Rab27B对食管鳞状细胞癌增殖和迁移能力的影响

Effect of Rab27B on growth and metastasis of esophageal squamous cell carcinoma

目的采用细胞功能实验探讨Rab27B对食管鳞状细胞癌(ESCC)细胞系表型的影响。方法收集100例福建医科大学附属协和医院胸外科行手术切除且经过严格病理诊断的食管鳞癌患者的肿瘤组织及癌旁正常食管上皮组织进行免疫组织化学染色;通过蛋白质印迹法(Western blot)实验对比不同ESCC细胞系与正常食管上皮中Rab27B的表达量,筛选适合实验的细胞系;通过慢病毒包装转染构建稳定的Rab27B过表达细胞株,进行细胞计数试剂盒(CCK-8)实验,Transwell实验及划痕实验等细胞功能实验,观察Rab27B在体外对ESCC细胞增殖及迁移的影响;通过裸鼠体表种瘤,观察Rab27B在体内对ESCC细胞增殖的影响。组间比较采用t检验。结果免疫组织化学染色结果显示,Rab27B在食管鳞癌组织中的表达与N分期(χ^(2)=8.726,P<0.05)、TNM(χ^(2)=8.036,P<0.01)分期及肿瘤分化程度(χ^(2)=8.707,P<0.05)密切相关;Western blot实验结果显示,Rab27B在食管鳞癌细胞系中表达均上调;通过对过表达稳转细胞株进行实验结果显示,在Rab27B过表达的情况下,体外细胞增殖能力增强,过表达组伤口愈合比例高于阴性对照组[KYSE140组(85±2)%比(81±1)%,t=3.098,P<0.05;KYSE9706组(68±2)%比(44±2)%,t=14.697,P<0.05],差异有统计学意义;过表达组细胞增殖数高于阴性对照组[KYSE140组0.701±0.031比0.620±0.044,t=3.607;1.311±0.045比1.180±0.066,t=2.840;1.826±0.063比1.556±0.052,t=6.900,P<0.05;KYSE9706组0.511±0.062比0.451±0.046,t=1.346;0.992±0.055比0.800±0.066,t=3.871;1.522±0.032比1.250±0.067,t=6.345,P值均<0.05],差异有统计学意义。过表达组迁移能力增强,细胞迁移实验中过表达组迁移数高于阴性对照组[KYSE140组1534±205比665±85,t=6.782,P<0.05;KYSE 9706组517±45比432±33,t=3.244,P<0.05]差异有统计学意义;裸鼠肿瘤实验结果显示过表达组裸鼠肿瘤重量高于阴性对照组裸鼠[(0.082±0.025)g比(0.142±0.037)g,t=2.845,P<0.05]差异有统计学意义。结论Rab27B在食管鳞癌组织中的表达与N分期、TNM分期及肿瘤分化程度密切相关;Rab27B在食管鳞癌细胞系中表达均上调;Rab27B对ESCC细胞的增殖及迁移起促进作用。

Objective Through cell function experiments,we drag on the effect of Rab27B on the phenotype of esophageal squamous cell carcinoma.Methods Collect tumor tissues and normal esophageal epithelial tissues adjacent to the cancer,which were taken from 100 esophageal squamous cell carcinoma(ESCC)patients who underwent surgical resection at the Thoracic Surgery Department of Fujian Medical University Affiliated Union Hospital and had undergone strict pathological diagnosis,for immunohistochemistry.Differentiate the ESCC cell lines with normal esophageal epithelium through Western blotting experiment.And select cell lines that suitable for the experiment via the expression level of Rab27B;Construct a stable Rab27B overexpression cell line through lentivirus packaging and transfection.Conduct cell counting kit-8(CCK-8)experiment,Transwell experiment and scratch test and other cell function experiments to observe the effect of Rab27B on ESCC;To observe the effect of Rab27B on ESCC cell proliferation in vivo,we inplant tumors on the surface of nude mice.We use t test for comparison between groups.Results The results of immunohistochemistry indicated that the expression of Rab27B in esophageal squamous cell carcinoma tissue had a close association with the N stage(χ^(2)=8.726,P<0.05),TNM(χ^(2)=8.036,P<0.01)stage and the degree of tumor differentiation(χ^(2)=8.707,P<0.05).In Western Blot experiment,it explained that Rab27B expression was up-regulated in ESCC lines.Experiments on overexpression stable cell lines depicted that cell proliferation in vitro was enhanced in the case of Rab27B overexpression,and the percentage of wound healing in the overexpression group was higher than that of the negative control group[KYSE140 group(85±2)%vs.(81±1)%,t=3.098,P<0.05;KYSE9706 group(68±2)%vs.(44±2)%,t=14.697,P<0.05];the number of cell proliferation in the overexpression group was higher than that of the negative control group[KYSE140 group 0.701±0.031 vs.0.620±0.044,t=3.607;1.311±0.045 vs.1.180±0.066,t=2.840;1.826±0.063 vs.1.556±0.052,t=6.900 or more P<0.05;KYSE9706 group 0.511±0.062 vs.0.451±0.046,t=1.346;0.992±0.055 vs.0.800±0.066,t=3.871;1.522±0.032 vs.1.250±0.067,t=6.345,above P<0.05],among which the difference is statistically significant.The migration ability of the overexpression group was enhanced.In the cell migration experiment,the migration number of the overexpression group was higher than that of the negative control group[KYSE140 group 1534±205 vs.665±85,t=6.782,P<0.05;KYSE 9706 group 517±45 vs.432±33,t=3.244,P<0.05],among which the difference is statistically significant.The nude mouse tumor experiment explained that the tumor weight of the overexpression group was higher than that of the negative control group[(0.082±0.025)g vs.(0.142±0.037)g,t=2.845,P<0.05].Conclusion The expression of Rab27B in ESCC tissue is closely related to the N stage,TNM stage and the degree of tumor differentiation.The expression of Rab27B in ESCC lines is up-regulated.Rab27B promotes the proliferation and metastasis of ESCC cells.