miR-302c在胃癌中表达及对胃癌细胞侵袭和迁移影响的机制研究
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摘要
[目的]探讨miR-302c对人胃癌细胞侵袭和迁移能力的影响及机制。[方法]实时荧光定量PCR法检测miR-302c在胃癌细胞系和正常胃黏膜上皮细胞中的表达情况。在SGC7901和AGS细胞中分别转染miR-302c mimic和inhibitor后,Tranwell实验检测细胞侵袭和迁移能力变化。实时荧光定量PCR和Western blot法检测转染后环磷酸腺苷反应元件结合蛋白1(CREB1)的变化情况。[结果]与永生化的正常胃黏膜上皮细胞GES-1相比,miR-302c在胃癌细胞系SGC7901、MKN28、N87、MKN45和AGS中表达降低。转染mimic和inhibitor后,miR-302c在SGC7901和AGS中表达分别明显上调和下调(P<0.001)。Transwell实验发现,上调miR-302c后,SGC7901细胞的侵袭和迁移能力明显降低,而下调miR-302c后,AGS细胞的侵袭和迁移能力明显增强。进一步研究发现,miR-302c可抑制CREB1的表达。功能挽救实验证明CREB1可部分恢复miR-302c上调对SGC7901细胞侵袭和迁移的抑制作用。[结论] miR-302c抑制胃癌细胞的侵袭和迁移,其机制可能是通过直接靶向CREB1。
[Purpose] To investigate the expression of miR-302c in gastric cancer(GC) cells and its relation to cell migration and invasion. [Methods] Real-time quantitative PCR(qRT-PCR) was used to detect miR-302c expression in GCSGC7901,MKN28,MKN45 and AGS cells and immortalized gastric epithelial cell line GES-1. Transwell assay was used to determine the migration and invasion abilities of SGC7901 and AGS cells transfected with miR-302c mimic and inhibitor,respectively. q RT-PCR and Western blot were used to detect the expression of CREB1 after transfection of miR-302c mimic and inhibitor in indicated cells. [Results] miR-302c was downregulated in GC cell lines compared with GES-1 cells. Overexpression of miR-302c significantly reduced migration,invasion and CREB1 expression of SGC7901 cells,while silencing of miR-302c significantly increased migration,invasion and CREB1 expression of AGS cells. Overexpression of CREB1 alleviated the decreased migration and invasion induced by miR-302c in SGC7901 cells. [Conclusion] mi R-302c is downregulated in gastric cancer cell lines and can suppress cancer cell migration and invasion by targeting CREB1.
[Purpose] To investigate the expression of miR-302c in gastric cancer(GC) cells and its relation to cell migration and invasion. [Methods] Real-time quantitative PCR(qRT-PCR) was used to detect miR-302c expression in GCSGC7901,MKN28,MKN45 and AGS cells and immortalized gastric epithelial cell line GES-1. Transwell assay was used to determine the migration and invasion abilities of SGC7901 and AGS cells transfected with miR-302c mimic and inhibitor,respectively. q RT-PCR and Western blot were used to detect the expression of CREB1 after transfection of miR-302c mimic and inhibitor in indicated cells. [Results] miR-302c was downregulated in GC cell lines compared with GES-1 cells. Overexpression of miR-302c significantly reduced migration,invasion and CREB1 expression of SGC7901 cells,while silencing of miR-302c significantly increased migration,invasion and CREB1 expression of AGS cells. Overexpression of CREB1 alleviated the decreased migration and invasion induced by miR-302c in SGC7901 cells. [Conclusion] mi R-302c is downregulated in gastric cancer cell lines and can suppress cancer cell migration and invasion by targeting CREB1.
引文
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[18]Yao WJ,Wang YL,Lu JG,et al.MicroRNA-506 inhibits esophageal cancer cell proliferation via targeting CREB1[J].Int J Clin Exp Pathol,2015,8(9):10868-10874.
[19]Peng B,Hu S,Jun Q,et al.MicroRNA-200b targets CREB1 and suppresses cell growth in human malignant glioma[J].Mol Cell Biochem,2013,379(1-2):51-58.
