高表达Per2基因的胶质瘤干细胞下调其基质金属蛋白酶2的研究
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摘要
目的构建Period2(Per2)基因过表达的U87胶质瘤干细胞,检测Per2基因过表达U87胶质瘤干细胞中基质金属蛋白酶2(MMP-2)的mRNA及蛋白的表达变化。方法以Per2基因过表达慢病毒转染U87胶质瘤干细胞,然后对U87胶质瘤干细胞进行转染,通过嘌呤霉素筛选,用real-time PCR和Western blot技术鉴定得到Per2基因过表达的U87胶质瘤干细胞,空载体转染的U87胶质瘤干细胞和U87胶质瘤干细胞未转染组作为对照。结果此次转染成功构建Per2基因过表达U87胶质瘤干细胞系,过表达组细胞内Per2基因的mRNA和蛋白表达量明显高于其他两组细胞,MMP-2 mRNA及蛋白表达量在Per2基因过表达U87胶质瘤干细胞内明显降低。结论 U87胶质瘤干细胞高表达Per2基因能够下调其MMP-2。
Objective To construct U87 glioma stem cells overexpressing the Period2(Per2) gene,and detection of m RNA and protein expression of matrix metalloproteinase-2 in U87 glioma stem cells overexpressing Per2 gene. Methods The lentivirus overexpressing Per2 gene was designed and synthesized,then U87 glioma stem cells were transfected,and U87 glioma stem cells overexpressing Per2 gene were identified by real-time PCR and Western-blot. Real-time PCR was used to identify the expression of MMP-2 in U87 glioma stem cells overexpressing Per2 gene and the expression of MMP-2 in U87 glial stem cells and normal U87 glial stem cells transfected with empty vector. Results The Per2 gene overexpressed U87 glioma stem cell line was successfully constructed. The expression of MMP-2 remarkably decreased in Per2 gene overexpressing U87 glioma stem cells. Conclusion High expression of Per2 gene in U87 glioma stem cells can down-regulate its matrix metalloproteinase 2.
Objective To construct U87 glioma stem cells overexpressing the Period2(Per2) gene,and detection of m RNA and protein expression of matrix metalloproteinase-2 in U87 glioma stem cells overexpressing Per2 gene. Methods The lentivirus overexpressing Per2 gene was designed and synthesized,then U87 glioma stem cells were transfected,and U87 glioma stem cells overexpressing Per2 gene were identified by real-time PCR and Western-blot. Real-time PCR was used to identify the expression of MMP-2 in U87 glioma stem cells overexpressing Per2 gene and the expression of MMP-2 in U87 glial stem cells and normal U87 glial stem cells transfected with empty vector. Results The Per2 gene overexpressed U87 glioma stem cell line was successfully constructed. The expression of MMP-2 remarkably decreased in Per2 gene overexpressing U87 glioma stem cells. Conclusion High expression of Per2 gene in U87 glioma stem cells can down-regulate its matrix metalloproteinase 2.
引文
[1] Hua H,Wang YQ,Wan CM,et al. Circadian gene m Per2 overexpression induces cancer cell apoptosis[J]. Cancer Sci,2006,97(7):589-596.
[2] Tsinkalovsky O,Filipski E,Rosenlund B,et al. Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow[J]. Exp Hematol,2006,34(9):1249-1261.
[3] Sharma VP,Anderson NT,Geusz ME. Circadian properties of cancer stem cells in glioma cell cultures and tumorspheres[J]. Cancer Lett,2014,345(1):65-74.
[4] Reya T,Morrison SJ,Clarke MF,et al. Stem cells,cancer,and cancer stem cells[J]. Nature,2001,414(6859):105-111.
[5] Lathia JD,Mack SC,Mulkearns-Hubert EE,et al.Cancer stem cells in glioblastoma[J]. Genes Dev,2015,29(12):1203-1217.
[6] Zhou WC,Ke SQ,Huang Z,et al. Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth[J]. Nat Cell Biol,2015,17(2):170-182.
[7] Tsinkalovsky O,Filipski E,Rosenlund B,et al. Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow[J]. Exp Hematol,2006,34(9):1249-1261.
[8] Sharma VP,Anderson NT,Geusz ME. Circadian properties of cancer stem cells in glioma cell cultures and tumorspheres[J]. Cancer Lett,2014,345(1):65-74.
