microRNA-708-5p对骨肉瘤细胞凋亡与迁移的作用及机制
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摘要
该文主要研究microRNA-708-5p(miR-708-5p)在骨肉瘤细胞中的表达及对细胞凋亡、迁移的影响及机制。该研究利用miRNA基因芯片筛选差异表达miRNA; qRT-PCR(quantitative Realtime PCR)检测miR-708-5p在骨肉瘤细胞株MG63和正常细胞hMSC、HS-5中的表达;通过阳离子脂质体介导法过表达miR-708-5p;分别用Hoechst 33258染色、流式细胞术、划痕实验、Transwell法检测凋亡和迁移;通过qRT-PCR检测miR-708-5p、ZEB1(Zinc?nger E-box binding homeobox 1)的RNA水平; Western blot检测E-cadherin、N-cadherin、ZEB1蛋白表达;利用TargetScan和双荧光素酶报告实验预测并验证miR-708-5p与ZEB1的靶向关系。结果显示, miR-708-5p在MG63中表达下调,恢复miR-708-5p表达水平可诱导MG63细胞凋亡并抑制迁移。Western blot结果显示,过表达miR-708-5p可上调E-cadherin,下调N-cadherin和ZEB1。双荧光素酶报告实验显示, miR-708-5p可直接靶向ZEB1。敲低ZEB1可抑制MG63迁移。该项研究结果表明, miR-708-5p可诱导骨肉瘤细胞凋亡,且通过靶向ZEB1来抑制迁移。
This article aimed to investigate the expression of microRNA-708-5p(miR-708-5p)in osteosarcoma cell and its effects on cell apoptosis, migration and its mechanism. MiRNA microarray was utilized to screen differential expressed miRNAs. MiR-708-5p expression level in MG63 cell line,normal cells hMSC and HS-5 were detected by quantitative Real-time PCR(qRT-PCR). MiR-708-5p was overexpressed in osteosarcoma cell MG63 using Lipofectamine 2000. Hoechst 33258 staining, flow cytometry(FCM) were utilized to measure cell apoptosis. Wound healing assay and Transwell assay were employed to measure migration ability. The RNA expression of miR-708-5p and ZEB1(Zinc finger E-box binding homeobox 1) were examined by qRT-PCR. Protein levels of E-cadherin, N-cadherin and ZEB1 were detected by means of Western blot. Targeting relationship between miR-708-5p and ZEB1 was predicted and validated using TargetScan and dual-luciferase reporter assay, respectively. The results indicated that miR-708-5p was lower expressed in MG63 cell compared to normal cells. Restoring miR-708-5p could induce cell apoptosis and inhibit migration. Showed by Western blot, protein levels of E-cadherin increased after miR-708-5 p overexpression while N-cadherin and ZEB1 decreased. Indicated by dual-luciferase reporter assay, miR-708-5p directly targeted ZEB1. What's more, knocking down ZEB1 can inhibit migration of MG63. These data demonstrated that miR-708-5p could induce osteosarcoma apoptosis and decrease migration ability by targeting ZEB1, resulting in inhibiting cell migration.
This article aimed to investigate the expression of microRNA-708-5p(miR-708-5p)in osteosarcoma cell and its effects on cell apoptosis, migration and its mechanism. MiRNA microarray was utilized to screen differential expressed miRNAs. MiR-708-5p expression level in MG63 cell line,normal cells hMSC and HS-5 were detected by quantitative Real-time PCR(qRT-PCR). MiR-708-5p was overexpressed in osteosarcoma cell MG63 using Lipofectamine 2000. Hoechst 33258 staining, flow cytometry(FCM) were utilized to measure cell apoptosis. Wound healing assay and Transwell assay were employed to measure migration ability. The RNA expression of miR-708-5p and ZEB1(Zinc finger E-box binding homeobox 1) were examined by qRT-PCR. Protein levels of E-cadherin, N-cadherin and ZEB1 were detected by means of Western blot. Targeting relationship between miR-708-5p and ZEB1 was predicted and validated using TargetScan and dual-luciferase reporter assay, respectively. The results indicated that miR-708-5p was lower expressed in MG63 cell compared to normal cells. Restoring miR-708-5p could induce cell apoptosis and inhibit migration. Showed by Western blot, protein levels of E-cadherin increased after miR-708-5 p overexpression while N-cadherin and ZEB1 decreased. Indicated by dual-luciferase reporter assay, miR-708-5p directly targeted ZEB1. What's more, knocking down ZEB1 can inhibit migration of MG63. These data demonstrated that miR-708-5p could induce osteosarcoma apoptosis and decrease migration ability by targeting ZEB1, resulting in inhibiting cell migration.
引文
1 Angulo P,Kaushik G,Subramaniam D,Dandawate P,Neville K,Chastain K,et al.Natural compounds targeting major cell signaling pathways:a novel paradigm for osteosarcoma therapy.J Hematol Oncol 2017;10(1):10.
