Fig. 4
miR-449a reduces cell proliferation and migration by suppressing Ccnd1, and neural phenotypes and apoptosis by suppressing GPR158 in mBTSC. a miR-449a reduces Ccnd1 levels in mBTSC: miR-449a antagomir (ant) treatment of mi449ahigh Rb/p53 mBTSC restores Ccnd1 expression, and conversely miR-449a mimic (mim) treatment of Pten/p53 miR-449alow mBTSC reduces Ccnd1 expression. Scr = scramble (b) Transient transfection of Rb/p53 mBTSC with a Ccnd1 expression vector results in twofold overexpression of Ccnd1 and increased cell proliferation, and c knockdown decreases it. d Forced Ccnd1 overexpression (transfection) antagonises miR-449a -mediated inhibition of cell proliferation. Top curve (grey) baseline Pten/p53 (miR-449alow), bottom curve miR-449a knockdown, and middle curve miR-449a kd + Ccnd1 restore. e Ccnd1 accelerates cell proliferation in a confluence assay: Pten/p53 cells (grey) grow faster than Rb/p53 cells (orange). Ccnd1 overexpression increases proliferation of Rb/p53 cells (red) which now proliferate faster than Pten/p53 cells. These grow slower than untransfected cells Rb/p53 cells (orange) when Ccnd1 is inhibited (black). f Inhibition of Ccnd1 reduces outgrowth of tumour sphere processes, demonstrating the role of Ccnd1 in cell proliferation and migration. G-S, effects of overexpression or inhibition of Gpr158 in Rb/p53 or Pten/p53 mBTSC (g) Gpr158 levels in naïve and Gpr158 transfected Rb/p53 or Pten/p53 mBTSC. h Gpr158 downregulates cell proliferation, i cell migration and j self-renewal proportionally in Rb/p53 or Pten/p53 mBTSC. A 2-fold decrease of tumour sphere forming cells was observed upon Gpr158 overexpression. BTSCs stably expressing Gpr158 = 1/6; BTSCs control = 1/3; i.e., requiring the presence of 3 cells to form 1 neurosphere in controls, vs. 6 cells to form a sphere in Gpr158 overexpressors (p = 0.02) n = 12; p = 0.02. k Suspension culture of mBTSCs in serum-free medium. Upon stable expression of Gpr158, mBTSC attach to the surface of the cell culture well, change morphology and involute/grow slower. l The Caspase-3/7 activity assay indicates that Gpr158 significantly increases apoptosis in mBTSC. m Knock-down of Gpr158 using siRNA in mouse BTSCs, confirmation of abolition of Gpr158 mRNA expression. Down-regulation of Gpr158 promotes cell proliferation (n), migration (o) and tumour sphere forming ability (p). q Stable expression of Gpr158 significantly upregulates expression of neural genes, assessed in a mouse neurogenesis qPCR profiler array, while siRNA knock-down of Gpr158 significantly reduces the expression of Map2, Sox2 and Pdgfra. r Stable knock-down of GPR158 in three human GBM primary cell lines cultured in serum-free medium, containing hBTSC reduces BTSC apoptosis. s Overexpression of GPR158 significantly increases apoptosis of human GBM primary cells (hBTSC). All figures: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 (Student’s t-test). Each assay was performed at least twice
