过表达肌球蛋白磷酸酶靶亚基1对前列腺癌细胞迁移侵袭能力的影响

Effect of overexpression of myosin phosphatase target subunit 1 on migration and invasion of prostate cancer cells

目的探讨前列腺癌细胞内肌球蛋白磷酸酶靶亚基1(MYPT1)的表达变化及其对侵袭转移能力的影响。方法设计合成过表达MYPT1基因的慢病毒质粒(pLenti6.3-MYPT1),转染前列腺癌PC3及DU145细胞48 h,设定对照组(转染对照pLenti6.3-Ctrl)和过表达组(转染pLenti6.3-MYPT1);采用细胞划痕和Transwell实验检测24 h及48 h细胞迁移和侵袭能力;免疫荧光(IF)染色检测细胞骨架变化;蛋白质印迹法(Western blot)和定量聚合酶链式反应(qPCR)检测细胞株(PC3、DU145)转染后MYPT1和上皮-间充质转化(EMT)相关分子标志物:N-钙黏蛋白(N-cadherin)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、锌指结构蛋白(Snail)和E盒结合锌指蛋白1(ZEB-1)的mRNA及蛋白相对表达水平。组间统计分析采用独立样本t检验。结果转染pLenti6.3-MYPT1后,PC3和DU145细胞内MYPT1相对表达水平明显上调,差有统计学意义(Western blot:0.834±0.055、0.849±0.044比0.487±0.026、0.526±0.045,t=8.098,7.212,P<0.01;qPCR:3.134±0.280、2.091±0.099比0.967±0.034、1.019±0.150,t=10.860,8.449,P<0.01)。细胞划痕和Transwell实验显示转染过表达质粒后细胞迁移(t=8.342,6.776,P<0.01)和侵袭(t=11.670,11.870,P<0.001)能力明显减弱。免疫荧光染色表明,与对照组比较,过表达MYPT1的细胞表面变光滑,丝状伪足和片状伪足减少,F-肌动蛋白(F-actin)从细胞质富集到细胞膜,着色变浅。两细胞株过表达组N-cadherin、MMP-2、MMP-9、Snail、ZEB-1显著低于对照组(P<0.05)。结论过表达MYPT1能抑制前列腺癌细胞的迁移侵袭能力,其作用机制可能与细胞骨架重构及EMT相关。

Objective To explore the effect of overexpression of myosin phosphatase target subunit 1(MYPT1)on the invasion and metastasis of prostate cancer cells.Methods MYPT1-overexpressed lentiviral vector(pLenti6.3-MYPT1)was designed and synthesized and transfected into PC3 and DU145 cells.The cells were divided into two groups:overexpression group(transfected with pLenti6.3-MYPT1)and control group(transfected with pLenti6.3-Ctrl).The changes of migration and invasion capacity were tested by cell scratch test and Transwell invasion test.The cytoskeleton changes of each group were tested by immunofluorescence staining.MYPT1 and molecular markers associated with epithelial-mesenchymal transition[EMT(N-cadherin,matrix metalloproteinase(MMP)-2,MMP-9,Snail,ZEB-1)]were detected by quantitative polymerase chain reaction(qPCR)and Western blotting.The t test was used for analysis between the two groups.Results Compared with control group,the expression of MYPT1 was obviously increased(Western blotting:0.834±0.055,0.849±0.044 vs.0.487±0.026,0.526±0.045,t=8.098,7.212,P<0.01;qPCR:3.134±0.280,2.091±0.099 vs.0.967±0.034,1.019±0.150,t=10.860,8.449,P<0.01).The cell scratch test and Transwell test revealed that the migration(t=8.342,6.776,P<0.01)and invasion(t=11.670,11.870,P<0.001)capacity of prostate cancer cells was notably decreased after transfecting with pLenti6.3-MYPT1.The immunofluorescence(IF)staining showed that the number of filamentous pseudopodia and patellar pseudopodia decreased,F-actin in the cytoskeleton was enriched from the cytoplasm to the cell membrane,and the color in the inclusion was relatively light after transfection.The qPCR and Western blotting exhibited that the EMT-associated markers,including N-cadherin,MMP-2,MMP-9,Snail,ZEB-1,were significantly decreased after MYPT1 overexpression(P<0.05).Conclusion Overexpression of MYPT1 inhibits the migration and invasion ability of prostate cancer cells,probably related to the cytoskeleton remodeling and the EMT process.