MicroRNA-34a inhibits human brain glioma cell growth by down-regulation of notch1

Abstract

The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.

Keywords

microRNA-34a / glioma / Notch1 / proliferation / apoptosis

References

[1] AllenJ.C.. Childhood brain tumors: current status of clinical trials in newly diagnosed and recurrent disease. Pediatr Clin North Am, 1985, 32(3): 633-651

[2] Van MeirE.G., HadjipanayisC.G., NordenA.D., et al.. Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma. CA Cancer J Clin, 2010, 60(3): 166-193

[3] YuJ., PoultonJ., HuangY.C., et al.. The hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity. PLoS One, 2008, 3(3): e1761

[4] JiangL., WuJ., ChenQ., et al.. Notch1 expression is upregulated in glioma and is associated with tumor progression. J Clin Neurosci, 2011, 18(3): 387-390

[5] WangZ., AhmadA., LiY., et al.. Targeting Notch to Eradicate Pancreatic Cancer Stem Cells for Cancer Therapy. Anticancer Res, 2011, 31(4): 1105-1113

[6] MieleL.. Notch signaling. Clin Cancer Res, 2006, 12(4): 1074-1079

[7] InuiM., MartelloG., PiccoloS.. MicroRNA control of signal transduction. Nat Rev Mol Cell Biol, 2010, 11(4): 252-263

[8] ChenC.Z., LiL., LodishH.F., et al.. MicroRNAs modulate hematopoietic lineage differentiation. Science, 2004, 303(5654): 83-86

[9] WelchC., ChenY., StallingsR.L.. MicroRNA-34a functions as a potential tumor suppressor by inducing apoptosis in neuroblastoma cells. Oncogene, 2007, 26(34): 5017-5022

[10] DuttaK.K., ZhongY., LiuY.T., et al.. Association of microRNA-34a overexpression with proliferation is cell type-dependent. Cancer Sci, 2007, 98(12): 1845-1852

[11] LiW.B., MaM.W., DongL.J., et al.. MicroRNA-34a targets notch1 and inhibits cell proliferation in glioblastoma multiforme. Cancer Biol Ther, 2011, 12(6): 477-483

[12] LivakK.J., SchmittgenT.D.. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25(4): 402-408

[13] ChioccaE.A., LawlerS.E.. The many functions of microRNAs in glioblastoma. World Neurosurg, 2010, 73(6): 598-601

[14] WongJ.W.. MicroRNA-induced silencing of glioma progression. J Neurosci, 2010, 30(11): 3868-3869

[15] HébertS.S., De StrooperB.. Alterations of the microRNA network cause neurodegenerative disease. Trends Neurosci, 2009, 32(4): 199-206

[16] DenliA.M., TopsB.B., PlasterkR.H., et al.. Processing of primary microRNAs by the Microprocessor complex. Nature, 2004, 432(7014): 231-235

[17] SassenS., MiskaE.A., CaldasC.. MicroRNA: implications for cancer. Virchows Arch, 2008, 452(1): 1-10

[18] ZhangB., PanX., CobbG.P., et al.. microRNAs as oncogenes and tumor suppressors. Dev Biol, 2007, 302(1): 1-12

[19] PangR.T., LeungC.O., YeT.M., et al.. MicroRNA-34a suppresses invasion through downregulation of Notch1 and Jagged1 in cervical carcinoma and choriocarcinoma cells. Carcinogenesis, 2010, 31(6): 1037-1044

[20] JiQ., HaoX., MengY., et al.. Restoration of tumor suppressor miR-34 inhibits human p53-mutant gastric cancer tumorspheres. BMC Cancer, 2008, 8: 266

[21] CarthewR.W.. Gene regulation by microRNAs. Curr Opin Genet Dev, 2006, 16(2): 203-208

[22] BerezikovE., GuryevV., van de BeltJ., et al.. Phylogenetic shadowing and computational identification of human microRNA genes. Cell, 2005, 120(1): 21-24

[23] BetelD., WilsonM., GabowA., et al.. The microRNA.org resource: targets and expression. Nucleic Acids Res, 2008, 36: D149-D153

[24] LiY., GuessousF., ZhangY., et al.. MicroRNA-34a inhibits glioblastoma growth by targeting multiple oncogenes. Cancer Res, 2009, 69(19): 7569-7576

[25] MilnerL.A., BigasA.. Notch as a mediator of cell fate determination in hematopoiesis: evidence and speculation. Blood, 1999, 93(8): 2431-2448

[26] ZhangX.P., ZhengG., ZouL., et al.. Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells. Mol Cell Biochem, 2008, 307(1–2): 101-108

[27] KatohM., KatohM.. Integrative genomic analyses on HES/HEY family: Notch-independent HES1, HES3 transcription in undifferentiated ES cells, and Notch-dependent HES1, HES5, HEY1, HEY2, HEYL transcription in fetal tissues, adult tissues, or cancer. Int J Oncol, 2007, 31(2): 461-466

[28] Zweidler-McKayP.A.. Notch signaling in pediatric malignancies. Curr Oncol Rep, 2008, 10(6): 459-468

[29] WangQ., LiH., LiuN., et al.. Correlative analyses of notch signaling with resveratrol-induced differentiation and apoptosis of human medulloblastoma cells. Neurosci Lett, 2008, 438(2): 168-173

[30] FanX., MikolaenkoI., ElhassanI., et al.. Notch1 and notch2 have opposite effects on embryonal brain tumor growth. Cancer Res, 2004, 64(21): 7787-7793

[31] Fèvre-MontangeM., ChampierJ., DurandA., et al.. Microarray gene expression profiling in meningiomas: differential expression according to grade or histopathological subtype. Int J Oncol, 2009, 35(6): 1395-1407

[32] MonkC.E., HutvagnerG., ArthurJ.S.. Regulation of miRNA transcription in macrophages in response to Candida albicans. PLoS One, 2010, 5(10): e13669

[33] JiQ., HaoX., ZhangM., et al.. MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells. PLoS One, 2009, 4(8): e6816