摘要
目的探讨罗格列酮(RG)对舒尼替尼(SU)耐药肾癌(RCC)细胞增殖和凋亡的影响及作用机制。方法采用递增SU浓度法(1、3、5、10、15μmol/L)培养建立耐药RCC细胞株(786-O/SR和A498/SR),转染短发夹RNA(shRNA)静默过氧化物酶体增殖物激活受体γ(PPARγ)表达。实验分为空白组(不加药物),sh(-)组(未转染shRNA)、shPPARγ组(转染shPPARγ)和shNC组(转染阴性对照),后3组给予30μmol/L RG。使用5-乙炔基-2’脱氧尿苷(EdU)染色、Transwell小室法和划痕实验分别检测RG对耐药细胞增殖、侵袭、迁移能力的影响。流式细胞术分析RG对细胞周期和细胞凋亡的影响,蛋白质印迹法(Western blot)检测视网膜母细胞瘤蛋白(pRb)、细胞周期素D1(Cyclin D1)、周期蛋白依赖激酶4和6(CDK4、CDK6)、周期蛋白依赖激酶抑制因子21和27(p21、p27)的表达。组间比较采用单因素方差分析。结果786-O/SR和A498/SR细胞sh(-)组、shPPARγ组、shNC组的EdU细胞阳性率[(23.0±2.0)%、(27.5±5.1)%;(31.0±2.2)%、(38.6±2.5)%;(22.3±3.6)%、(25.3±2.4)%比(52.0±3.6)%、(55.3±6.8)%,F=73.457、27.212,P<0.01];侵袭细胞数[(47.0±7.8)、(72.0±9.0)个;(90.6±11.2)、(104.0±9.6)个;(54.0±13.5)、(66.0±7.5)个比(129.6±11.7)、(169.6±18.1)个,F=34.236、48.367,P<0.01];细胞迁移率[(25.6±5.7)%、(25.7±2.1)%;(39.9±5.8)%、(38.6±5.4)%;(25.3±4.7)%、(28.8±3.9)%比(59.3±11.4)%、(63.5±8.7)%,F=13.557、29.859,P<0.01]均低于空白组,其中shPPARγ组均高于sh(-)组和shNC组。G1期细胞比例sh(-)组高于空白组[(69.4±1.3)%、(73.1±2.2)%比(50.3±2.3)%、(52.0±3.6)%,F=34.815、22.033,P<0.01];S期细胞比例sh(-)组低于空白组[(16.2±1.1)%、(14.1±1.7)%比(34.4±2.1)%、(33.0±1.4)%,F=91.784、58.237,P<0.01]。sh(-)组pRb(0.17±0.2、0.14±0.09比0.37±0.02、0.32±0.02,F=71.950、83.872,P<0.01)、Cyclin D1(0.21±0.09、0.21±0.02比0.45±0.06、0.65±0.04,F=20.865、48.571,P<0.01)、CDK4(0.34±0.02、0.27±0.02比0.53±0.03、0.47±0.03,F=46.726、72.617,P<0.01)、CDK6蛋白表达量(0.38±0.01、0.25±0.08比0.59±0.02、0.44±0.03,F=40.428、55.998,P<0.01)低于空白组,而p21(0.57±0.05、0.41±0.03比0.17±0.02、0.17±0.02,F=80.713、77.589,P<0.01)、p27蛋白表达量(0.67±0.02、0.50±0.02比0.23±0.03、0.16±0.02,F=35.688、69.329,P<0.01)高于空白组。786-O/SR细胞凋亡率空白组低于sh(-)组[(10.2±1.7)%比(25.5±2.1)%,F=35.768,P<0.01],RG对A498/SR细胞凋亡无影响。结论RG由PPARγ依赖及非依赖途径共同参与调节抑制SU耐药RCC细胞的增殖和侵袭。
Objective To investigate the effects of peroxisome proliferator-activated receptorγ(PPARγ)ligand rosiglitazone(RG)on proliferation and apoptosis of sunitinib-resistant renal carcinoma(RCC)cells and explore its mechanism.Methods SU resistant RCC cell lines(786-O/SR and A498/SR)were established by increasing concentration culture of SU(1,3,5,10 and 15μmol/L),then transfected with short hairpin RNA(shRNA)to silence PPARγexpression.The experiment was divided into blank group(no drug added),sh(-)group(no shRNA transfected),shPPARγgroup(transfected shPPARγ)and shNC group(transfected negative control).The last three groups were given 30μmol/L RG.The effects of RG on the proliferation,invasion and migration of drug-resistant cells were determined by 5-acetyne-2′deoxyuridine(EdU)staining,Transwell cell assay and scratch assay,respectively.The effects of RG on cell cycle and apoptosis were analyzed by flow cytometry.The