TMZ inhibited mammalian target of rapamycin (mTOR) pathways through miR-128 targeting.

<p>(A) Schematic diagram of potential miR-128-targeted sites in the 3’-untranslated region (UTR) of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. (B) Effects of miR-128 on 3’-UTR luciferase activity of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. To examine the effect of miR-128, different doses of miR-128-expressing plasmids were cotransfected with 500 ng pmiRGlo-3’-UTR or mutant 3’-UTR of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. Luciferase activity was measured in these cells 24 h after transfection. Effects of miR-128 overexpression on mRNA (C) and protein (D) expressions of <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes. After cells were respectively transfected with the indicated dose of miR-128-expressing plasmids for 24 h, relative mRNA and protein levels of the <i>mTOR</i>, <i>RICTOR</i>, <i>IGF1</i>, and <i>PIK3R1</i> genes were analyzed through real-time PCR and immunoblotting assays. Data are the mean ± SD of three experiments. * <i>p</i> < 0.05. (E) miR-128 is involved in TMZ-reduced mTOR signaling. (F) The effects of IGF-1 stimulation on miR-128-targeted genes expression levels. (G) IGF-1 stimulation attenuated miR-128-induced cytotoxicity. After cells were transfected with 750 ng of miR-128-expressing plasmids or 100 nM of a miR-128 inhibitor, 400 μM of TMZ was added for another 24 h. For IGF-1 stimulation assays, cells were treated with 200ng/mL of IGF-1 recombinant proteins for 24 h or transfected with 500 ng of miR-128-expressing plasmids combined with 200ng/mL of IGF-1 treatment for 24 h. Protein levels of mTOR, RICTOR, IGF1, and PIK3R1 were analyzed using immunoblotting assays. Cell viability was measured using the MTT assay. Data are the mean ± SD of three experiments. * <i>p</i> < 0.05.