过表达上皮膜蛋白1对结直肠癌SW-480细胞生物学行为的影响及其可能的机制

Putative role of epithelial membrane protein-1 in the pathogenesis of colorectal carcinoma

目的:探讨上皮膜蛋白1(epithelial membrane protejn-1,EMP1)在人结直肠癌(colorectal carcinoma,CRC)组织中的表达水平及其过表达对CRC SW-480细胞增殖、凋亡和侵袭的影响。方法:免疫组织化学、Western blotting方法检测63例CRC组织及31例癌旁组织中EMP1的表达水平,分析EMP1表达与CRC临床病理参数的关系。慢病毒介导将pLenti6-EMP1质粒转染SW-480细胞,建立EMP1过表达细胞,转染plenti6/V5-DEST空白质粒为对照。Real-time PCR及Western blotting检测转染后SW-480细胞株中EMP1的表达,MTT、流式细胞术及Transwell实验分别检测EMP1过表达对SW-480细胞增殖、凋亡及侵袭能力的影响。结果:EMP1蛋白在人CRC组织的表达显著低于癌旁组织(0.257±0.022 vs 0.863±0.086,P<0.05),并且其表达水平在不同T分期、有无淋巴结转移、临床分期以及组织分级组间表达有显著差异(P<0.05),而与患者年龄、性别、肿瘤部位无关(P>0.05)。成功构建EMP1过表达的LeEMP1细胞。与LeEmpty细胞相比,LeEMP1细胞的增殖能力显著下降[(60.94±4.04)%vs(100.00±0.00)%,P<0.05],凋亡率显著升高[(12.10±1.30)%vs(3.10±0.60)%,P<0.05]、侵袭转移能力显著降低[穿膜细胞数:(87.00±12.00)vs(178.00±21.00)个,P<0.05]。LeEMP1细胞Caspase-9表达显著高于LeEmpty细胞(0.764±0.073 vs 0.231±0.029,P<0.05),VEGFC表达显著降低(0.185±0.022 vs 0.663±0.065,P<0.05)。结论:人CRC组织中EMP1蛋白表达明显减低,过表达EMP1能够抑制CRC SW-480细胞的恶性生物学行为,其可能通过调控Caspase-9和VEGFC的表达来发挥作用。

Objective： To elucidate the putative involvement of epithelial membrane protein-1（ EMP1） in the pathogenesis of colorectal carcinoma（ CRC）. Methods： Tumor（ n = 63） and peri-tumor（ n = 31） tissue specimens were collected from 63 patients with CRC. EMP1 protein content in these specimens was assessed by immunohistochemistry and Western blotting. The relationship between EMP1 protein content in the tumor tissue and the pathologic grade of the lesions was analyzed. To further evaluate the putative role for EMP1 in the development of CRC,we also performed analysis in vitro. To this end,CRC SW-480 cells were transfected with an EMP1 lentiviral vector pLenti6-EMP1 or a control vector plenti6 /V5-DEST. EMP1 mRNA abundance and protein content in transfected cells were determined by quantitative realtime PCR and Western blotting respectively. Effects of EMP1 overexpression on the proliferation,apoptosis and invasion of SW-480 cells were evaluated by methyl thiazolyl tetrazoliun（ MTT） assay,flow cytometry（ FCM） and transwell invasion assays,respectively. Results： EMP1 protein content was significantly lower in CRC tissue than in peritumor tissues（ P 0. 05）. The level of EMP1 protein was not correlated with gender,age,and tumor location（ P 0. 05） but was positively correlated with lymph node metastasis,clinic stage and histological grade of the lesions（ P 0. 05）. Compared with SW-480 cells transfected with the control vector,SW-480 cells overexpressing EMP1 had a lower survival fraction（ [60. 94 ±4. 04]% vs [100. 00 ± 0. 00]%,P 0. 05）,a higher cell apoptosis rate（ [12. 10 ± 1. 30]% vs [3. 10 ± 0. 60]%,P 0. 05）,a decreased invasive capacity（ [178. 00 ± 21. 00] vs [87. 00 ± 12. 00],P 0. 05）,higher caspase-9 protein content（ 0. 764 ± 0. 073 vs 0. 231 ± 0. 029,P 0. 05） and lower VEGFC protein content（ 0. 663 ± 0. 065 vs 0. 185 ±0. 022,P 0. 05）. Conclusion： EMP1 protein content is significantly lower in the CRC tissue than in the non-CRC tissue. Overexpression of EMP1 in CRC cells in vitro results in a significant inhibition of invasive activity through regulating the expression of caspase-9 and VEGF-C. These findings suggest that EMP1 is involved in the pathogenesis of colorectal carcinoma.