Ginsenoside Rh2 inhibits glioma cell proliferation by targeting microRNA-128

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.

Figure 1

Chemical structure of ginsenoside Rh2 (20R-form).

Figure 2

Validation of miRNA expression by quantitative real-time PCR. (A) The expressions of up-regulated miR-128 and miR-15b, as well as the down-regulated miR-21 and miR-25 in U251 cells were verified. The results of quantitative RT-PCR for the four selected miRNAs were consistent with the miRNA array results. (B) The expressions of miR-128 and miR-21 in human T98MG and A172 glioma cells were determined. The consistent results in these two giloma cells were observed as compared to U251 cells. The cells were treated with 12 μg/mL Rh2 for 24 h. Data were expressed as means±SEM of 3 independent experiments. Fold change of miRNA expression was presented in log 2 scale.

Figure 3

Transfection of miR-128 inhibitor significantly suppressed Rh2-induced miR-128 over-expression in U281 cells (A), T98MG cells (B), and A172 cells (C). The effects of miR-128 inhibitor transfection on Rh2-induced miR-128 overexpression in human U251, T98MG and A172 glioma cells were determined using qRT-PCR. The cells were transfected with 50 nmol/L miR-128 inhibitor or inhibitor control for 48 h, and then with Rh2 (12 μg/mL) for 24 h. The experiment was performed three times, each in triplicate. Data were expressed as means±SEM. ^bP<0.05 compared with control. ^eP<0.05 compared with Rh2 alone or miR-128 inhibitor control+Rh2.

Figure 4

Rh2 inhibited viability of human glioma U251 cells determined using a CCK-8 assay. (A) Dose-dependent response. U251 cells were treated with Rh2 (3, 6, 12 and 25 μg/mL) for 48 h. (B) Time-dependent response. U251 cells were treated with Rh2 (12 μg/mL) for 12, 24, 48 and 72 h. The results represented three independent experiments. Data were expressed as means±SEM. ^bP<0.05 compared with control.

Figure 5

Transfection of miR-128 inhibitor prevented Rh2-induced cytotoxicity in glioma U251 cells. U251 cells were transfected with 50 nmol/L miR-128 inhibitor to knockdown miR-128. A miR-128 inhibitor control was used as negative control. At 48 h after transfection, the cells were treated with 12 μg/mL Rh2 for 24 or 48 h. Cell proliferation was determined using CCK-8 assay. ^bP<0.05 compared with Rh2+miR-128 inhibitor. The experiment was performed three times, each in triplicate.

Figure 6

Transfection of miR-128 inhibitor inhibited Rh2-induced apoptosis in U251 cells. The cells were transfected with 50 nmol/L miR-128 inhibitor or inhibitor control for 48 h and then with Rh2 (12 μg/mL) for 48 h. Apoptosis was evaluated by annexin V and propidium iodide staining. The LR and UR quadrants indicated the percentage of early and late apoptotic cells, respectively, in control (A), Rh2 alone (B), Rh2+miR-128 inhibitor control (C) and Rh2+miR-128 inhibitor group (D). The experiment was performed three times, each in triplicate. Each point represented the means±SEM. There were significant differences between Rh2 alone or Rh2+miR-128 inhibitor control and control group, as well as between Rh2 alone and Rh2+miR-128 inhibitor group (P<0.05).

Figure 7

Transfection of miR-128 inhibitor inhibited Rh2-induced caspase 3 activation in U251 cells. (A) Transfection of miR-128 inhibitor inhibited the caspase 3 activation induced by treatment with 6, 12, and 25 μg/mL Rh2 for 48 h. The cells were transfected with 50 nmol/L miR-128 inhibitor or inhibitor control for 48 h, and then with Rh2 for 48 h. (B) Transfection of miR-128 inhibitor inhibited the caspase 3 activation induced by treatment with 12 μg/mL Rh2. The cells were transfected with 50 nmol/L miR-128 inhibitor or inhibitor control for 48 h, and then with Rh2 for 24, 48, and 72 h. Caspase 3 avtivity was determined using colorimetric assay. The experiment was performed three times each in triplicate. Mean±SEM. ^bP<0.05 compared with control or miR-128 inhibitor+Rh2.

Figure 8

Rh2 suppressed the activity of luciferase reporter containing the E2F3a-3′-UTR and the level of E2F3a protein in U251 cells. (A) Rh2 inhibited the luciferase activity of E2F3a-3′-UTR. The activity of luciferase reporter containing the E2F3a-3′-UTR sequence was measured by TECAN Genios multifunctional microplate reader. The cells were transfected with 50 nmol/L E2F3a 3′-UTR plasmid or empty E2F3a 3′-UTR plasmid for 48 h using Lipofectamine 2000 (Invitrogen). After transfection, the cells were treated with Rh2 for 24 h, and the luciferase assay was performed. The experiment was performed three times, each in triplicate. Mean±SEM. ^bP<0.05, ^cP<0.01 compared to E2F3a 3′-UTR plasmid alone. (B) Transfection of miR-128 inhibitor prevented Rh2-induced inhibition of E2F3a protein expression in U251 cells, as compared with Rh2+miR-128 inhibitor control. The cells were transfected with 50 nmol/L miR-128 inhibitor for 48 h and then with 12 μg/mL Rh2 for 24 h. β-actin was used as a loading control in Western blot. ^bP<0.05 as compared to control or Rh2+miR-128 inhibitor.

Figure 9

Sequence of miR-128 and its target site in the 3′-UTR of E2F3a.