[9] Wong JC,Chan SK,Schaeffer DF,et al. Absence of MMP2 expression correlates with poor clinical outcomes in rectal cancer,and is distinct from MMP1-related outcomes in colon cancer[J]. Clin Cancer Res,2011,17(12):4167-4176.
[10]刘文斌.人脑胶质母细胞瘤干细胞生物钟Per2基因的节律性研究[D].银川:宁夏医科大学,2018.
[11] Park MJ,Kwak HJ,Lee HC,et al. Nerve growth factor induces endothelial cell invasion and cord formation by promoting matrix metalloproteinase-2 expression through the phosphatidylinositol 3-kinase/Akt signaling pathway and AP-2 transcription factor[J].J Biol Chem,2007,282(42):30485-30496.
[12] Forsyth PA,Wong H,Laing TD,et al. Gelatinase-A(MMP-2),Gelatinase-B(MMP-9)and membrane type matrix metalloproteinase-1(MT1-MMP)are involved in different aspects of the pathophysiology of malignant gliomas[J]. Br J Cancer,1999,79(11/12):1828-1835.
[13]吴艳林,蔡政,张明智,等.双氢青蒿素抑制胶质瘤干细胞侵袭迁移的机制[J].中国组织工程研究,2016,20(6):765-770.
[14] El Cheikh R,Bernard S,El Khatib N. Modeling circadian clock-cell cycle interaction effects on cell population growth rates[J]. J Theor Biol,2014,363:318-331.
[2] Tsinkalovsky O,Filipski E,Rosenlund B,et al. Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow[J]. Exp Hematol,2006,34(9):1249-1261.
[3] Sharma VP,Anderson NT,Geusz ME. Circadian properties of cancer stem cells in glioma cell cultures and tumorspheres[J]. Cancer Lett,2014,345(1):65-74.
[4] Reya T,Morrison SJ,Clarke MF,et al. Stem cells,cancer,and cancer stem cells[J]. Nature,2001,414(6859):105-111.
[5] Lathia JD,Mack SC,Mulkearns-Hubert EE,et al.Cancer stem cells in glioblastoma[J]. Genes Dev,2015,29(12):1203-1217.
[6] Zhou WC,Ke SQ,Huang Z,et al. Periostin secreted by glioblastoma stem cells recruits M2 tumour-associated macrophages and promotes malignant growth[J]. Nat Cell Biol,2015,17(2):170-182.
[7] Tsinkalovsky O,Filipski E,Rosenlund B,et al. Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow[J]. Exp Hematol,2006,34(9):1249-1261.
[8] Sharma VP,Anderson NT,Geusz ME. Circadian properties of cancer stem cells in glioma cell cultures and tumorspheres[J]. Cancer Lett,2014,345(1):65-74.
[9] Wong JC,Chan SK,Schaeffer DF,et al. Absence of MMP2 expression correlates with poor clinical outcomes in rectal cancer,and is distinct from MMP1-related outcomes in colon cancer[J]. Clin Cancer Res,2011,17(12):4167-4176.
[10]刘文斌.人脑胶质母细胞瘤干细胞生物钟Per2基因的节律性研究[D].银川:宁夏医科大学,2018.
[11] Park MJ,Kwak HJ,Lee HC,et al. Nerve growth factor induces endothelial cell invasion and cord formation by promoting matrix metalloproteinase-2 expression through the phosphatidylinositol 3-kinase/Akt signaling pathway and AP-2 transcription factor[J].J Biol Chem,2007,282(42):30485-30496.
[12] Forsyth PA,Wong H,Laing TD,et al. Gelatinase-A(MMP-2),Gelatinase-B(MMP-9)and membrane type matrix metalloproteinase-1(MT1-MMP)are involved in different aspects of the pathophysiology of malignant gliomas[J]. Br J Cancer,1999,79(11/12):1828-1835.
[13]吴艳林,蔡政,张明智,等.双氢青蒿素抑制胶质瘤干细胞侵袭迁移的机制[J].中国组织工程研究,2016,20(6):765-770.
[14] El Cheikh R,Bernard S,El Khatib N. Modeling circadian clock-cell cycle interaction effects on cell population growth rates[J]. J Theor Biol,2014,363:318-331.