Effects Of MiR-584 On The Invasion And Migration Of Human Glioblastoma U251 Cells

Backgrounds and ObjectivesMicroRNAs are noncoding RNAs that post-transcriptionally regulate gene expression.Existing extensive in animal and plant.More and more reseach show that microRNAs plays an important role in human tumor, regulate and control the apoptosis,proliferation,and other biological behaviour of the tumor cells.lt brightly that microRNAs will be a potential signal of diagnosis,treat and judge the effect of the tumor. Studies have confirmed that microRNAs is closely associated with tumors.The purpose of this experiment is to explore the effects of miR-584 expression on the invasion and migration of glioblastoma U251 cells in vitro.Methods(1)Cell culture conditions The human glioblastoma U251 cell lines were maintained as monolayers in medium comprising DMEM containing 10% heat inactivated fetal calf serum and incubated in 37℃,5%CO2 and saturated humidity.Cell transfection was conducted when cell convergence rate was 60% 70%.The transfaction of the U251 cells steps to follow Lipofectamine2000 instruction,5μl of siRNA per well.(2)Chemosynthetic miR-584-3p was transfected into glioblastoma cells under the induction of Lipofectamine 2000. The cells were divided into control group(no transfection), mimics NC group(transfected with negative control sequence of miR-584-3p mimics), miR-584-3p mimics group(transfected with miR-584-3p mimics),inhibitor NC(transfected with negative control sequence of miR-584-3p inhibitor), miR-584-3p inhibitor group(transfected with miR-584-3p inhibitor).(3) microRNA extraction and reverse transcription MicroRNA was extracted from cells by Trizol 48h after transfection.Reaction conditions:37℃ for 15min,98℃ for 5min,4℃ to maintain.The reverse transcription product were stored in the 20℃ refrigerator.(4)The expression of miR-584-3p was detected by Real-time PCR.The reverse transcription reaction was performed using PrimeScript RT Reagent Kit with gDNA Eraser, according to the manufacturer’s instructions where u6 as internal.Quantitative values were obtained by the threshold cycle (CT) value. Relative mean fold change in expression ratios was calculated by the 2-△△CT method.(5)Cell proliferation was detected by CCK-8 proliferation assay.(6)The invasive and migratory effects of glioblastoma cell were measured by Wound healing and Transwell assays.ResultsCompared with mimics negative control group and control group, the expression of miR-584-3p in miR-584-3p mimics group was upregulated by 100 times(P<0.05). Compared with mimics negative control group and control group, the cell proliferation ability of miR-584-3p mimics group showed no significant change(P>0.05), but the cellular migratory and invasive activity of miR-584-3p mimics group were down-regulated significantly(P<0.05). Compared with inhibitor negative control group and control group, the expression of miR-584-3p in miR-584-3p inhibitor group was down-regulated by 20 times(P<0.05). Compared with inhibitor negative control group and control group, the cell proliferation ability of miR-584-3p inhibitor group showed no significant change(P>0.05), the cellular migration and invasive activity in miR-584-3p inhibitor group were up-regulated significantly(P<0.05). ConclusionsThe migration and invasion of glioblastoma cells can be inhibited by the over-expression of miR-584-3p, while the transfection of miR-584-3p inhibitor can enhance the migration and invasion of glioblastoma cells. Thus,miR-584-3p may serve as a attractive target of gene therapy for glioblastoma.