2 Jaffe N,Puri A,Gelderblom H.Osteosarcoma:evolution of treatment paradigms.Sarcoma 2013;2013:1-7.
3 Meazza C,Scanagatta P.Metastatic osteosarcoma:a challenging multidisciplinary treatment.Expert Rev Anticancer Ther 2016;16(5):543-56.
4 Isakoff MS,Bielack SS,Meltzer P,Gorlick R.Osteosarcoma:current treatment and a collaborative pathway to success.J Clin Oncol 2015;33(27):3029-35.
5 Bartel DP.Metazoan microRNAs.Cell 2018;173(1):20-51.
6 David P.Bartel.MicroRNAs:Genomics,biogenesis,mechanism,and function.Cell 2004;116(2):281-97.
7 Ambros V.The functions of animal microRNAs.Nature 2004;431(7006):350-5.
8 Calin GA,Croce CM.MicroRNA signatures in human cancers.Nat Rev Cancer 2006;6(11):857-66.
9 Jones KB,Salah Z,Del Mare S,Galasso M,Gaudio E,Nuovo GJ,et al.miRNA signatures associate with pathogenesis and progression of osteosarcoma.Cancer Res 2012;72(7):1865-77.
10 Xinyu Tan,Shicai Fan,Wen Wu and Yin Zhang.MicroRNA-26a inhibits osteosarcoma cell proliferation by targeting IGF-1.Bone Research 2015;3:15033.
11 Zhang L,Lv Z,Xu J,Chen C,Ge Q,Li P,et al.MicroRNA-134inhibits osteosarcoma angiogenesis and proliferation by targeting the VEGFA/VEGFR1 pathway.FEBS J 2018;285(7):1359-71.
12 Niu G,Li B,Sun J,Sun L.miR-454 is down-regulated in osteosarcomas and suppresses cell proliferation and invasion by directly targeting c-Met.Cell Prolif 2015;48(3):348-55.
13 Liu T,Wu X,Chen T,Luo Z,Hu X.Downregulation of DNMT3A by miR-708-5p Inhibits Lung Cancer Stem Cell-like Phenotypes through Repressing Wnt/beta-catenin Signaling.Clin Cancer Res 2018,24(7):1748-60.
14 Ryu S,McDonnell K,Choi H,Gao D,Hahn M,Joshi N,et al.Suppression of miRNA-708 by polycomb group promotes metastases by calcium-induced cell migration Cancer Cell 2013;23(1):63-76.
15 Saini S,Yamamura S,Majid S,Shahryari V,Hirata H,Tanaka Y,et al.MicroRNA-708 induces apoptosis and suppresses tumorigenicity in renal cancer cells.Cancer Res 2011;71(19):6208-19.
16 Delsin LEA,Roberto GM,Fedatto PF,Engel EE,Scrideli CA,Tone LG,et al.Downregulated adhesion-associated microRNAs as prognostic predictors in childhood osteosarcoma.Pathol Oncol Res 2019;25(1):11-20.
17 Jaffe N.Osteosarcoma:review of the past,impact on the future.The American experience.Cancer Treat Res 2009;152:239-62.
18郑颖,王刚阳,陈瑞玲,蔡郑东,华莹奇.骨肉瘤多药耐药的研究进展.现代生物医学进展(Zheng Ying,Wang Gangyang,Chen Ruiling,Cai Zhengdong,Hhua Yingqi.The research progress of osteosarcoma multi-resistance.Progress in Modern Biomedicine)2017;17(33):6570-3.
19 Alvarez-Garcia I,Miska EA.MicroRNA functions in animal development and human disease.Development 2005;132(21):4653-62.
20 Bartel DP.MicroRNAs:target recognition and regulatory functions.Cell 2009;136(2):215-33.
21 Fabian MR,Sonenberg N,Filipowicz W.Regulation of mRNAtranslation and stability by microRNAs.Annu Rev Biochem2010;79:351-79.
22 Lin KT,Yeh YM,Chuang CM,Yang SY,Chang JW,Sun SP,et al.Glucocorticoids mediate induction of microRNA-708 to suppress ovarian cancer metastasis through targeting Rap1B.Nat Commun 2015;6:5917.
24 Wu X,Liu T,Fang O,Dong W,Zhang F,Leach L,et al.MicroRNA-708-5p acts as a therapeutic agent against metastatic lung cancer.Oncotarget 2016;7(3):2417-32.
25 Gambera S,Abarrategi A,Rodriguez-Milla MA,Mulero F,Menendez ST,Rodriguez R,et al.Role of activator protein-1complex on the phenotype of human osteosarcomas generated from mesenchymal stem cells.Stem Cells 2018;36(10):1487-500.
26 Kong D,Li Y,Wang Z,Sarkar FH.Cancer stem cells and epithelial-to-mesenchymal transition(EMT)-phenotypic cells:Are they cousins or twins?Cancers(Basel)2011;3(1):716-29.
27 Shen A,Zhang Y,Yang H,Xu R,Huang G.Overexpression of ZEB1 relates to metastasis and invasion in osteosarcoma.J Surg Oncol 2012;105(8):830-4.
