miR-183对滑膜肉瘤细胞株SW982转移潜能及EGR1表达影响研究
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摘要
目的探讨微小RNA-183(miRNA-183)对人滑膜肉瘤细胞株SW982转移潜能的影响,并分析miR-183对早期生长反应因子1(early growth response,EGR1)表达影响的研究。方法培养人滑膜肉瘤细胞株SW982细胞,采用脂质体转染方法转染miR-183mimics、inhibitor和negative control,并分为miR-183 mimics模拟物组(mimics组),miR-183inhibitor抑制剂组(inhibitor组),miR-183negative control阴性对照组(NC)以及正常对照组(normal)。流式细胞术检测各组细胞转染效率;MTT法检测各组细胞转染前后细胞增殖率的改变;Transwell实验检验各组转染前后SW982细胞迁移和侵袭能力的变化;蛋白质印迹法检测各组转染前后EGR1蛋白表达量的变化;双荧光素酶报告基因实验验证EGR1是否为miR-183靶基因。结果转染miR-183mimics、inhibitor和negative control后,SW982细胞中miR-183的相对含量明显改变;与正常对照组(1.16±0.52)相比,mimics组(75.30±9.90)miR-183含量明显增加(P<0.001),inhibitor组(0.22±0.09)miR-183含量明显降低(P<0.001),NC组(1.06±0.98)miR-183含量无明显变化,P=0.637。MTT法测得转染后24、48、72和96h,SW982细胞增殖率无明显变化。Transwell实验结果显示,在迁移实验中,mimics组和inhibitor组穿膜细胞数分别为(108.61±4.94)和(54.36±4.03)个,P<0.001。在侵袭实验中,mimics组和inhibitor组穿膜细胞数分别为(79.13±5.77)和(20.87±3.17)个,P<0.001。在EGR1表达分析中,转染48h后mimics组中EGR1的相对表达量(286.59±32.77)较正常对照组(373.00±38.40)明显降低,P<0.001;inhibitor组中EGR1的相对表达量(496.93±42.61)较正常对照组明显增高,P=0.012;双荧光素酶报告基因实验表明,miR-183特异性靶向EGR1基因。结论转染miR-183mimics后,miR-183表达增加,SW982细胞转移潜能增强,EGR1表达降低;转染miR-183inhibitor后,miR-183表达降低,SW982细胞转移潜能降低,EGR1表达增加。EGR1可作为miR-183的潜在靶点。
OBJECTIVE To investigate the influence on metastasis potential of synovial sarcoma via regulating the expression of microRNA-183 in human synovial sarcoma cells line SW982 and analyze the effects of miR-183 on EGR1.METHODS We cultured the human synovial sarcoma cells line SW982.We changed the expression of miR-183 by transfecting the miR-183 mimics,inhibitor and NC to SW982 cells line,and divided the cells into four groups:miR-183 mimics group,miR-183 inhibitor group,miR-183 negative control group and normal group.Transfection efficiency was detected by RT-PCR and FACS.MTT was used to detect the cell proliferation after transfection.Transwell model was used to exam the change of migratory and invasive abilities after transfection.Western blot was used to analyze the expression of EGR1 after transfection.Dual-luciferase report assay was used to identify whether EGR1 was the direct target of miR-183.RESULTS Real-time PCR analysis revealed that the expression of miR-183 was higher in mimics group(75.30±9.90)compared to the normal groups(1.16±0.52,P<0.001).The miR-183 expression in inhibitor(0.22±0.09)was lower than that of normal groups(P<0.001).In normal and negative control(NC)groups(1.06±0.98),the result was similar(P=0.637).Twenty-four hours,48,72 and 96hafter transfection,the proliferation of SW982 cells had no significant effect.Transwell analysis revealed that in migratory study the number of SW982 cells penetrated the membrane in mimics and inhibitors group were(108.61±4.94)and(54.36±4.03),respectively(P<0.001).In invasive study,the number of SW982 cells penetrated this membrane in mimics and inhibitors group were(79.13±5.77)and(20.87±3.17),respectively(P<0.001).The expression of EGR1 was detected by western blotting,the result showed that in mimics group(286.59±32.77)the relative expression of EGR1 was dramatically decreased compared with inhibitor group(373.00±38.40,P<0.001).In inhibitor group(496.93±42.61)the expression of EGR1 was dramatically increased compared with mimics group(P=0.012).We further identified EGR1 as a direct target of miR-183 by the dual-luciferase report assay.CONCLUSIONS After the transfection of miR-183 mimics,the expression of miR-183 was increased and the metastasis potential of SW982 cells was increased while the expression of EGR1 reduced.After the transfection of miR-183 inhibitor,the expression of miR-183 was reduced and the metastasis potential of SW982 cells was reduced while the expression of EGR1 increased.EGR1is a potential target of miR-183.
