LASS2/TMSG-1基因沉默对人前列腺癌细胞生物学功能的影响及其机制  

Influence of LASS2/TMSG-1 gene silencing on the biological function of human prostate cancer cell and its mechanism

作　　者：徐晓艳[1,2] 

机构地区：[1]内蒙古医科大学附属医院病理科,呼和浩特010059 [2]内蒙古医科大学基础医学院病理学教研室,呼和浩特010059

出　　处：《临床与实验病理学杂志》2014年第2期153-158,共6页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金(81201854)

摘　　要：目的探讨LASS2/TMSG-1基因沉默对PC-3M-2B4细胞增殖、迁移及侵袭的影响及其作用机制。方法将实验分为3组:空白对照组(仅转染PBS的PC-3M-2B4细胞)、无关阴性对照siRNA组及LASS2/TMSG-1 siRNA转染组。首先针对LASS2/TMSG-1基因,设计并合成siRNA;在脂质体介导下瞬时转染人前列腺癌PC-3M-2B4细胞24 h后分别采用Western blot和RT-PCR技术检测细胞内LASS2/TMSG-1蛋白及ATP6L mRNA表达水平;使用BCECF AM H+敏感荧光探针检测转染24、36、48、96 h后3组细胞的细胞内H+浓度;用MTT法检测细胞增殖;划痕修复实验和Transwell体外侵袭实验分别检测PC-3M-2B4细胞的迁移和侵袭能力。结果转染LASS2/TMSG-1 siRNA 24 h后,LASS2/TMSG-1 siRNA组LASS2/TMSG-1蛋白表达与对照组相比下降65%,ATP6L mRNA表达水平与对照组比较提高75%。通过BCECF AM荧光探针检测LASS2/TMSG-1 siRNA转染组细胞内H+浓度明显下降,且与空白对照组及无关阴性对照siRNA组相比,差异有统计学意义(P<0.05)。MTT法检测显示,PC-3M-2B4细胞在转染24、48 h后,3组细胞生长速度无明显差异(P>0.05);转染72 h后LASS2/TMSG-1 siRNA转染组细胞生长速度明显升高,与空白对照组、无关阴性对照siRNA组比较,差异均有统计学意义(P均<0.05)。划痕修复试验和Transwell体外侵袭实验显示,转染LASS2/TMSG-1 siRNA 24 h后,与空白对照组及无关阴性对照siRNA组相比,能显著促进细胞迁移和侵袭能力(P<0.05)。结论体外合成的siRNA能有效沉默LASS2/TMSG-1基因表达,同时上调V-ATPase质子泵中关键性亚基—ATP6L mRNA的表达水平,且基因沉默后其细胞内H+浓度下降,提示LASS2/TMSG-1基因可能与ATP6L相互作用、共同参与对细胞内pH值的影响,导致肿瘤细胞内外微环境的改变并影响肿瘤细胞的生物学行为,如增殖、迁移及侵袭能力。Purpose To investigate the LASS2/TMSG-1 on the expression and metastasis of human prostate carcinoma cell line （PC= 3M-2B4）, a prostate cancer cell line with high expression of LASS2/TMSG-1 and its mechanism. Methods PC-3M-2B4 cells were cultured and divided into three groups： the blank control group, cells transfected with nonsense siRNA group, and cells transfected with specific LASS2/TMSG-1 siRNA. The expression of LASS2/TMSG-1 protein and ATP6L mRNA was detected by Western blot and RT-PCR before and after the transfection, respectively. The concentration of intracellular hydrogen ion was detected with pH-sensitive fluorescence probe BCECF AM. Proliferation of PC-3M-2B4 cells was detected by MTT assay. Furthermore, the in vitro invasion and migragation capacity of PC-3M-2B4 cells were tested by wound migration assay and in vitro invasion assay. Results After the transfec- tion with siRNA-LASS2/TMSG-1 for 24 hours, the expression level of LASS2/TMSG-1 protein decreased 65% than the blank control group, while the expression level of ATP6L mRNA increased 75% as compared with the blank control group. After PC-3M-2B4 cells was transfected with siRNA-LASS2/TMSG-1, its intracellular hydrogenion concentration significantly decreased, with significant differ- ences compared with the blank control group and nonsense siRNA group （ P 〈 0. 05）. And the proliferation, invasion and migration in vitro were significantly higher than the blank control group and nonsense siRNA group （P 〈 0. 05 ）. Conclusion siRNA-LASS2/ TMSG-1 can silence the expression of LASS2/TMSG-1 protein, and at the same time increase the expression of ATP6L mRNA （the key subunit of V-ATPase proton pump）. And the hydrogen ion concentration within the cell decreases after the silencing of LASS2/TMSG- 1, which indicates that LASS2/TMSG-1 may influence intracellular pH by interaction with ATP6L and lead to the change of tumor mi- cro-environment. All these further affect the biological behavior of tumor cells such as proliferation, invas

关 键 词：前列腺肿瘤 PC-3M-2B4 LASS2 TMSG-1 SIRNA 

分 类 号：R737.25[医药卫生—肿瘤]