Nan Wu1, Guo-cai Wu, Rong Hu, Mei Li, Hua Feng. 1. Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing, China.
Abstract
AIM: To examine the influence of ginsenoside Rh2 (Rh2), a triterpene saponin extracted from the traditional medicinal plant ginseng, on the expression of miRNAs in human glioma cells. METHODS: The expression profile of miRNA (miR) was analyzed in human U251, T98MG and A172 glioma cells using a miRNA array and quantitative real-time PCR. Cell viability was assessed using a colorimetric assay (cell counting kit-8). Transfection of miR-128 was performed using Lipofectamine 2000. Caspase 3 activity was determined using a caspase colorimetric assay kit. Apoptosis was assessed using annexin V and propidium iodide staining. Protein expression was determined with Western blot analysis. miRNA-128 targeting activity was measured using a luciferase reporter assay. RESULTS: In U251 cells treated with Rh2 (12 μg/mL), 14 of 452 human miRNAs were up-regulated and 12 were down-regulated as detected with the miRNA array assay. The up-regulation of miR-128 by Rh2 was further verified in human U251, T98MG and A172 cells using quantitative real-time PCR. In U251 cells, transfection of a miR-128 inhibitor (50 nmol/L) prevented the overexpression of miR-128 by Rh2, and significantly blunted Rh2-induced cytotoxicity, apoptosis, caspase 3 activation, transcriptional activation of E2F3a, a miR-128 target gene, as well as E2F3a protein expression. CONCLUSION: The anti-proliferative effect of Rh2 in human glioma cells was mediated in part through up-regulation of miRNA-128 expression.
AIM: To examine the influence of ginsenoside Rh2 (Rh2), a triterpene saponin extracted from the traditional medicinal plant ginseng, on the expression of miRNAs in humanglioma cells. METHODS: The expression profile of miRNA (miR) was analyzed in human U251, T98MG and A172 glioma cells using a miRNA array and quantitative real-time PCR. Cell viability was assessed using a colorimetric assay (cell counting kit-8). Transfection of miR-128 was performed using Lipofectamine 2000. Caspase 3 activity was determined using a caspase colorimetric assay kit. Apoptosis was assessed using annexin V and propidium iodide staining. Protein expression was determined with Western blot analysis. miRNA-128 targeting activity was measured using a luciferase reporter assay. RESULTS: In U251 cells treated with Rh2 (12 μg/mL), 14 of 452 human miRNAs were up-regulated and 12 were down-regulated as detected with the miRNA array assay. The up-regulation of miR-128 by Rh2 was further verified in human U251, T98MG and A172 cells using quantitative real-time PCR. In U251 cells, transfection of a miR-128 inhibitor (50 nmol/L) prevented the overexpression of miR-128 by Rh2, and significantly blunted Rh2-induced cytotoxicity, apoptosis, caspase 3 activation, transcriptional activation of E2F3a, a miR-128 target gene, as well as E2F3a protein expression. CONCLUSION: The anti-proliferative effect of Rh2 in humanglioma cells was mediated in part through up-regulation of miRNA-128 expression.
Authors: George Adrian Calin; Cinzia Sevignani; Calin Dan Dumitru; Terry Hyslop; Evan Noch; Sai Yendamuri; Masayoshi Shimizu; Sashi Rattan; Florencia Bullrich; Massimo Negrini; Carlo M Croce Journal: Proc Natl Acad Sci U S A Date: 2004-02-18 Impact factor: 11.205