Sun Guo-Gui, Wang Ya-Di, Cui Da-Wei, Cheng Yun-Jie, Hu Wan-Ning
Guo-Gui Sun, Wan-Ning Hu, Department of Chemoradiotherapy, Tangshan People's Hospital, Tangshan 063000, Hebei Province, China.
World J Gastroenterol. 2014 Apr 14;20(14):4001-10. doi: 10.3748/wjg.v20.i14.4001.
To determine the expression and function of epithelial membrane protein 1 (EMP1) in colorectal carcinoma.
Colorectal samples were taken from cancer lesions and adjacent normal tissue in colorectal cancer patients immediately after endoscopic biopsy. A portion of the sample was either fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry or stored in liquid nitrogen for Western blot. In order to determine protein expression of EMP1 in colorectal cancer (n = 63) and normal tissue (n = 31), semi-quantitative immunohistochemistry and Western blot were utilized. For in vitro studies, the human colorectal cancer cell line SW-480 was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. Recombinant lentivirus mediated overexpression of EMP1 in SW-480 cells was quantified by real-time reverse transcription-polymerase chain reaction and Western blot. Control SW-480 cells were transfected with an empty vector. To further study the effect of EMP1 overexpression in SW-480 cells, cell proliferation, apoptosis, migration and invasion assays were conducted.
Expression of EMP1 was significantly lower in colorectal cancer tissue than in normal tissue using both immunohistochemistry (39.7% vs 90.3% of tissues, P < 0.05) and Western blot (0.126 ± 0.022 vs 0.632 ± 0.053, P < 0.05). The level of EMP1 protein expression was not correlated with gender, age, or tumor location. Decreased expression of EMP1 was significantly correlated with T stage, lymph node metastasis, clinic stage, and histological grade in patients with colorectal cancer (P < 0.05). According to Kaplan-Meier analysis, low EMP1 expression correlated significantly with poor overall five-year survival (34.2% vs 64.0% survival, P < 0.05). SW-480 cells transfected with EMP1 had a lower survival fraction, higher cell apoptosis (12.1% ± 1.3% vs 3.1% ± 0.6%, P < 0.05), a significant decrease in migration and invasion (124.0 ± 17.0 and 87.0 ± 12.0, respectively vs 213.0 ± 29.0 and 178.0 ± 21.0, respectively, P < 0.05), higher caspase-9 (0.635 ± 0.063 vs 0.315 ± 0.032, P < 0.05), and lower VEGFC protein expression (0.229 ± 0.021 vs 0.519 ± 0.055, P < 0.05) relative to cells not transfected with EMP1.
Low EMP1 expression in colorectal cancer is associated with increased disease severity, suggesting that EMP1 may be a negative regulator of colorectal cancer.
确定上皮膜蛋白1（EMP1）在结直肠癌中的表达及功能。
在内镜活检后立即从结直肠癌患者的癌灶及相邻正常组织采集结直肠样本。一部分样本用4%多聚甲醛固定并石蜡包埋用于免疫组织化学，或储存在液氮中用于蛋白质印迹法。为确定EMP1在结直肠癌（n = 63）和正常组织（n = 31）中的蛋白表达，采用了半定量免疫组织化学和蛋白质印迹法。对于体外研究，人结直肠癌细胞系SW - 480在补充有10%胎牛血清的RPMI - 1640培养基中培养。通过实时逆转录 - 聚合酶链反应和蛋白质印迹法定量重组慢病毒介导的EMP1在SW - 480细胞中的过表达。对照SW - 480细胞用空载体转染。为进一步研究EMP1过表达对SW - 480细胞的影响，进行了细胞增殖、凋亡、迁移和侵袭实验。
采用免疫组织化学（组织阳性率分别为39.7%和90.3%，P < 0.05）和蛋白质印迹法（蛋白表达水平分别为0.126 ± 0.022和0.632 ± 0.053，P < 0.05）均显示，结直肠癌组织中EMP1的表达明显低于正常组织。EMP1蛋白表达水平与性别、年龄或肿瘤位置无关。结直肠癌患者中EMP1表达降低与T分期、淋巴结转移、临床分期和组织学分级显著相关（P < 0.05）。根据Kaplan - Meier分析，EMP1低表达与总体五年生存率低显著相关（生存率分别为34.2%和64.0%，P < 0.05）。与未转染EMP1的细胞相比，转染EMP1的SW - 480细胞存活分数更低，细胞凋亡率更高（分别为12.1% ± 1.3%和3.1% ± 0.6%，P < 0.05），迁移和侵袭能力显著降低（迁移分别为124.0 ± 17.0和213.0 ± 29.0，侵袭分别为87.0 ± 12.0和178.0 ± 21.0，P < 0.05），半胱天冬酶 - 9水平更高（0.635 ± 0.063和0.315 ± 0.032，P < 0.05），VEGFC蛋白表达更低（0.229 ± 0.021和0.519 ± 0.055，P < 0.05）。
结直肠癌中EMP1低表达与疾病严重程度增加相关，提示EMP1可能是结直肠癌的负调节因子。
