X连锁核糖体S6激酶4在乳腺癌发生发展中的作用及其机制
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摘要
目的探讨X连锁核糖体S6激酶4(RSK4)在乳腺癌发生发展的作用及其机制。方法 (1)检测乳腺癌组织及癌旁正常组织、乳腺癌细胞(T47D、ZR-75-1、SK-BR-3、MDA-MB-231、MDA-MB-436、MCF-7)及乳腺正常细胞MCF-10A中RSK4 mRNA及蛋白的表达水平。(2)将MDA-MB-231细胞分为实验组、阴性对照组和空白组,其中实验组、阴性对照组分别转染过表达RSK4的慢病毒载体、不含RSK4的慢病毒载体,空白组未进行转染。检测3组细胞RSK4 mRNA及蛋白、蛋白激酶(AKT)、磷酸化AKT(p-AKT)、细胞外信号调节激酶(ERK)、磷酸化ERK(p-ERK)、E-钙黏蛋白和波形蛋白的表达水平,以及细胞迁移和侵袭能力。(3)检测MDA-MB-231细胞及去甲基化的MDA-MB-231细胞的甲基化程度,比较两种细胞RSK4 mRNA的表达水平。(4)将12只裸鼠随机分为实验组及对照组各6只,分别在裸鼠乳房脂肪垫中注射实验组、阴性对照组MDA-MB-231细胞混悬液,比较两组接种后1～6周的瘤体体积及6周后的瘤体重量。结果 (1)乳腺癌组织RSK4 mRNA及蛋白的表达水平均低于癌旁正常组织(均P <0. 05)。MCF-10A细胞的RSK4 mRNA及蛋白表达水平均高于6种乳腺癌细胞(均P <0. 05)。在6种乳腺癌细胞中,MCF-7细胞的RSK4 mRNA及蛋白表达水平最高,MDA-MB-231细胞的RSK4 mRNA及蛋白表达水平最低(均P <0. 05)。(2)与阴性对照组和空白组比较,实验组细胞的RSK4 mRNA及蛋白、E-钙黏蛋白表达水平升高,细胞迁移数、细胞侵袭数以及p-AKT、p-ERK、波形蛋白表达水平降低(均P <0. 05)。(3)去甲基化的MDA-MB-231细胞中RSK4 mRNA表达水平高于MDA-MB-231细胞(P <0. 05)。(4)在第3、4、5、6周,实验组裸鼠的体重均高于对照组(均P <0. 05),在第2、3、4、5、6周,实验组裸鼠的瘤体体积均小于对照组(均P <0. 05)。结论 RSK4乳腺癌的发生发展过程中起抑制作用,其在乳腺癌中低表达可能由其启动子异常甲基化引起的。RSK4可能通过AKT/ERK信号通路调节乳腺癌细胞的上皮间质转化过程。
Objective To investigate the role of X-linked ribosomal S6 kinase 4( RSK4) in the carcinogenesis and development of breast cancer and its mechanism. Methods( 1) Expression levels of RSK4 mRNA and protein in breast cancer tissues,adjacent normal tissues,breast cancer cells( including T47D,ZR-75-1,SK-BR-3,MDA-MB-231,MDA-MB-436,and MCF-7),and normal breast cells MCF-10A were detected.( 2) MDA-MB-231 cells were divided into experimental group,negative control group,or blank group,among which experimental group and negative control group were transfected by lentivirus expression vectors of RSK4 and lentivirus vectors without RSK4,respectively,and blank group was not transfected. The expression level of RSK4 mRNA,RSK4 protein,protein kinase( AKT),phosphorylated AKT( p-AKT),extracellular signal-regulated kinase( ERK),phosphorylated ERK( p-ERK),E-cadherin,and vimentin in the three groups were detected,and the cell migration and invasion ability in the three groups were also detected.( 3) The methylation levels of MDA-MB-231 cells and demethylated MDA-MB-231 cells were detected,and the expression levels of RSK4 mRNA were compared between the two cells.( 4) Twelve nude mice were randomly divided into experimental group or control group,with 6 mice in each group,and MDA-MB-231 cells suspension liquid of the experimental group and the negative control group were injected into the breast fat pad of the two groups,respectively,and tumor volume from 1 to 6 weeks after inoculation and the tumor weight 6 weeks after inoculation were compared between the two groups. Results( 1) Expression levels of RSK4 mRNA and protein were lower in breast cancer tissues than in adjacent normal tissues( P < 0. 05). The expression levels of RSK4 mRNA and protein in MCF-10A cells were higher than those in 6 breast cancer cells( P < 0. 05). Among the 6 breast cancer cells,the highest expression level of RSK4 mRNA and protein occurred in MCF-7 cells and the lowest in MDA-MB-231 cells( all P < 0. 05).( 2) Compared with the negative control group and the blank group,the experimental group obtained higher E-cadherin,RSK4 mRNA,and protein expression,but lower cell migration,cell invasion,and p-AKT,p-ERK,and vimentin expression( P < 0. 05).( 3) The expression level of RSK4 mRNA in demethylated MDA-MB-231 cells was higher than that in MDA-MB-231 cells( P < 0. 05).