[20]Rao M,Zhu Y,Zhou Y,et al.MicroRNA-122 inhibits proliferation and invasion in gastric cancer by targeting CREB1[J].Am J Cancer Res,2017,7(2):323-333.
[2]Van Cutsem E,Sagaert X,Topal B,et al.Gastric cancer[J].Lancet,2016,388(10060):2654-2664.
[3]Bartel DP.MicroRNAs:target recognition and regulatory functions[J].Cell,2009,136(2):215-233.
[4]Lei C,Du Feng,Sun Lina,et al.miR-143 and miR-145inhibit gastric cancer cell migration and metastasis by suppressing MYO6[J].Cell Death Dis,2017,8(10):e3101.
[5]Li X,Zhang Y,Shi Y,et al.MicroRNA-107,an oncogene microRNA that regulates tumor invasion and metastasis by targeting DICER1 in gastric cancer[J].J Cell Mol Med,2011,15(9):1887-1895.
[6]Zhang Z,Li Z,Li Y,et al.MicroRNA and signaling pathways in gastric cancer[J].Cancer Gene Ther,2014,21(8):305-316.
[7]Gao Z,Zhu X,Dou Y.The miR-302/367 cluster:a comprehensive update on its evolution and functions[J].Open Biol,2015,5(12):150138.
[8]Yang L,Guo Y,Liu X,et al.The tumor suppressive miR-302c-3p inhibits migration and invasion of hepatocellular carcinoma cells by targeting TRAF4[J].J Cancer,2018,9(15):2693-2701.
[9]Leivonen SK,Makela R,Ostling P,et al.Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines[J].Oncogene,2009,28(44):3926-3936.
[10]Wang Y,Wei Y,Tong H,et al.MiR-302c-3p suppresses invasion and proliferation of glioma cells via down-regulating metadherin(MTDH)expression[J].Cancer Biol Ther,2015,16(9):1308-1315.
[11]Iorio MV,Croce CM.Micro RNA dysregulation in cancer:diagnostics,monitoring and therapeutics.A comprehensive review[J].Embo Mol Med,2012,4(3):143-159.
[12]Zhang H,Li Y,Lai M.The microRNA network and tumor metastasis[J].Oncogene,2010,29(7):937-948.
[13]Li Y,Huo J,Pan X,et al.MicroRNA 302b-3p/302c-3p/302d-3p inhibits epithelial-mesenchymal transition and promotes apoptosis in human endometrial carcinoma cells[J].Oncol Targets Ther,2018,11(3):1275-1284.
[14]Ma W,Liu B,Li J,et al.MicroRNA-302c represses epithelial-mesenchymal transition and metastasis by targeting transcription factor AP-4 in colorectal cancer[J].Biomed Pharmacother,2018,105(1):670-676.
[15]Mayr B,Montminy M.Transcriptional regulation by the phosphorylation-dependent factor CREB[J].Nat Rev Mol Cell Biol,2001,2(8):599-609.
[16]Conkright MD,Montminy M.CREB:the unindicted cancer co-conspirator[J].Trends Cell Biol,2005,15(9):457-459.
[17]Wang YW,Chen X,Gao JW,et al.High expression of cAMP-responsive element-binding protein 1(CREB1)is associated with metastasis,tumor stage and poor outcome in gastric cancer[J].Oncotarget,2015,6(12):10646-10657.
[18]Yao WJ,Wang YL,Lu JG,et al.MicroRNA-506 inhibits esophageal cancer cell proliferation via targeting CREB1[J].Int J Clin Exp Pathol,2015,8(9):10868-10874.
[19]Peng B,Hu S,Jun Q,et al.MicroRNA-200b targets CREB1 and suppresses cell growth in human malignant glioma[J].Mol Cell Biochem,2013,379(1-2):51-58.
[20]Rao M,Zhu Y,Zhou Y,et al.MicroRNA-122 inhibits proliferation and invasion in gastric cancer by targeting CREB1[J].Am J Cancer Res,2017,7(2):323-333.