2 Jaffe N,Puri A,Gelderblom H.Osteosarcoma:evolution of treatment paradigms.Sarcoma 2013;2013:1-7.
3 Meazza C,Scanagatta P.Metastatic osteosarcoma:a challenging multidisciplinary treatment.Expert Rev Anticancer Ther 2016;16(5):543-56.
4 Isakoff MS,Bielack SS,Meltzer P,Gorlick R.Osteosarcoma:current treatment and a collaborative pathway to success.J Clin Oncol 2015;33(27):3029-35.
5 Bartel DP.Metazoan microRNAs.Cell 2018;173(1):20-51.
6 David P.Bartel.MicroRNAs:Genomics,biogenesis,mechanism,and function.Cell 2004;116(2):281-97.
7 Ambros V.The functions of animal microRNAs.Nature 2004;431(7006):350-5.
8 Calin GA,Croce CM.MicroRNA signatures in human cancers.Nat Rev Cancer 2006;6(11):857-66.
9 Jones KB,Salah Z,Del Mare S,Galasso M,Gaudio E,Nuovo GJ,et al.miRNA signatures associate with pathogenesis and progression of osteosarcoma.Cancer Res 2012;72(7):1865-77.
10 Xinyu Tan,Shicai Fan,Wen Wu and Yin Zhang.MicroRNA-26a inhibits osteosarcoma cell proliferation by targeting IGF-1.Bone Research 2015;3:15033.
11 Zhang L,Lv Z,Xu J,Chen C,Ge Q,Li P,et al.MicroRNA-134inhibits osteosarcoma angiogenesis and proliferation by targeting the VEGFA/VEGFR1 pathway.FEBS J 2018;285(7):1359-71.
12 Niu G,Li B,Sun J,Sun L.miR-454 is down-regulated in osteosarcomas and suppresses cell proliferation and invasion by directly targeting c-Met.Cell Prolif 2015;48(3):348-55.
13 Liu T,Wu X,Chen T,Luo Z,Hu X.Downregulation of DNMT3A by miR-708-5p Inhibits Lung Cancer Stem Cell-like Phenotypes through Repressing Wnt/beta-catenin Signaling.Clin Cancer Res 2018,24(7):1748-60.
14 Ryu S,McDonnell K,Choi H,Gao D,Hahn M,Joshi N,et al.Suppression of miRNA-708 by polycomb group promotes metastases by calcium-induced cell migration Cancer Cell 2013;23(1):63-76.
15 Saini S,Yamamura S,Majid S,Shahryari V,Hirata H,Tanaka Y,et al.MicroRNA-708 induces apoptosis and suppresses tumorigenicity in renal cancer cells.Cancer Res 2011;71(19):6208-19.
16 Delsin LEA,Roberto GM,Fedatto PF,Engel EE,Scrideli CA,Tone LG,et al.Downregulated adhesion-associated microRNAs as prognostic predictors in childhood osteosarcoma.Pathol Oncol Res 2019;25(1):11-20.
17 Jaffe N.Osteosarcoma:review of the past,impact on the future.The American experience.Cancer Treat Res 2009;152:239-62.
18郑颖,王刚阳,陈瑞玲,蔡郑东,华莹奇.骨肉瘤多药耐药的研究进展.现代生物医学进展(Zheng Ying,Wang Gangyang,Chen Ruiling,Cai Zhengdong,Hhua Yingqi.The research progress of osteosarcoma multi-resistance.Progress in Modern Biomedicine)2017;17(33):6570-3.
19 Alvarez-Garcia I,Miska EA.MicroRNA functions in animal development and human disease.Development 2005;132(21):4653-62.
20 Bartel DP.MicroRNAs:target recognition and regulatory functions.Cell 2009;136(2):215-33.
21 Fabian MR,Sonenberg N,Filipowicz W.Regulation of mRNAtranslation and stability by microRNAs.Annu Rev Biochem2010;79:351-79.
22 Lin KT,Yeh YM,Chuang CM,Yang SY,Chang JW,Sun SP,et al.Glucocorticoids mediate induction of microRNA-708 to suppress ovarian cancer metastasis through targeting Rap1B.Nat Commun 2015;6:5917.
24 Wu X,Liu T,Fang O,Dong W,Zhang F,Leach L,et al.MicroRNA-708-5p acts as a therapeutic agent against metastatic lung cancer.Oncotarget 2016;7(3):2417-32.
25 Gambera S,Abarrategi A,Rodriguez-Milla MA,Mulero F,Menendez ST,Rodriguez R,et al.Role of activator protein-1complex on the phenotype of human osteosarcomas generated from mesenchymal stem cells.Stem Cells 2018;36(10):1487-500.
26 Kong D,Li Y,Wang Z,Sarkar FH.Cancer stem cells and epithelial-to-mesenchymal transition(EMT)-phenotypic cells:Are they cousins or twins?Cancers(Basel)2011;3(1):716-29.
27 Shen A,Zhang Y,Yang H,Xu R,Huang G.Overexpression of ZEB1 relates to metastasis and invasion in osteosarcoma.J Surg Oncol 2012;105(8):830-4.