expressions of retinoblastoma protein(pRb),cyclin D1(Cyclin D1),cyclin dependent kinases 4 and 6(CDK4,CDK6),and cyclin dependent kinase inhibitors 21 and 27(p21,p27)were detected by Western blotting.One-way analysis of variance was used for comparison between groups.Results For the sh(-),shPPARγand shNC groups of 786-O/SR and A498/SR cells,the positive rates of EdU cells[(23.0±2.0)%,(27.5±5.1)%;(31.0±2.2)%,(38.6±2.5)%;(22.3±3.6)%,(25.3±2.4)%vs.(52.0±3.6)%,(55.3±6.8)%,F=73.457,27.212,P<0.01],the number of invasive cells[(47.0±7.8),(72.0±9.0)cells;(90.6±11.2),(104.0±9.6)cells;(54.0±13.5),(66.0±7.5)cells vs.(129.6±11.7),(169.6±18.1)cells,F=34.236,48.367,P<0.01],the cell migration rate[(25.6±5.7)%,(25.7±2.1)%;(39.9±5.8)%,(38.6±5.4)%;(25.3±4.7)%,(28.8±3.9)%vs.(59.3±11.4)%,(63.5±8.7)%,F=13.557,29.859,P<0.01]were all lower than that of blank group,and shPPARγgroups were all higher than sh(-)groups and shNC groups.For sh(-)group,the proportion of G1 phase cells was higher than blank group[(69.4±1.3)%,(73.1±2.2)%vs.(50.3±2.3)%,(52.0±3.6)%,F=34.815,22.033,P<0.01],and the proportion of S-phase cells was lower than blank group[(16.2±1.1)%,(14.1±1.7)%vs.(34.4±2.1)%,(33.0±1.4)%,F=91.784,58.237,P<0.01].The protein expression level of pRb(0.17±0.2,0.14±0.09 vs.0.37±0.02,0.32±0.02,F=71.950,83.872,P<0.01),Cyclin D1(0.21±0.09,0.21±0.02 vs.0.45±0.06,0.65±0.04,F=20.865,48.571,P<0.01),CDK4(0.34±0.02,0.27±0.02 vs.0.53±0.03,0.47±0.03,F=46.726,72.617,P<0.01),CDK6(0.38±0.01,0.25±0.08 vs.0.59±0.02,0.44±0.03,F=40.428,55.998,P<0.01)of sh(-)group were lower than that of blank group,while p21(0.57±0.05,0.41±0.03 vs.0.17±0.02,0.17±0.02,F=80.713,77.589,P<0.01),p27(0.67±0.02,0.50±0.02 vs.0.23±0.03,0.16±0.02,F=35.688,69.329,P<0.01)proteins were higher than that of blank group.The apoptosis rate of 786-O/SR cells in blank group was lower than that in sh(-)group[(10.2±1.7)%vs.(25.5±2.1)%,F=35.768,P<0.01],but RG had no effect on A498/SR cell apoptosis.Conclusion RG inhibits the proliferation and invasion of SU resistant RCC cells by PPARγ-dependent and independent pathways.
作者
杨风光
许清江
魏永宝
彭俊铭
张弛
陈平舟
阮君山
Yang Fengguang;Xu Qingjiang;Wei Yongbao;Peng Junming;Zhang Chi;Chen Pingzhou;Ruan Junshan(Department of Urology,Fujian Provincial Hospital of Fujian Provincial College of Fujian Medical University,Fuzhou 350001,China)
出处
《中华实验外科杂志》
CAS
北大核心
2023年第10期2051-2054,共4页
Chinese Journal of Experimental Surgery
基金
福建省自然科学基金面上项目(2019J01181)。
关键词
肾细胞癌
过氧化酶增殖物激活受体γ
增殖
舒尼替尼耐药
Renal cell carcinoma
Peroxisome proliferator activated receptor-γ
Proliferation
Sunitinib resistance
