摘要
目的:采用RNA干涉技术沉默液泡型ATP酶c亚基ATP6V0C的表达,探索ATP6V0C在前列腺癌侵袭中的分子作用机制,并研究其与LASS2/TMSG1的关系。方法与结果:ATP6V0C在高转移潜能人前列腺癌细胞系(PC-3M-1E8和PC-3M)中的表达明显高于低转移潜能人前列腺癌细胞系(PC-3M-2B4和PC-3),选择ATP6V0C表达量最高的PC-3M-1E8细胞进行基因沉默实验,发现ATP6V0C的siRNA转染PC-3M-1E8后,其基质金属蛋白酶2(matrix metalloprotein-2,MMP-2)和基质金属蛋白酶9(MMP-9)蛋白的表达量及MMP-2的分泌量均无明显变化,但上清液中MMP-9的活性明显减弱,与空白对照组及阴性对照组相比,差异有统计学意义(P<0.05);干扰组细胞外氢离子浓度及液泡型ATPase的活性明显降低(P<0.05);细胞划痕修复实验及transwell体外侵袭实验结果显示ATP6V0C siRNA干扰组细胞的体外迁移及侵袭能力明显减弱(P<0.05)。PC-3M-1E8细胞转染ATP6V0C siRNA后,LASS2/TMSG1的表达明显减弱(P<0.05)。免疫荧光双重染色结果显示ATP6V0C蛋白与LASS2/TMSG1共定位于胞浆和胞膜,干扰组共定位信号与对照组相比明显减弱。结论:特异性siRNA沉默ATP6V0C能抑制人前列腺癌细胞的迁移和侵袭能力,其机制与ATP6V0C沉默后抑制V-ATPase的活性,使细胞外氢离子浓度降低,抑制MMP-9的激活,从而影响细胞外基质的降解和重塑有关。靶向siRNA干扰ATP6V0C的表达后,可能通过反馈调节机制抑制LASS2/TMSG1的表达,但其具体分子机制尚待进一步研究。
Objective： Vacuolar ATPase（ V-ATPase） was found within the membranes and internal organelles of a vast array of eukaryotic cells,and was related to various kinds of highly metastatic tumors.LASS2/TMSG1 gene was a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3 M in 1999 by our laboratory. It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of V-ATPase（ ATP6 V0 C）. In this study,To use RNA interference to suppress the expression of ATP6 V0 C and try to further investigate the molecular mechanism of ATP6 V0 C in tumor metastasis and its relationship with LASS2/TMSG1 gene. Methods and Results： The expression level of ATP6 V0 C mRNA and protein in high metastatic potential prostate cancer cell lines（ PC-3 M-1 E8 and PC-3 M） was significantly higher than that in low metastatic potential prostate cancer cell lines（ PC-3 M-2 B4 and PC-3）,the expression level in PC-3 M-1 E8 being the highest. Follow-up tests selected PC-3 M-1 E8 cells for gene silencing. The expression and secretion of MMP-2 and the expression of MMP-9 in ATP6 V0 C siRNA transfected PC-3 M-1 E8 cells displayed no obvious change,but the activity of secreted MMP-9 was abated noticeably compared with the controls（ P 〈 0. 05）. Extracellular hydrogen ion concentration and V-ATPase activity in interference group were both reduced significantly compared with the controls（ P 〈 0. 05）. The migration and invasion capacity of ATP6 V0 C siRNA interfered cells in vitro were diminished significantly compared with the controls（ P 〈 0. 05）. Furthermore,a dramatic reduction of LASS2/TMSG1 mRNA and protein level after transfection of siRNA in PC-3 M-1 E8 cells was discovered（ P 〈 0. 05）. Confocal immunofluorescence showed a vast co-localization of ATP6 V0 C protein and LASS2/TMSG1 protein in plasma and membrane. The co-localization signals of control group were much stronger than those of interference group. Conclusion： Specific siRNA silencing of ATP6 V0 C gene inhibits the invasion of human prostate cancer cells in vitro by mechanism of inhibiting V-ATPase activity and then reducing the extracellular hydrogen ion concentration,inhibiting MMP-9 activation and affecting ECM degradation and reconstruction. Meanwhile,ATP6 V0 C and LASS2/TMSG1 have interaction and it is likely that ATP6 V0 C functions as a feedback regulator of LASS2/TMSG1.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2017年第6期937-947,共11页
Journal of Peking University:Health Sciences
基金
国家自然科学基金(81572533)资助~~
