RNA沉默LASS2基因促进人前列腺癌细胞体外侵袭及其机制研究

Silencing of tumor metastasis suppressor gene 1promotes invasion of prostate cancer cell in vitro and its molecular mechanisms

目的:研究特异性沉默人源性长寿保障基因2(homosapiens longevity assurance homologue 2,LASS2,亦称TMSG1)的小干扰RNA(small interfering RNA,siRNA)对人前列腺癌细胞系低转移潜能亚系PC-3M-2B4的体外侵袭能力的影响及相关机制。方法:通过脂质体转染法将特异性沉默LASS2基因的siRNA转染入PC-3M-2B4细胞。分别用实时荧光定量PCR(real-time fluorogenetic quantitative PCR,RFQ-PCR)和Western blot方法检测LASS2siRNA对PC-3M-2B4细胞LASS2基因和蛋白表达的抑制作用,筛选有效的siRNA片段;运用液泡型ATPases(vacuo-lar H+-ATPases,V-ATPases)活性测定试剂盒来检测PC-3M-2B4细胞的V-ATPases活性;运用BCECF氢离子敏感荧光探针检测PC-3M-2B4细胞外氢离子浓度;采用Western blot方法分析PC-3M-2B4细胞的基质金属蛋白酶2(ma-trix metalloproteinase 2,MMP-2)的表达及分泌情况;应用明胶酶谱法检测PC-3M-2B4细胞上清液中MMP-2的活性;利用细胞划痕修复实验和Transwell法检测细胞体外迁移及侵袭能力。结果:通过RFQ-PCR和Western blot筛选后,siRNA-2能显著抑制PC-3M-2B4细胞LASS2基因mRNA和蛋白的表达,对mRNA表达的抑制率可达84.5%,对蛋白表达的抑制率可达60%。PC-3M-2B4细胞转染LASS2 siRNA-2后,其V-ATPases的活性及细胞外氢离子浓度明显升高,与空白对照组相比差异有统计学意义(P<0.05);MMP-2蛋白的表达及分泌无明显变化,但分泌的MMP-2的活性形式增加,与空白对照组相比明显提高,差异有统计学意义(P<0.05);且细胞的体外迁移及侵袭能力明显高于空白对照组(P<0.05)。结论:特异性siRNA沉默LASS2基因能促进人前列腺癌细胞体外侵袭能力,其机制与沉默LASS2基因后激活V-ATPase,从而使细胞外氢离子浓度升高,进而激活MMP-2有关,提示LASS2基因是一新的肿瘤转移抑制基因。

Objective：To explore the effect of small interference RNA（siRNA） targeting homosapiens longevity assurance homologue 2（LASS2,or TMSG1） on the invasion of PC-3M-2B4（a variant subline of human prostate carcinoma cell line PC-3M with low metastatic potential） and its molecular mechanisms.Methods： PC-3M-2B4 cells were transfected with siRNA by using lipofectamine 2000.The expression of LASS2 mRNA and protein was detected after transfection by real-time fluorogentic quantitative PCR（RFQ-PCR） and Western blot to screen the effective siRNA fragment.The V-ATPase activity of PC-3M-2B4 cells was detected by V-ATPase activity assay kit.The concentration of extracellular hydrogen ion was measured by pH-sensitive fluorescence probe bis-carboxyethyl-carboxyfluorescein（BCECF）.The matrix metalloproteinase-2（MMP-2） protein in the supernatant and cells was analyzed by Western blot.The activity of MMP-2 was examined by Gelatin zymography.Furthermore,the migration and invasion of cells were evaluated by in vitro wound migration assay and invasion assay.Results： RFQ-PCR and Wes-tern blot revealed dramatic reduction（84.5% and 60%） in the levels of LASS2 mRNA and protein after transfection of siRNA-2 in PC-3M-2B4 cells.The V-ATPases activity and extracellular hydrogen ion concentration were significantly increased in PC-3M-2B4 cells transfected with the siRNA-2 compared with other control groups（P0.05）;There were no differences in the expression and secretion of MMP-2 protein between LASS2-siRNA treated cells and other control groups.However,the activity of MMP-2 was up-regulated in LASS2-siRNA treated cells compared with other control groups（P0.05）;and the capacity for migration and invasion in LASS2-siRNA treated cells was significantly higher than in other control groups（P0.05）.Conclusion： Silencing of LASS2 can promote invasion of prostate cancer cells in vitro through the increase of the V-ATPases activity、extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2,indicating that LASS2 is a novel tumor metastasis suppressor gene.