OBJECTIVE To investigate the influence on metastasis potential of synovial sarcoma via regulating the expression of microRNA-183 in human synovial sarcoma cells line SW982 and analyze the effects of miR-183 on EGR1.METHODS We cultured the human synovial sarcoma cells line SW982.We changed the expression of miR-183 by transfecting the miR-183 mimics,inhibitor and NC to SW982 cells line,and divided the cells into four groups:miR-183 mimics group,miR-183 inhibitor group,miR-183 negative control group and normal group.Transfection efficiency was detected by RT-PCR and FACS.MTT was used to detect the cell proliferation after transfection.Transwell model was used to exam the change of migratory and invasive abilities after transfection.Western blot was used to analyze the expression of EGR1 after transfection.Dual-luciferase report assay was used to identify whether EGR1 was the direct target of miR-183.RESULTS Real-time PCR analysis revealed that the expression of miR-183 was higher in mimics group(75.30±9.90)compared to the normal groups(1.16±0.52,P<0.001).The miR-183 expression in inhibitor(0.22±0.09)was lower than that of normal groups(P<0.001).In normal and negative control(NC)groups(1.06±0.98),the result was similar(P=0.637).Twenty-four hours,48,72 and 96hafter transfection,the proliferation of SW982 cells had no significant effect.Transwell analysis revealed that in migratory study the number of SW982 cells penetrated the membrane in mimics and inhibitors group were(108.61±4.94)and(54.36±4.03),respectively(P<0.001).In invasive study,the number of SW982 cells penetrated this membrane in mimics and inhibitors group were(79.13±5.77)and(20.87±3.17),respectively(P<0.001).The expression of EGR1 was detected by western blotting,the result showed that in mimics group(286.59±32.77)the relative expression of EGR1 was dramatically decreased compared with inhibitor group(373.00±38.40,P<0.001).In inhibitor group(496.93±42.61)the expression of EGR1 was dramatically increased compared with mimics group(P=0.012).We further identified EGR1 as a direct target of miR-183 by the dual-luciferase report assay.CONCLUSIONS After the transfection of miR-183 mimics,the expression of miR-183 was increased and the metastasis potential of SW982 cells was increased while the expression of EGR1 reduced.After the transfection of miR-183 inhibitor,the expression of miR-183 was reduced and the metastasis potential of SW982 cells was reduced while the expression of EGR1 increased.EGR1is a potential target of miR-183.
引文
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[13]Wang J,Wang X,Li Z,et al.MicroRNA‐183suppresses retinoblastoma cell growth,invasion and migration by targeting LRP6[J].FEBS J,2014,281(5):1355-1365.
[14]魏明辉,张宗敏,李琳,等.喉鳞癌及癌旁正常组织miRNA差异表达分析[J].中华肿瘤防治杂志,2014,21(7):506-508,513.
[15]Sarver AL,Li L,Subramanian S.MicroRNA miR-183functions as an oncogene by targeting the transcription factor EGR1and promoting tumor cell migration[J].Cancer Res,2010,70(23):9570-9580.
[16]Wang G,Mao W,Zheng S.MicroRNA-183regulates Ezrin expression in lung cancer cells[J].FEBS letters,2008,582(25):3663-3668.
[17]Spaapen F,van den Akker GG,Caron MM,et al.The Immediate Early Gene Product EGR1and Polycomb Group Proteins Interact in Epigenetic Programming during Chondrogenesis[J].PLoS One,2013,8(3):e58083.
[18]王臻,王佳玉,徐海荣,等.肢体软组织肉瘤临床诊疗专家共识的解读[J].临床肿瘤学杂志,2014,32(7):637-648.
[19]Torres-Mora JM,Fritchie KJ,Robinson SI,et al.Molecular Genetics of Soft-Tissue Sarcomas:A Brief Overview for Clinical Oncologists[J].Cancer J,2014,20(Issue):73-79.
