The Influence Of Silencing The Expression Of TPD52 In Glioma Cells And MiRNA-34a Modulat The Cell Cycle By Targeting TPD52 In Glioma U87 Cells

Obiective(1)Explore TPD52 genes in glioma and normal brain tissue and the expression level of U87 cells;(2)Silence after TPD52 gene expression on glioma U87 cell proliferation,cycle,and the effect of apoptosis;(3)Silence after TPD52 gene expression on glioma U87 attacks,the effects of migration;(4)Explore the mi RNA-34 a gliomas in the organization and expression level of U87 cells;(5)To explore the mi RNA-34 a of glioma cells in cell cycle regulation;(6)Explore the mi RNA-34 a through targeted TPD52 glioma U87 cell cycle adjustment.Methods(1)Collected in 12 cases of gliomas specimens,12 patients with traumatic brain injuries were normal brain biopsy specimens,using fluorescent quantitative PCR detection TPD52 m RNA expression,using Western blot detection TPD52 protein expression.Application of fluorescence quantitative PCR to detect the expression of micrornas-34 a quantity.(2)Will be carrying the sh RNA-tpd52(inhibiting tpd52 nucleotide sequence),sh RNA-NC(negative control sequence of nucleotides)slow virus in vitro transfection U87 glioma cell line.Fluorescence microscope after 48 h transfection expressing GFP cells,using fluorescence quantitative PCR,Western blot detection respectively TPD52 m RNA and protein expression,determined by MTT test cell proliferation ability,flow cytometry to detect the cell cycle distribution,Annexin V and PI double flow cytometry to detect cell apoptosis,scratch test cell migration ability,Transwell experimental detection cell invasive ability.(3)Mi RNA34 a simulation and micrornas-34 a inhibitor transfection into U87 cells,using fluorescence quantitative PCR detection turn and then the expression of cell micrornas-34 a level.Refer to relevant literature and the use of biological information analysis and prediction of network TPD52 3,UTR is the role of micrornas-34 a target,adopts the luciferase report experiments verify whether TPD52 micrornas-34 a target genes.U87 cells treated with different factors,using the fluorescent quantitative PCR detection TPD52 and related protein m RNA expression level;Using Western blot test TPD52 protein expression levels and related proteins;Using flow cytometry instrument detection U87 cell of the cell cycle.Result(1)Fluorescence quantitative PCR detection results showed tpd52 m RNA ?-? grade glioma group(? CT0.73 + /-0.47),U87 cell(? CT0.74 + /-0.32)expression levels were significantly higher than those in the normal brain tissue(? CT1.92 + /-0.42)(P < 0.05);And the U87 cells and ?-? grade glioma tissues tpd52 m RNA there was no statistically significant difference expression level.Western blot test results show that TPD52 quantity of protein expression in ?-? grade glioma group,expression level of U87 cells significantly higher than the normal brain tissue expression level.(2)Lentivirus U87 cell transfection rate through carrying the GFP expression to evaluate,through cell count under fluorescent microscope cell transfection rate of > 90%.Compared with sh RNA-NC group and blank control group,sh RNA-tpd52 group cell tpd52 m RNA and protein expression quantity decreased significantly(P < 0.01),and cell proliferation capacity decreased obviously,the difference was statistically significant(P < 0.05);Cell cycle block in G0 / G1 phase and cell apoptosis rate increased significantly(P < 0.05),the ability of cell migration decreased obviously difference was statistically significant(P < 0.05);Cell invasion ability decreased obviously difference was statistically significant(P < 0.05).(3)Fluorescence quantitative PCR detection results show the mi RNA-34 a ?-? grade glioma group(? CT3.26 + /-0.24),U87 cell(? CT3.45 + /-0.12)expression level was lower than that in normal brain tissue(? CT1.94 + /-0.40)(P < 0.05);U87 cells and ? ? grade glioma tissues of micrornas-there was no statistically significant difference 34 a expression level.(4)U87 cells after transfection,the cycle experiments found that transfection of micrornas-34 a analog can obviously make U87 cell for the number of cells in S phase proportion significantly reduced to 16.12%,P < 0.05,and transfection of micrornas-34 a inhibitor can obviously make U87 cell for the number of cells in S phase proportion increased 43.56%,P < 0.05).Dual luciferase reporter gene transfection experiment found the mi RNA-34 a analog can obviously make TPD52 U87 cells 3,UTR activity significantly decreased 32%,P < 0.05,and transfection of micrornas-34 a inhibitor can obviously make TPD52 U87 cells 3,UTR activity significantly increased to 58%,P < 0.05).conclusion(1)TPD52 in glioma tissues expression level is higher than the normal brain tissue.(2)Silence tpd52 gene expression,inhibits the proliferation of glioma cells,promote cell apoptosis,block the cell cycle.(3)Silence tpd52 gene expression,inhibits glioma cell migration ability and the ability.(4)Micrornas-34 a in glioma tissues expression level lower than the normal brain tissue.(5)Micrornas-34 a can adjust the cell cycle of glioma cells,inhibition of glioma cells S phase proportion.(6)Micrornas-34 a has a directly targeted TPD52 glioma cell cycle adjustment.