摘要
目的 :探讨磷脂酰肌醇4,5-二磷酸5磷酸酶A(phosphatidylinositol 4,5-bisphosphate 5-phosphatase A,PIB5PA)对人黑素瘤Mel-FH细胞迁移和侵袭能力的影响。方法 :构建携带4-羟基三苯氧胺(4-hydroxytamoxifen,4-OHT)可调控基因表达系统的重组真核表达载体pF-5xUAS-SV40-PIB5PA,并将其感染人黑素瘤Mel-FH细胞,经嘌呤霉素和潮霉素B的双重筛选,成功获得稳定感染的Mel-FH.PIB5PA亚克隆细胞。应用蛋白质印迹法检测4-OHT诱导Mel-FH.PIB5PA细胞表达PIB5PA蛋白的最佳浓度和最适时间,应用细胞划痕实验和Transwell小室法检测Mel-FH.PIB5PA细胞的迁移和侵袭能力,蛋白质印迹法检测Mel-FH.PIB5PA细胞中磷酸化蛋白激酶B(phospho-protein kinase B,p-PKB,又称p-AKT)、磷酸化黏着斑激酶(phospho-focal adhesion kinase,p-FAK)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、MMP-9、组织金属蛋白酶抑制剂-1(tissue inhibitor of matrix metalloproteinase-1,TIMP-1)和TIMP-2蛋白的表达。结果 :10 nmol/L 4-OHT处理黑素瘤MelFH.PIB5PA细胞16 h后可稳定诱导PIB5PA蛋白过表达。与未经4-OHT处理的Mel-FH.PIB5PA细胞比较,诱导PIB5PA过表达能显著抑制Mel-FH.PIB5PA细胞的迁移和侵袭能力(P<0.05),并下调p-AKT、p-FAK、MMP-2、MMP-9、TIMP-1、TIMP-2、MMP-2/TIMP-2和MMP-9/TIMP-1蛋白的表达水平(P<0.05)。结论 :外源性过表达PIB5PA可明显抑制人黑素瘤Mel-FH细胞的体外迁移和侵袭能力,其机制可能与蛋白激酶AKT和FAK的活性下调,从而下调MMP-2/TIMP-2和MMP-9/TIMP-1的相对表达水平有关。
Objective： To investigate the effects of phosphatidylinositol 4, 5-bisphosphate 5-phosphatase A （PIB5PA） on the migration and invasion of human melanoma MeI-FH cells. Methods： Eukaryotic expression recombinant vector pF-5xUAS-SV40-PIB5PA which carried a 4-hydroxytamoxifen （4-OHT）-inducible lentiviral expression system was constructed and infected into melanoma MeI-FH cells. MeI-FH,PIBSPA cells were successfully obtained via dual antibiotic selection of puromycin and hygromycin B. The optimum concentration and acting time of 4-OHT on the expression of PIB5PA of Mel- FH.PIB5PA cells were detected by Western blotting. The migration and invasion of MeI-FH.PIB5PA cells were determined by wound healing and Transwell chamber assay, respectively. The expression levels of phospho-protein kinase B （p-AKT）, phospho-focal adhesion kinase （p-FAK）, matrix metalloproteinase-2 （MMP-2）, MMP-9, tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） and TIMP-2 were measured by Western blotting. Results： The addition of 10 nmol/L 4-OHT for 16 h readily induced PIB5PA over- expression in MeI-FH.PIB5PA cells. The exogenous expression of PIB5PA significantly inhibited migration and invasion of MeI-FH.PIB5PA cells as compared with MeI-FH.PIB5PA cells without treatment of 4-OHT （P 〈 0.05）, and reduced the expression levels of p-AKT, p-FAK, MMP-2, MMP-9, TIMP-1, TIMP-2, MMP-2/ TIMP-2 and MMP-9/TIMP-1 proteins （all P 〈 0.05）. Conclusion： Over-expression of PIB5PA can inhibit the abilities of migration and invasion of human melanoma MeI-FH cells in vitro, which may be associated with inactivity of AKT and FAK, and down-regulation of the relative expression levels of MMP-2/TIMP-2 and MMP-9/TIMP-1.
出处
《肿瘤》
CAS
CSCD
北大核心
2014年第6期487-493,共7页
Tumor
基金
国家自然科学基金资助项目(编号:81301738)