[20]Marino ALF,Evangelista AF,Vieira RA,et al.MicroRNA expression as risk biomarker of breast cancer metastasis:apilot retrospective case-cohort study[J].BMC Cancer,2014,14(1):739.
[2]Moskwa P,Buffa FM,Pan Y,et al.miR-182-mediated downregulation of BRCA1impacts DNA repair and sensitivity to PARP inhibitors[J].Mol Cell,2011,41(2):210-220.
[3]Renner M,Czwan E,Hartmann W,et al.MicroRNA profiling of primary high-grade soft tissue sarcomas[J].Genes Chromosomes Cancer,2012,51(11):982-996.
[4]Li J,Fu H,Xu C,et al.miR-183inhibits TGF-β1-induced apoptosis by downregulation of PDCD4expression in human hepatocellular carcinoma cells[J].BMC Cancer,2010,10(1):354.
[5]Azrak SS,Ginel-Picardo A,Drosten M,et al.Reversible,interrelated mRNA and miRNA expression patterns in the transcriptome of Rasless fibroblasts:functional and mechanistic implications[J].BMC Genomics,2013,14(1):731.
[6]Ren LH,Chen WX,Li S,et al.MicroRNA-183promotes proliferation and invasion in oesophageal squamous cell carcinoma by targeting programmed cell death 4[J].J Cancer,2014,111(10):2003-2013.
[7]Zhao DY,Jacobs KM,Karvas RM,et al.The Egr1transcription factor contributes to radiation-induced apoptosis in the mouse hippocampus and intestinal crypts[J].Cancer Res,2014,74(19Supplement):3941.
[8]Fang L,Min L,Lin Y,et al.Downregulation of stathmin expression is mediated directly by Egr1and associated with p53activity in lung cancer cell line A549[J].Cell Sign,2010,22(1):166-173.
[9]Su L,Sampaio AV,Jones KB,et al.Deconstruction of the SS18-SSX fusion oncoprotein complex:insights into disease etiology and therapeutics[J].Cancer cell,2012,21(3):333-347.
[10]Su L,Cheng H,Sampaio AV,et al.EGR1reactivation by histone deacetylase inhibitors promotes synovial sarcoma cell death through the PTEN tumor suppressor[J].Oncogene,2010,29(30):4352-4361.
[11]葛丽娜,尚官敏,李英,等.原发性肺滑膜肉瘤伴顽固低血糖症一例报告并文献复习[J].中华肿瘤防治杂志,2013,20(16):1284-1286.
[12]Tanaka H,Sasayama T,Tanaka K,et al.MicroRNA-183upregulates HIF-1αby targeting isocitrate dehydrogenase 2(IDH2)in glioma cells[J].J Neuro Oncol,2013,111(3):273-283.
[13]Wang J,Wang X,Li Z,et al.MicroRNA‐183suppresses retinoblastoma cell growth,invasion and migration by targeting LRP6[J].FEBS J,2014,281(5):1355-1365.
[14]魏明辉,张宗敏,李琳,等.喉鳞癌及癌旁正常组织miRNA差异表达分析[J].中华肿瘤防治杂志,2014,21(7):506-508,513.
[15]Sarver AL,Li L,Subramanian S.MicroRNA miR-183functions as an oncogene by targeting the transcription factor EGR1and promoting tumor cell migration[J].Cancer Res,2010,70(23):9570-9580.
[16]Wang G,Mao W,Zheng S.MicroRNA-183regulates Ezrin expression in lung cancer cells[J].FEBS letters,2008,582(25):3663-3668.
[17]Spaapen F,van den Akker GG,Caron MM,et al.The Immediate Early Gene Product EGR1and Polycomb Group Proteins Interact in Epigenetic Programming during Chondrogenesis[J].PLoS One,2013,8(3):e58083.
[18]王臻,王佳玉,徐海荣,等.肢体软组织肉瘤临床诊疗专家共识的解读[J].临床肿瘤学杂志,2014,32(7):637-648.
[19]Torres-Mora JM,Fritchie KJ,Robinson SI,et al.Molecular Genetics of Soft-Tissue Sarcomas:A Brief Overview for Clinical Oncologists[J].Cancer J,2014,20(Issue):73-79.
[20]Marino ALF,Evangelista AF,Vieira RA,et al.MicroRNA expression as risk biomarker of breast cancer metastasis:apilot retrospective case-cohort study[J].BMC Cancer,2014,14(1):739.