过表达miR-497通过靶向作用于神经调节蛋白受体降解蛋白1(Nrdp1)促进U87胶质瘤细胞的增殖
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摘要
目的在U87人脑胶质瘤细胞中过表达miR-497,探讨miR-497对人脑胶质瘤细胞增殖的影响。方法包装阴性对照(NC)及miR-497慢病毒;构建U87-NC及U87-miR-497细胞系;荧光素酶报告基因实验检测miR-497在U87细胞中与神经调节蛋白受体降解蛋白1(Nrdp1)的靶向关系;集落形成实验检测细胞增殖能力、流式细胞术检测细胞周期变化;Western blot法检测Nrdp1、AKT及磷酸化的AKT(p-AKT)蛋白表达水平。结果成功包装p GLV3/H1-NC及p GLV3/H1-miR-497慢病毒;构建稳定的U87-NC及U87-miR-497细胞系;过表达miR-497的U87-miR-497细胞较对照组U87-NC细胞集落形成能力增强;荧光素酶报告基因实验证实miR-497在U87细胞中靶向作用于Nrdp1;在稳定感染细胞中,过表达miR-497后Nrdp1蛋白水平降低,p-AKT蛋白水平增加,而AKT蛋白未见明显改变。结论过表达miR-497通过靶向作用于Nrdp1促进U87胶质瘤细胞的增殖。
Objective To investigate the effect of miR-497 over-expression on the proliferation of U87 human glioma cells. Methods We packaged both p GLV3/H1-NC lentivirus as a negative control group and p GLV3/H1-miR-497 lentivirus as an experimental group,and then constructed U87-NC and U87-miR-497 cell lines,respectively. The relationship between miR-497 and neuregulin receptor degradation protein 1( Nrdp1) was analyzed by luciferase reporter assay in U87 cells; cell colony formation assay was used to detect cell proliferation and flow cytometry to detect cell cycle; the expressions of Nrdp1,AKT and phosphorylated AKT( p-AKT) were determined by Western blotting. Results We successfully packaged p GLV3/H1-NC and p GLV3/H1-miR-497 lentivirus,and obtained stable U87-NC and U87-miR-497 cell lines. When miR-497 was over-expressed in U87 cells,the cell colony formation ability was enhanced compared with the control group U87-NC. The luciferase reporter assay confirmed that miR-497 targeted Nrdp1 in U87 cells. In the stable infected cells,the level of Nrdp1 protein decreased and p-AKT protein increased,while the AKT protein did not change significantly after over-expression of miR-497. Conclusion Over-expression of miR-497 promotes the proliferation of glioma cells U87 by targeting Nrdp1.
Objective To investigate the effect of miR-497 over-expression on the proliferation of U87 human glioma cells. Methods We packaged both p GLV3/H1-NC lentivirus as a negative control group and p GLV3/H1-miR-497 lentivirus as an experimental group,and then constructed U87-NC and U87-miR-497 cell lines,respectively. The relationship between miR-497 and neuregulin receptor degradation protein 1( Nrdp1) was analyzed by luciferase reporter assay in U87 cells; cell colony formation assay was used to detect cell proliferation and flow cytometry to detect cell cycle; the expressions of Nrdp1,AKT and phosphorylated AKT( p-AKT) were determined by Western blotting. Results We successfully packaged p GLV3/H1-NC and p GLV3/H1-miR-497 lentivirus,and obtained stable U87-NC and U87-miR-497 cell lines. When miR-497 was over-expressed in U87 cells,the cell colony formation ability was enhanced compared with the control group U87-NC. The luciferase reporter assay confirmed that miR-497 targeted Nrdp1 in U87 cells. In the stable infected cells,the level of Nrdp1 protein decreased and p-AKT protein increased,while the AKT protein did not change significantly after over-expression of miR-497. Conclusion Over-expression of miR-497 promotes the proliferation of glioma cells U87 by targeting Nrdp1.
引文
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[19]Wu Y,Wang L,Bao H,et al.Nrdp1S,short variant of Nrdp1,inhibits human glioma progression by increasing Nrdp1-mediated Erb B3 ubiquitination and degradation[J].J Cell Mol Med,2016,20(3):422-429.
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[23]Shi H,Gong H,Cao K,et al.Nrdp1-mediated Erb B3 degradation inhibits glioma cell migration and invasion by reducing cytoplasmic localization of p27(Kip1)[J].J Neurooncol,2015,124(3):357-364.
[24]Shi H,Du J,Wang L,et al.Lower expression of Nrdp1 in human glioma contributes tumor progression by reducing apoptosis[J].IUBMB Life,2014,66(10):704-710.
[25]Meng Q,Xia C,Fang J,et al.Role of PI3K and AKT specific isoforms in ovarian cancer cell migration,invasion and proliferation through the p70S6K1 pathway[J].Cell Signal,2006,18(12):2262-2271.
[2]Gabriely G,Wurdinger T,Kesari S,et al.microRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators[J].Mol Cell Biol,2008,28(17):5369-5380.