( 4) Weight of the nude mice was lower in the experimental group than in the control group from the 3 rd week to the 6 th week,and tumor volume of the nude mice in the experimental group was smaller in the experimental group than in the control group from the 2 nd week to the 6 th week( all P < 0. 05).Conclusion RSK4 might play an inhibiting role in the carcinogenesis and development of breast cancer,the low expression of RSK4 in breast cancer may be caused by abnormal methylation of its promoter. RSK4 might regulate epithelial-mesenchymal transition in breast cancer cells via the AKT/ERK signaling pathway.
Objective To investigate the role of X-linked ribosomal S6 kinase 4( RSK4) in the carcinogenesis and development of breast cancer and its mechanism. Methods( 1) Expression levels of RSK4 mRNA and protein in breast cancer tissues,adjacent normal tissues,breast cancer cells( including T47D,ZR-75-1,SK-BR-3,MDA-MB-231,MDA-MB-436,and MCF-7),and normal breast cells MCF-10A were detected.( 2) MDA-MB-231 cells were divided into experimental group,negative control group,or blank group,among which experimental group and negative control group were transfected by lentivirus expression vectors of RSK4 and lentivirus vectors without RSK4,respectively,and blank group was not transfected. The expression level of RSK4 mRNA,RSK4 protein,protein kinase( AKT),phosphorylated AKT( p-AKT),extracellular signal-regulated kinase( ERK),phosphorylated ERK( p-ERK),E-cadherin,and vimentin in the three groups were detected,and the cell migration and invasion ability in the three groups were also detected.( 3) The methylation levels of MDA-MB-231 cells and demethylated MDA-MB-231 cells were detected,and the expression levels of RSK4 mRNA were compared between the two cells.( 4) Twelve nude mice were randomly divided into experimental group or control group,with 6 mice in each group,and MDA-MB-231 cells suspension liquid of the experimental group and the negative control group were injected into the breast fat pad of the two groups,respectively,and tumor volume from 1 to 6 weeks after inoculation and the tumor weight 6 weeks after inoculation were compared between the two groups. Results( 1) Expression levels of RSK4 mRNA and protein were lower in breast cancer tissues than in adjacent normal tissues( P < 0. 05). The expression levels of RSK4 mRNA and protein in MCF-10A cells were higher than those in 6 breast cancer cells( P < 0. 05). Among the 6 breast cancer cells,the highest expression level of RSK4 mRNA and protein occurred in MCF-7 cells and the lowest in MDA-MB-231 cells( all P < 0. 05).( 2) Compared with the negative control group and the blank group,the experimental group obtained higher E-cadherin,RSK4 mRNA,and protein expression,but lower cell migration,cell invasion,and p-AKT,p-ERK,and vimentin expression( P < 0. 05).( 3) The expression level of RSK4 mRNA in demethylated MDA-MB-231 cells was higher than that in MDA-MB-231 cells( P < 0. 05).( 4) Weight of the nude mice was lower in the experimental group than in the control group from the 3 rd week to the 6 th week,and tumor volume of the nude mice in the experimental group was smaller in the experimental group than in the control group from the 2 nd week to the 6 th week( all P < 0. 05).Conclusion RSK4 might play an inhibiting role in the carcinogenesis and development of breast cancer,the low expression of RSK4 in breast cancer may be caused by abnormal methylation of its promoter. RSK4 might regulate epithelial-mesenchymal transition in breast cancer cells via the AKT/ERK signaling pathway.
