摘要
目的:探讨干扰线粒体关键蛋白亲环素D(CypD)对骨肉瘤MG63细胞增殖、迁移、凋亡等生物学行为的影响。方法:构建3种转染不同siRNA的MG63细胞系,分别为CypD-siRNA1、CypD-siRNA2和CypD-siRNA3组,采用qRT-PCR和Western blot检测3组细胞中CypD的表达水平,筛选用于后续实验的CypD-siRNA。将MG63细胞分为阴性对照组(转染siRNA-NC)和敲减组(转染CypD-siRNA2)。采用CCK-8法和平板克隆实验检测细胞的增殖能力,Transwell小室实验检测细胞的迁移能力;采用MitoSOX Red检测线粒体活性氧(mROS)水平,钙黄绿素-钴评价线粒体通透性转换孔的开放状态;采用流式细胞术检测细胞凋亡情况,Western blot法检测细胞迁移和凋亡相关蛋白的表达。结果:与CypD-siRNA1组和CypD-siRNA3组相比,CypD-siRNA2组中CypD mRNA和蛋白表达水平下降(P<0.05)。与阴性对照组相比,敲减组细胞克隆形成数和迁移数减少,细胞增殖能力下降,细胞凋亡率升高,N-cadherin和Vimentin蛋白表达水平降低(P<0.05),Bax/Bcl-2、Cleaved Caspase 3/Caspase 3以及E-cadherin和细胞色素C蛋白表达水平升高(P<0.05)。敲减组细胞mROS水平高于阴性对照组,而线粒体通透性转换孔开放程度低于阴性对照组(P<0.05)。结论:敲减CypD表达可上调MG63细胞氧化应激水平,抑制其增殖、迁移能力,并通过线粒体途径诱导其凋亡。
Aim:To investigate the effects of interference with the key mitochondrial protein cyclophilin D(CypD)on the proliferation,migration and apoptosis of osteosarcoma MG63 cells.Methods:Three MG63 cell lines transfected with different siRNA knockdowns of CypD were constructed,namely CypD-siRNA1,CypD-siRNA2 and CypD-siRNA3 groups,and the expression of CypD in the 3 groups was detected by qRT-PCR and Western blot,and CypD-siRNA was screened for subsequent experiments.MG63 cells were divided into negative control group(transfected with siRNA-NC)and knockdown group(transfected with CypD-siRNA2).The proliferation ability of MG63 cells was detected by CCK-8 assay and plate cloning assay,and the migration ability of MG63 cells was detected by Transwell chamber assay.MitoSOX Red was used to detect the content of mitochondrial reactive oxygen species,and calcein and cobalt were used to evaluate the open state of mitochondrial permeability transition pores.The apoptosis was detected by flow cytometry.The expressions of proteins related to migration and apoptosis of MG63 cells were detected by Western blot.Results:Compared with CypD-siRNA1 and CypD-siRNA3 group,the mRNA and protein expression levels of CypD in CypD-siRNA2 group were decreased(P<0.05).Compared with negative control group,the number of clones and migration cells and proliferation ability decreased,apoptosis rate increased,N-cadherin and Vimentin protein expression levels decreased(P<0.05),and Bax/Bcl-2,Cleaved Caspase 3/Caspase 3,E-cadherin and Cytochrome C protein expression levels increased(P<0.05).The level of mitochondrial reactive oxygen species in knockdown group was higher than that in negative control group,and the openness of mitochondrial permeability transition pore was lower than that in negative control group(P<0.05).Conclusion:CypD knockdown can up-regulate the oxidative stress level,inhibit proliferation and migration ability,and induce apoptosis of MG63 cells through mitochondrial pathway.
作者
刘一
夏德佳
赵柔
王旭彤
赵岳
马玉芳
王文军
张玲
LIU Yi;XIA Dejia;ZHAO Rou;WANG Xutong;ZHAO Yue;MA Yufang;WANG Wenjun;ZHANG Ling(Department of Maternal,Child and Adolescent Health,School of Public Health,North China University of Science and Technology,Tangshan,Hebei 063000;Department of Nursing for Maternal and Child,School of Nursing,Jining Medical University,Jining,Shandong 272000;Clinical laboratory,Jinan Great Master Diagnostics Co.Ltd,Jinan 250104;Department of Laboratory Medicine,the Affiliated Hospital of Jining Medical University,Jining,Shandong 272000;Department of Maternal,Child and Adolescent Health,School of Public Health,Binzhou Medical University,Yantai,Shandong 264003;Party and Government Office,Weifang Nursing Vocational College,Weifang,Shandong 262500)
出处
《郑州大学学报(医学版)》
CAS
北大核心
2024年第5期602-607,共6页
Journal of Zhengzhou University(Medical Sciences)
基金
山东省自然科学基金项目(ZR2021QH294)
济宁医学院贺林院士新医学临床转化工作站科研基金项目(JYHL2022MS02)。
关键词
骨肉瘤
亲环素D
线粒体通透性转换孔
生物学行为
MG63细胞
osteosarcoma
cyclophilin D
mitochondrial permeability transition pore
biological behavior
MG63 cell