Overexpression of miR-382-5p Overexpression Mediated Downregulation of PTEN on Malignant Biological Behavior of Human Glioma U251 Cells

ZHAO Shu-peng, JI Ying-hua, GUAN Qing-kai, MA Ji-wei, JIN Cai-ling, WANG Xiang-yang, ZHAO Xin-li

Abstract

To investigate the effect of overexpression of miR-382-5p overexpression on malignant biological behavior of human glioma U251 cells.  Methods  U251 cells were transfected with miR-382-5pmimic. Then miR-382-5p and PTEN mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) after transfection. Used bioinformatics to predicted the presence of base binding sites between miR-382-5p and PTEN, and constructed PTEN pcDNA vector overexpression plasmid was constructed. Luciluciase reporting experiment was used to detect the targeting relationship between miR-382-5p and PTEN. Cells were randomly divided into four groups: control group, mimics group, pc-PTEN group and mimics+pc-PTEN group for follow-up experiments. RT-PCR was carried out to detect the level of PTEN mRNA in each group. Cell proliferation was detected by clone formation method. The mRNA levels of Ki67, Survivin and c-Myc were detected by RT-PCR. Transwell experiment was used to assayed cell invasion ability. The expression levels of E-cadherin, N-cadherin and Vimentin were determined by Western blot.  Results  Results showed that miR-382-5p directly targeted PTEN. Compared with the control group, miR-382-5p and c-Myc mRNA levels and E-cadherin protein level were increased (P<0.05), PTEN, Ki67 and Survivin mRNA levels were decreased (P<0.05), cell clonal formation rate and cell invasion number were decreased (P<0.05), N-cadherin and Vimentin protein levels were decreased (P<0.05) in the mimics group; In pc-PTEN group, miR-382-5p mRNA and c-Myc mRNA levels and E-cadherin protein level were decreased (P<0.05), PTEN, Ki67 and Survivin mRNA levels were increased (P<0.05), cell clonal formation rate and cell invasion number were increased (P<0.05), N-cadherin and Vimentin protein levels were increased (P<0.05). Compared with pc-PTEN group, PTEN, Ki67 and Survivin mRNA levels, the cell clone formation rate, the number of invasion cells and the N-cadherin and Vimentin levels of mimics+PC-PTEN group decreased significantly (P<0.05), while the c-Myc mRNA level and E-cadherin protein level increased significantly (P<0.05).  Conclusion  Overexpression of miR-382-5p mediates the downregulation of PTEN expression, causing the inhibition of the proliferation, invasion, growth and EMT of U251 glioma cells.

 

Keywords: Glioma, miR-382-5p, PTEN / EMT


Full Text:

PDF


References


OSTROM Q T， GITTLEMAN H， FULOP J， et al. Cbtrus statistical report: primary brain and central nervous system tumors diagnosed in the united states in 2008−2012. Neuro Oncol，2015，17(Suppl 4): iv1–iv62.

OSTROM Q T， GITTLEMAN H， STETSON L， et al. Epidemiology of gliomas. Cancer Treat Res，2015，163: 1–14.

WANG J， CHEN C， YAN X， et al. The role of miR-382-5p in glioma cell proliferation, migration and invasion. Onco Targets Ther，2019，12: 4993–5002.

ZHANG Y， CHEN J， XUE Q， et al. Prognostic significance of micrornas in glioma: a systematic review and meta-analysis. Biomed Res Int， 2019， 2019:4015969[2020-02-24]. https://doi.org/10.1155/2019/4015969.

VERHAAK R G， HOADLEY K A， PURDOM E， et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell，2010，17(1): 98–110.

SCHWARZENBACH H， NISHIDA N， CALIN G A， et al. Clinical relevance of circulating cell-free micrornas in cancer. Nat Rev Clin Oncol，2014，11(3): 145–156.

PANNER A， CRANE C A， WENG C， et al. A novel PTEN-dependent link to ubiquitination controls flips stability and trail sensitivity in glioblastoma multiforme. Cancer Res，2009，69(20): 7911–7916.

FILIPPI-CHIELA E C， VILLODRE E S， ZAMIN L L， et al. Autophagy interplay with apoptosis and cell cycle regulation in the growth inhibiting effect of resveratrol in glioma cells. PLoS One， 2011， 6(6):e20849[2019-08-07]. https://doi.org/10.1371/journal.pone.0020849.

NIIKURA N， IWAMOTO T， MASUDA S， et al. Immunohistochemical Ki67 labeling index has similar proliferation predictive power to various gene signatures in breast cancer. Cancer Sci，2012，103(8): 1508–1512.

KIM W， RYU J， KIM J E. CCAR2/DBC1 and Hsp60 positively regulate expression of Survivin in neuroblastoma cells. Int J Mol Sci，2019，20(1): 131.

VARUGHESE R K， TORP S H. Survivin and gliomas: a literature review. Oncol Lett，2016，12(3): 1679–1686.

DANG C V， O'DONNELL K A， ZELLER K I， et al. The c-Myc target gene network. Semin Cancer Biol，2006，16(4): 253–264.

DING Z， LIU X， LIU Y， et al. Expression of far upstream element (fuse) binding protein 1 in human glioma is correlated with c-Myc and cell proliferation. Mol Carcinog，2015，54(5): 405–415.

CUDDAPAH V A， ROBEL S， WATKINS S， et al. A neurocentric perspective on glioma invasion. Nat Rev Neurosci，2014，15(7): 455–465.

MEHRAN R， NILSSON M， KHAJAVI M， et al. Tumor endothelial markers define novel subsets of cancer-specific circulating endothelial cells associated with antitumor efficacy. Cancer Res，2014，74(10): 2731–2741.

LI X， LIU Y， GRANBERG K J， et al. Two mature products of miR-491 coordinate to suppress key cancer hallmarks in glioblastoma. Oncogene， 2015，34(13): 1619–1628.

THIERY J P， SLEEMAN J P. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol，2006，7(2): 131–142.

SCANLON C S， VAN TUBERGEN E A， INGLEHART R C， et al. Biomarkers of epithelial-mesenchymal transition in squamous cell carcinoma. J Dent Res，2013，92(2): 114–121.


Refbacks

  • There are currently no refbacks.