MiR-210 up-regulation inhibits proliferation and induces apoptosis in glioma cells by targeting SIN3A

Abstract

Background: The aim of this study was to determine whether miR-210 can affect the apoptosis and proliferation of human U251 glioma cells from down-regulating SIN3A.

Material and methods: The expression of miRNA-210 was detected by quantitative real-time PCR in normal brain tissue and glioma samples. The apoptosis and proliferation ability of U251 cells were analyzed by MTT and flow cytometry assay after anti-miR-210 transfection. For the regulation mechanism analysis of miR-210, TargetScan, PicTar, and microRNA were selected to predict some potential target genes of miR-210. The predicted gene was identified to be the direct and specific target gene of miR-210 by luciferase activities assay and Western blot. RNA interference technology was used to confirm that the apoptosis and proliferation effects of miR-210 were directly induced by SIN3A.

Results: The expression of miR-210 increased significantly in glioma in comparison with normal brain tissue. The silence of miR-210 expression could inhibit the proliferation of U251 cells and induce the apoptosis. Mechanism analysis revealed that SIN3A was a specific and direct target gene of miR-210. The siRNA-SIN3A could down-regulate the expression of SIN3A protein, which was up-regulated in U251 cells by anti-miR-210 transfection, and our experiments found that silence of SIN3A could inhibit the apoptosis and sharply increase the proliferation of U251 cells. The regulation effects of anti-miR-210 on apoptosis and proliferation can be reversed respectively by the expression silence of SIN3A.

Conclusions: Aberrantly expressed miR-210 regulates human U251 glioma cells apoptosis and proliferation partly through directly down-regulating SIN3A protein expression. This might offer a new potential therapeutic stratagem for glioma.

Figure 1

qRT-PCR analysis for the expression of miR-210 and SIN3A in brain glioma tissue and U251 cells. Compared to paracancerous tissue, the expression of miR-210 was higher in brain glioma tissue and U251 cells (P<0.05), and SIN3A showed significant down-regulation (P<0.05). *, ^# P<0.05 vs. paracancerous tissue.

Figure 2

(A) Impact of SIN3A gene expression changes on the proliferation ability of U251 cells. (B) Impact of SIN3A gene expression changes the apoptosis rate of U251 cells. (C) Western blot analysis for SIN3A and cleaved Caspase3 expression of U251 cells treated with anti-miR-210 and siRNA-SIN3A transfection. GAPDH was used as a reference control. (D) quantitative analysis of the relative protein levels of SIN3A and cleaved Caspase3 normalized to those of GAPDH was shown. *, ** P <0.05 vs. control U251 cells; ^#, ^## P <0.05 vs. U251 cells with anti-miR-210. IDV is the abbreviation for “integrated density values”, which is calculated by computerized image analysis system (Fluor Chen 2.0) and normalized with that of GAPDH.

Figure 3

(A) 3′UTR of SIN3A is a target of miR-210 predicted by TargetScan, microRNA and PicTar. (B) The luciferase reporter assay results with each bar representing values from three independent experiments. The transfection efficiency was normalized by co-transfected renilla luciferase and the light units were calculated by relative luciferase activity of firefly to renilla. * P<0.05. (C) Representative image of the protein level of SIN3A. GAPDH was used as a reference control. (D) quantitative analysis of the relative protein levels of SIN3A normalized to those of GAPDH was shown. Data were mean ± SD of three independent experiments. * P<0.05. ^# P<0.05.