引文
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[9]Li Q,Jiang Y,Wei W,et al.Frequent epigenetic inactivation of RSK4 by promoter methylation in cancerous and non-cancerous tissues of breast cancer[J].Med Oncol,2014,31(1):793.
[10]Pradella D,Naro C,Sette C,et al.EMT and stemness:flexible processes tuned by alternative splicing in development and cancer progression[J].Mol Cancer,2017,16(1):8.
[11]Huber MA,Azoitei N,Baumann B,et al.NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression[J].J Clin Invest,2004,114(4):569-581.
[12]Zhu L,Zhang S,Huan X,et al.Down-regulation of TRAF4targeting RSK4 inhibits proliferation,invasion and metastasis in breast cancer xenografts[J].Biochem Biophys Res Commun,2018,500(3):810-816.
[13]Rafiee M,Keramati MR,Ayatollahi H,et al.Down-regulation of ribosomal S6 kinase RPS6KA6 in acute myeloid leukemia patients[J].Cell J,2016,18(2):159-164.
[14]Cyprian FS,Al-Farsi HF,Vranic S,et al.Epstein-Barr virus and human papillomaviruses interactions and their roles in the initiation of epithelial-mesenchymal transition and cancer progression[J].Front Oncol,2018,8:111.
[2]Zhu J,Li QY,Liu JL,et al.RSK4 knockdown promotes proliferation,migration and metastasis of human breast adenocarcinoma cells[J].Oncol Rep,2015,34(6):3 156-3 162.
[3]卞雪春,余艳兰,赵平,等.结直肠癌组织RSK4、p53蛋白的表达及临床病理意义[J].现代生物医学进展,2018,18(6):1 102-1 106.
[4]杨斌,张建武.核糖体S6激酶4在胰腺癌组织中的表达及其对预后评估的价值[J].现代医学,2017,45(3):439-443.
[5]Dewdney SB,Rimel BJ,Thaker PH,et al.Aberrant methylation of the X-Linked ribosomal S6 kinase RPS6KA6(RSK4)in endometrial cancers[J].Clin Cancer Res,2011,17(8):2 120-2 129.
[6]Arechavaleta-Velasco F,Zeferino-Toquero M,Estrada-Moscoso I,et al.Ribosomal S6 kinase 4(RSK4)expression in ovarian tumors and its regulation by antineoplastic drugs in ovarian cancer cell lines[J].Med Oncol,2016,33(2):11.
[7]李秋云.RSK4与BRCA1基因异常甲基化在乳腺癌发病机制中作用的研究[D].南宁:广西医科大学,2013.
[8]唐林,陈巍魏,管晓翔.AJCC第8版乳腺癌分期系统更新的解读[J].临床肿瘤学杂志,2017,22(11):1 038-1 040.
[9]Li Q,Jiang Y,Wei W,et al.Frequent epigenetic inactivation of RSK4 by promoter methylation in cancerous and non-cancerous tissues of breast cancer[J].Med Oncol,2014,31(1):793.
[10]Pradella D,Naro C,Sette C,et al.EMT and stemness:flexible processes tuned by alternative splicing in development and cancer progression[J].Mol Cancer,2017,16(1):8.
[11]Huber MA,Azoitei N,Baumann B,et al.NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression[J].J Clin Invest,2004,114(4):569-581.
[12]Zhu L,Zhang S,Huan X,et al.Down-regulation of TRAF4targeting RSK4 inhibits proliferation,invasion and metastasis in breast cancer xenografts[J].Biochem Biophys Res Commun,2018,500(3):810-816.
[13]Rafiee M,Keramati MR,Ayatollahi H,et al.Down-regulation of ribosomal S6 kinase RPS6KA6 in acute myeloid leukemia patients[J].Cell J,2016,18(2):159-164.
[14]Cyprian FS,Al-Farsi HF,Vranic S,et al.Epstein-Barr virus and human papillomaviruses interactions and their roles in the initiation of epithelial-mesenchymal transition and cancer progression[J].Front Oncol,2018,8:111.