沉默泡膜蛋白1对SW480细胞增殖与侵袭影响的研究

Effect of vacuole membrane protein-1 shRNA on proliferation and invasion of colorectal cancer SW480 cells

目的泡膜蛋白1(vacuole membrane protein-1,VMP1)是近年来新发现的一个重要功能的抑癌基因。为明确VMP1在结直肠癌中的功能和意义,本研究通过慢病毒载体沉默结直肠癌细胞系SW480中VMP1基因的表达,探讨VMP1基因沉默后对结直肠癌细胞增殖、凋亡及迁移的调控作用。方法应用RT-PCR及蛋白质印迹法检测5种结直肠癌细胞系中VMP1的表达状况;设计人VMP1基因的shRNA慢病毒载体,在293T细胞中包装病毒并感染VMP1高表达的结直肠癌细胞系,运用蛋白质印迹法鉴定其对VMP1基因的沉默效果;VMP1基因沉默后,通过MTT法和软琼脂克隆形成实验检测其对细胞增殖能力的影响,并应用流式细胞技术检测细胞凋亡;运用Transwell迁移实验检测细胞的体外侵袭迁移能力。结果 VMP1在5种细胞系中存在差异性表达。具有低转移潜能的HT-29和SW480细胞与具有高转移潜能的LoVo、RKO和SW620细胞相比,有更高的VMP1表达水平,其中mRNA表达差异有统计学意义,t=3.88,P=0.005;而蛋白表达差异有统计学意义,t=14.29,P<0.001。选取低转移细胞株中VMP1表达相对较高的SW480细胞作为研究对象,构建VMP1shRNA慢病毒载体沉默SW480细胞中VMP1的表达。蛋白质印迹结果提示,VMP1稳定沉默的SW480细胞构建成功。在稳定沉默VMP1基因后,MTT法显示SW480细胞的增殖速度在24和48h显著升高,F值分别为79.77和12.35,P值分别为<0.001和0.025。软琼脂克隆形成实验结果显示,SW480细胞的集落数目显著增多,F=75.23,P<0.001。二者均表明SW480细胞增殖能力增强。流式细胞技术检测显示,VMP1基因沉默后可抑制SW480细胞凋亡,F=84.44,P<0.001。Transwell迁移实验结果显示,VMP1沉默后SW480迁移能力增强,F=155.90,P<0.001。结论 VMP1基因可能与结直肠癌的细胞增殖、凋亡与转移有关,进而参与了结直肠癌的发生、发展。

OBJECTIVE Vacuole membrane protein-1 （VMP1） is confirmed as tumor suppressor gene with many important functions in recent years. The objective of this study was to explore the role of VMP1 in colorectal carcinoma （CRC）,the shRNA lentiviral vector was constructed to silence VMP1 expression in CRC cells, and the proliferation, apoptosis and migration regulation of CRC cells were further evaluated. METHODS RT-PCR and Western-blot were used to detect VMP1 expression profile in 5 different CRC cell lines in order to confirm the target cell line. In vitro, the lentivical Vector was constructed to silence VMP1 expression in SW480 cells. Methyl thiazolyl tetrazolium （MTT） and soft agar assay were used to detect cell proliferation. Cell apoptosis and cell migration after VMP1 knockdown were measured by the method of flow cytometry and transwell assay. RESULTS The expression of VMP1 in protein and mRNA level in different cell lines of colorectal cancer were detected, among which the highly metastatic SW620,RKO and LoVo cell lines showed significant lower VMP1 expression than that in the low metastatic HT-29 and SW480 cell lines wherein both the expression of mRNA（t=3.88,P=0. 005） and protein （t=14.29,P〈0. 001） had significant difference. After stably silencing VMP1 in SW480, MTT assay showed cell growth was significantly increased at 24 and 48 hours, the difference was statistically significant （F24 h = 79.77, P〈0. 001, F48 h = 12. 35, P = 0. 025）. Clonogenic assay showed that the number of shVMP1 colonies was also significantly increased （F= 75.23,P〈0. 001）. The above data indicated that the proliferation of SW480 cells increased obviously after downregulation of VMPl,together with an induction of anti-apoptosis effect on SW480 cells（F= 84.44,P〈0. 001）. Moreover, SW480 cells with VMP1 silencing gained significant aggressive potential in migration than other tested cells（F= 155.90,P〈0. 001）. CONCLUSION VMP1 may be closely associated with CRC cell proliferation, apoptosis and migration,which indicates that it plays a pivotal role in CRC development and progression.