PIB5PA对人黑素瘤细胞迁移和侵袭能力的影响

发布时间：2019-03-29 12:21

【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(P0.05),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(P0.05)。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。
[Abstract]:Aim: to investigate the effects of phosphatidylinositol 4,5-diphosphate 5-phosphatase A (phosphatidylinositol 4, 5-bisphosphate 5-phosphatase A, PIB5PA on migration and invasion of human melanoma Mel-FH cells. Methods: the recombinant eukaryotic expression vector pF-5xUAS-SV40-PIB5PA, carrying 4-hydroxytamoxifen (4-hydroxytamoxifen)-regulated gene expression system was constructed and infected with human melanoma Mel-FH cells. Stable Mel-FH.PIB5PA subclone cells were successfully obtained by double screening of puromycin and hygromycin B. The optimal concentration and time of expression of PIB5PA protein in Mel-FH.PIB5PA cells induced by 4-OHT were detected by Western blot. The migration and invasion ability of Mel-FH.PIB5PA cells were detected by cell scratch test and Transwell chamber assay. Western blot assay was used to detect phosphorylated protein kinase B (phospho-protein kinase B), phosphorylated focal adhesion kinase (phospho-focal adhesion kinase,p-FAK), matrix metalloproteinases-2 (matrix metalloproteinase-2,) in Mel-FH.PIB5PA cells. Expression of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (tissue inhibitor of matrix metalloproteinase-1,TIMP-1 (TIMP-1) and TIMP-2 protein. Results: the overexpression of PIB5PA protein was stably induced in melanoma MelFH.PIB5PA cells treated with 10 nmol/L 4-OHT for 16 h. Compared with non-4-OHT-treated Mel-FH.PIB5PA cells, the over-expression of PIB5PA significantly inhibited the migration and invasion ability of Mel-FH.PIB5PA cells (P0.05), and down-regulated the migration and invasiveness of Mel-FH.PIB5PA cells (P < 0.05), and down-regulated the expression of MMP-2, MMP-9, TIMP-1. Expression level of TIMP-2,MMP-2/TIMP-2 and MMP-9/TIMP-1 protein (P0.05). Conclusion: exogenous overexpression of PIB5PA can significantly inhibit the migration and invasion of human melanoma Mel-FH cells in vitro, and its mechanism may be related to the down-regulation of protein kinase AKT and FAK activities. Thus, the relative expression level of MMP-2/TIMP-2 and MMP-9/TIMP-1 was down-regulated.
【作者单位】：安徽医科大学免疫学教研室;澳大利亚纽卡斯尔大学肿瘤与免疫实验室;
【基金】：国家自然科学基金资助项目(编号:81301738)
【分类号】：R739.5

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