[3]Hwang H W,Mendell J T.microRNAs in cell proliferation,cell death,and tumorigenesis[J].Br J Cancer,2006,94(6):776-780.
[4]Yang L,Li Q,Wang Q,et al.Silencing of miRNA-218 promotes migration and invasion of breast cancer via Slit2-Robo1 pathway[J].Biomed Pharmacother,2012,66(7):535-540.
[5]Bush N A,Chang S M,Berger M S,et al.Current and future strategies for treatment of glioma[J].Neurosurg Rev,2016,40(1):1-14.
[6]Wang L,Wei B,Hu G,et al.Gene expression analyses to explore the biomarkers and therapeutic targets for gliomas[J].Neurol Sci,2015,36(3):403-409.
[7]Birk H S,Han S J,Butowski N A,et al.Treatment options for recurrent high-grade gliomas[J].CNS Oncol,2017,6(1):61-70.
[8]Ghotme K A,Barreto G E,Echeverria V,et al.Gliomas:New perspectives in diagnosis,treatment and prognosis[J].Curr Top Med Chem,2017,17(12):1438-1447.
[9]Zhang Y,Han D,Wei W,et al.miR-218 inhibited growth and metabolism of human glioblastoma cells by directly targeting E2F2[J].Cell Mol Neurobiol,2015,35(8):1165-1173.
[10]Liu Y,Zhang B,Wen C,et al.Overexpressed miRNA-134b inhibits proliferation and invasion of CD133+U87 glioma stem cells[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2017,33(5):637-642.
[11]Wu Z,Han Y,Li Y,et al.miR-218-5p inhibits the stem cell properties and invasive ability of the A2B5+CD133-subgroup of human glioma stem cells[J].Oncol Rep,2016,35(2):869-877.
[12]Zhang Y,Zhang Z,Li Z,et al.microRNA-497 inhibits the proliferation,migration and invasion of human bladder transitional cell carcinoma cells by targeting E2F3[J].Oncol Rep,2016,36(3):1293-1300.
[13]Zhang L,Yu Z,Xian Y,et al.microRNA-497 inhibits cell proliferation and induces apoptosis by targeting YAP1 in human hepatocellular carcinoma[J].FEBS Open Bio,2016,6(2):155-164.
[14]Wu D,Niu X,Pan H,et al.MicroRNA-497 targets hepatoma-derived growth factor and suppresses human prostate cancer cell motility[J].Mol Med Rep,2016,13(3):2287-2292.
[15]Soriano A,Paris-Coderch L,Jubierre L,et al.microRNA-497impairs the growth of chemoresistant neuroblastoma cells by targeting cell cycle,survival and vascular permeability genes[J].Oncotarget,2016,7(8):9271-9287.
[16]Yang F,Wang W,Yuan L,et al.Overexpressed microRNA-93inhibits the proliferation and promotes apoptosis of A172 glioma cells[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2014,30(4):342-345.
[17]Lan J,Xue Y,Chen H,et al.Hypoxia-induced miR-497 decreases glioma cell sensitivity to TMZ by inhibiting apoptosis[J].FEBS Lett,2014,588(18):3333-3339.
[18]Jiang Y,Meng Q,Qi J,et al.miR-497 promotes metastasis of colorectal cancer cells through Nrdp1 inhibition[J].Tumour Biol,2015,36(10):7641-7647.
[19]Wu Y,Wang L,Bao H,et al.Nrdp1S,short variant of Nrdp1,inhibits human glioma progression by increasing Nrdp1-mediated Erb B3 ubiquitination and degradation[J].J Cell Mol Med,2016,20(3):422-429.
[20]Maddirevula S,Anuppalle M,Huh T L,et al.Nrdp1 governs differentiation of the melanocyte lineage via Erbb3b signaling in the zebrafish embryogenesis[J].Biochem Biophys Res Commun,2011,409(3):454-458.
[21]Fry W H,Simion C,Sweeney C,et al.Quantity control of the Erb B3 receptor tyrosine kinase at the endoplasmic reticulum[J].Mol Cell Biol,2011,31(14):3009-3018.
[22]Yao Y L,Shao J,Zhang C,et al.Proliferation of colorectal cancer is promoted by two signaling transduction expression patterns:Erb B2/Erb B3/AKT and MET/Erb B3/MAPK[J/OL].PLo S One,2013,8(10):e78086.doi:10.1371/journal.pone.0078086.e Collection 2013.
[23]Shi H,Gong H,Cao K,et al.Nrdp1-mediated Erb B3 degradation inhibits glioma cell migration and invasion by reducing cytoplasmic localization of p27(Kip1)[J].J Neurooncol,2015,124(3):357-364.
[24]Shi H,Du J,Wang L,et al.Lower expression of Nrdp1 in human glioma contributes tumor progression by reducing apoptosis[J].IUBMB Life,2014,66(10):704-710.
[25]Meng Q,Xia C,Fang J,et al.Role of PI3K and AKT specific isoforms in ovarian cancer cell migration,invasion and proliferation through the p70S6K1 pathway[J].Cell Signal,2006,18(12):2262-2271.