MiR-29a Inhibits Glioma Tumorigenesis through a Negative Feedback Loop of TRAF4/Akt Signaling

Figure 4

MiR-29a-3p directly targets TRAF4. (a) The wild-type 3’-UTR of TRAF4 contains one predicted miR-29a-3p binding site in green. The mutagenesis nucleotides are indicated in red. (b) Dual-luciferase reporter assay. A172 cells were transfected with wild-type 3’-UTR-reporter or mutant 3’-UTR reporter together with control or miR-29a mimics. Relative firefly luciferase expression was normalized to renilla luciferase. (c) A172 or U251 cells were transfected with miR-29a mimics or inhibitors. 48h later, cells were harvested for qRT-PCR to detect the mRNA level of TRAF4. (d) Protein expression analysis of TRAF4 and p-Akt was performed by western blot in A172 and U251 cells treated as (c). (e) Protein expression analysis of TRAF4 was performed by western blot in noncancerous brain tissues and glioma samples with different tumor grades (Grade I+II and Grade III+IV). (f) Linear correlation between miR-29a and TRAF4 expressions in noncancerous brain tissues and glioma samples. Statistical analysis was performed by Pearson’s correlation analysis. : P<0.05; : P<0.01 versus control group.

(a)

(b)

(c)

(d)

(e)

(f)