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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 an important tumor suppressor gene newly discovered in recent years. In order to clarify the function and significance of VMP1 in colorectal cancer, we studied the effect of silencing VMP1 gene on the proliferation, apoptosis and migration of colorectal cancer cells by lentiviral vector silencing the expression of VMP1 in colorectal cancer cell line SW480. effect. Methods The expression of VMP1 in five colorectal cancer cell lines was detected by RT-PCR and Western blotting. The shRNA lentiviral vector containing human VMP1 gene was designed and packaged in 293T cells and infected with colorectal cancer cell line with high VMP1 expression The silencing effect of VMP1 gene on VMP1 gene expression was detected by Western blotting. The silencing of VMP1 gene was used to detect the effect of VMP1 gene silencing on cell proliferation by MTT assay and soft agar colony formation assay. Flow cytometry was used to detect cell apoptosis. Migration assay to detect cell invasion and migration in vitro. Results VMP1 was differentially expressed in five cell lines. HT-29 and SW480 cells with low metastatic potentials had higher VMP1 expression levels than LoVo, RKO and SW620 cells with high metastatic potentials, and the mRNA expression differences were statistically significant (t = 3.88, P = 0.005) ; While the protein expression difference was statistically significant, t = 14.29, P <0.001. SW480 cells with relatively high expression of VMP1 in low-metastatic cell lines were selected as research objects and VMP1 shRNA lentiviral vector was constructed to silence the expression of VMP1 in SW480 cells. Western blot results suggest that VMP1 stably silenced SW480 cells were successfully constructed. After stable silencing of VMP1 gene, MTT assay showed that the proliferation rate of SW480 cells was significantly increased at 24 and 48 hours, with F values of 79.77 and 12.35, respectively, with P values of <0.001 and 0.025, respectively. The results of soft agar colony formation assay showed that the number of colonies of SW480 cells increased significantly, F = 75.23, P <0.001. Both showed that the proliferation of SW480 cells increased. Flow cytometry showed that VMP1 gene silencing could inhibit SW480 cell apoptosis, F = 84.44, P <0.001. The results of Transwell migration showed that the migration ability of SW480 after VMP1 silencing was enhanced, F = 155.90, P <0.001. Conclusion VMP1 gene may be involved in cell proliferation, apoptosis and metastasis of colorectal cancer, which may be involved in the occurrence and development of colorectal cancer.