摘要
目的探讨基质硬度对骨肉瘤细胞侵袭的影响及分子机制。方法按照不同配比合成硬度差异聚丙烯酰胺水凝胶,并通过万能力学测试仪检测确认其基质硬度。通过光镜观察不同基质硬度培养条件下细胞形态差异。通过免疫荧光检测不同硬度条件下细胞骨架排列和心肌蛋白相关转录因子-A(MRTF-A)表达及亚细胞分布。通过Transwell侵袭实验观察基质硬度及MRTF-A抑制剂CCG203971对MG-63细胞侵袭的影响。应用实时荧光定量聚合酶联反应(RT-qPCR)和蛋白印迹法(Western blot)的方法检测基质金属蛋白酶(MMP)-2和MMP-9 mRNA和蛋白的表达。计量资料的两两比较采用t检验。结果成功构建不同硬度聚丙烯酰胺水凝胶。高硬度组细胞更倾向于铺展和梭形,肌动蛋白的聚合更明显。高硬度组MG-63细胞侵袭能力明显高于低硬度组[(247.6±25.40)个比(130.3±23.3)个,t=-11.820,P<0.01],并促进MRTF-A核转位。而MRTF-A抑制剂CCG-203971干预后硬基质组穿膜细胞数低于无干预硬基质组[(140.5±21.7)比(247.6±25.4)个,t=11.100,P<0.01]。硬基质组MG-63细胞较软基质组MMP-2和MMP-9 RNA及蛋白水平表达均显著增加(MMP-2 RNA水平:0.89±0.02、0.33±0.01,t=22.450,P<0.01;MMP-9 RNA水平:0.92±0.03、0.41±0.02,t=19.171,P<0.01;MMP-2蛋白水平:0.63±0.04、0.31±0.03,t=7.942,P<0.05;MMP-9蛋白水平:0.69±0.06、0.41±0.02,t=6.822,P<0.05),而MRTF-A抑制剂CCG-203971可以显著阻断基质硬度诱导的MMP-2和MMP-9 RNA及蛋白水平表达增加(MMP-2 RNA水平:0.73±0.03、0.29±0.02,t=6.954,P<0.01;MMP-9 RNA水平:0.91±0.04、0.38±0.03,t=29.362,P<0.01;MMP-2蛋白水平:0.66±0.03、0.29±0.02,t=8.342,P<0.05;MMP-9蛋白水平:0.72±0.02、0.38±0.01,t=7.362,P<0.05)。结论细胞外基质硬度增加通过影响MRTF-A转录调控影响骨肉瘤MG-63细胞侵袭能力。
Objective To investigate the effect of matrix stiffness on invasion of osteosarcoma MG-63 cells.Methods Polyacrylamide hydrogels with different stiffness were fabricated and tested by Instron universal testing machine.Morphological changes of MG-63 cells in different matrix stiffness were observed under light microscope.The remodeling of cytoskeleton and nuclear translocation of myocardin-related transcription factors A(MRTF-A)were confirmed by immunofluorescence.The Transwell invasion assay was performed to understand the effect of hydrogels on invasion of osteosarcoma cells.RT-qPCR and Western blotting were used to determine the expression of matrix metalloproteinase(MMP)-2 and MMP-9.Results Polyacrylamide hydrogel with various stiffness can be fabricated.Cell morphology can be affected by matrix stiffness.Actin polymerization and nuclear translocation of MRTF-A were regulated by matrix stiffness,which promoted invasion of osteosarcoma cells.In addition,the nuclear accumulation of MRTF-A was significantly inhibited by CCG203971,as well as the invasion effect and MMP-2/MMP-9 expression.Conclusion Matrix stiffness of the osteosarcoma microenvironment regulates invasion through translocation of MRTF-A,which may contribute to cancer progression.
作者
秦亮
廖晖
戴俊
刘洪娟
赵利波
梁爽
刘晓峰
Qin Liang;Liao Hui;Dai Jun;Liu Hongjuan;Zhao Libo;Liang Shuang;Liu Xiaofeng(Department of Orthopedics,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;Department of Orthopedics,the Second Affiliated Hospital of Soochow University,Suzhou 215000,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第8期1550-1553,共4页
Chinese Journal of Experimental Surgery
基金
湖北省科技计划项目(2019CFB412)。
关键词
基质硬度
骨肉瘤
侵袭
Matrix stiffness
Osteosarcoma
Invasion
