expression of HER4 in lung cancer and adjacent normal lung tissues were detected by real-time fluorescence quantitative PCR(RT-qPCR).The correlation between HER4 and clinicopathological characteristics of patients was analyzed.The relationship between HER4 and NSCLC prognosis was analyzed by Kaplan-Meier survival curve.After HER4-siRNA was transfected into NSCLC cell line H522,the proliferation,migration,invasion and apoptosis of NSCLC cells were detected by methyl thiazolyl tetrazolium(MTT)method and Transwell method and flow cytometry.Western blot was used to detect the expression of HER4 and the related protein of mesenchymal epithelial transformation(EMT).Results Compared with normal lung tissue adjacent to cancer,the expression of HER4 mRNA in NSCLC(3.80±0.64)was significantly higher than that in normal lung tissues(2.07±0.57),and the difference was statistically significant(P<0.05).The expression level of HER4 mRNA in H522 cells(1.45±0.12)was significantly higher than that in normal lung epithelial cells BEAS-2B(0.78±0.06),(P<0.05).NSCLC patients were divided into the high expression group and low expression group,according to the median HER4 expression.And there were significant difference on TNM stage and lymph node metastasis between the two groups(P<0.05).Kaplan-Meier analysis found that the overall survival of the low expression group was(40.14±2.58)months,higher than that of the high expression group(30.45±2.58)months(P<0.05).And the 5-year overall survival rate of the low expression group was 38.1%(16/42),significantly higher than that of the high expression group 23.8%(10/42),(P<0.05).After transfection with HER4 siRNA,the expression level of HER4 mRNA in the HER4-siRNA group(0.52±0.04)was lower than that in the blank group(1.00±0.01)and negative group(0.96±0.06),which was detected by RT-qPCR.Western blot also showed that the expression of HER4 protein in the HER4-siRNA group(0.64±0.04)was lower than that in the blank group(1.16±0.09)and the negative group(1.12±0.08),(P<0.05).The results of MTT assay showed that the proliferation activity of H522 cells in the HER4-siRNA group was lower than that in the blank group and the negative group(P<0.05).Transwell assay showed that compared to the blank group and the negative group,the number of migration and invasion cells in the HER4-siRNA group were significantly reduced with statistical differences(P<0.05).Western blot results showed that the expression levels of E-cadherin in the HER4-siRNA group were significantly higher,while the expression levels of N-cadherin and Vimentin in the HER4-siRNA group were significantly lower than those in the blank group and the negative group(P<0.05).The results of flow cytometry showed that the apoptosis rate in the HER4-siRNA group was(12.48±1.02)%,significantly higher than that in the blank group(2.25±0.28)%and the negative group(2.69±0.35)%,(P<0.05).Conclusion HER4 may be a potential new target for the prognosis and treatment of NSCLC.While the specific mechanism of HER4 in the proliferation,migration,invasion,and apoptosis of NSCLC cells remains to be further studied.
作者
王晚萍
边海波
王夏珍
刘亮
魏丁
Wang Wanping;Bian Haibo;Wang Xiazhen;Liu Liang;Wei Ding(Department of Respiratory and Critical Care Medicine,Changzhi People′s Hospital,Changzhi 046000,China)
出处
《中国临床实用医学》
2020年第4期1-7,共7页
China Clinical Practical Medicine
基金
CSCO-齐鲁肿瘤研究基金会(Y-Q201802-035)。
关键词
非小细胞肺癌
人表皮生长因子受体4
肺癌
恶性肿瘤
Non-small cell lung cancer
Human epidermal growth factor receptor 4
Lung cancer
Malignant tumor
作者简介
通信作者:王晚萍,Email:researchbao123@163.com。
