MiR-125b acts as an oncogene in glioblastoma cells and inhibits cell apoptosis through p53 and p38MAPK-independent pathways

Abstract

Background: We have recently identified miR-125b upregulation in glioblastoma (GMB). The aim of this study is to determine the correlation between miR-125b expression and malignant grades of glioma and the genes targeted by miR-125b.

Methods: Real-time PCR was employed to measure the expression level of miR-125b. Cell viability was evaluated by cell growth and colony formation in soft-agar assays. Cell apoptosis was determined by Hoechst 33342 staining and AnnexinV-FITC assay. The Luciferase assay was used to confirm the actual binding sites of p38MAPK mRNA. Western blot was used to detect the gene expression level.

Results: The expression level of miR-125b is positively correlated with the malignant grade of glioma. Ectopic expression of miR-125b promotes the proliferation of GMB cells. Knockdown of endogenous miR-125b inhibits cell proliferation and promotes cell apoptosis. Further studies reveal that p53 is regulated by miR-125b. However, downregulation of the endogenous miR-125b also results in p53-independent apoptotic pathway leading to apoptosis in p53 mutated U251 cells and p53 knockdown U87 cells. Moreover, p38MAPK is also regulated by miR-125b and downregulation of miR-125b activates the p38MAPK-induced mitochondria apoptotic pathway.

Conclusion: High-level expression of miR-125b is associated with poor outcomes of GMB. MiR-125b may have an oncogenic role in GMB cells by promoting cell proliferation and inhibiting apoptosis.

Figure 1

Expression of miR-125b in glioma samples and C6, U87 and U251 cells. The malignant grade of glioma was evaluated according to WHO criteria as described in Materials and Methods. Samples of ID 1–7 were normal brain tissues from patients with noncancerous disease and were used as the controls; ID 8–16 from pilocytic astrocytomas classified to WHO I; ID 17–26 from astrocytoma classified to WHO II; ID 27–35 from anaplastic astrocytomas classified to WHO III; and ID 36–48 from Glioblastoma Multiforme classified to WHO IV. Each sample was divided by a dashed line. Total RNA was isolated from the glioma tissues and glioma cells of C6, U87 and U251 and real-time PCR was performed to analyse the expression of miR-125b as described in Materials and Methods. The relative expression of miR-125b was expressed as the ratio of the expression level miR-125b to that of U6. *P<0.05 and **P<0.01, as compared with the control group.

Figure 2

Effects of miR-125b on the cell proliferation of glioblastoma. (A) Morphological observation. C6, U87 and U251 cells were treated with NC-DP, 125b-DP or 125b-AS, respectively. After incubation for 48 h, the cell morphology was observed under the microscope. (B) Determination of cell proliferation by MTT assay. C6, U87 and U251 cells were transfected with NC-DP, 125b-DP, 125b-AS or the negative antisense oligonucleotides (NC-AS), respectively. After incubation for 48 h, cell proliferation rates were analysed by MTT assay. The growth rates of cells transfected with NC-DP were defined as 1.0. **P<0.01, as compared with NC-DP group. (C) Cell-cycle assay. U87 cells were transfected with NC-DP or 125b-DP or 125b-AS, respectively. After being cultured for 48 h, cells were fixed and stained with propidium iodide and cell cycle was analysed by flow cytometry. Representative analysis of three independent experiments is shown. Statistically significant differences of S phase between the groups of 125b-DP and NC-DP or between 125b-AS and NC-DP group were observed: *P<0.05; **P<0.01. (D) Colony formation assay. U87 cells were transfected with NC-DP or 125b-DP or 125b-AS, respectively. After incubation for 7 days, the colonies formed by the transfected cells were stained with crystal violet and the number of the colonies was counted. The top panel was a representative sample from each group, while low panel indicated the quantitive results of colony formation assay. Statistically significant differences between the groups of 125b-DP and NC-DP or between 125b-AS and NC-DP group were observed: **P<0.01.

Figure 3

Knockdown of miR-125b induces apoptosis in U87 cells. U87 cells were transfected with NC-DP, 125b-DP or 125b-AS, respectively. After incubation for 72 h, cells were fixed and apoptosis was detected by Hoechst 33342 stain, TUNEL and Annexin V-FITC/PI analysis, respectively. (A) Observation of nuclear morphology. Cells were stained with Hoechst 33342 and nuclear morphology was observed under a fluorescence microscope. (B) TUNEL analysis of the apoptosis cells. TUNEL analysis was performed as described in Materials and Methods and Fluorescence microscope was used to analyse the apoptosis cells. (C) The externalisation of phosphatidylserine during the progression of apoptosis was detected. Early apoptotic cells (Annexin V+/PI−) are in the right lower quadrant. The apoptotic cells are indicated by white arrows.

Figure 4

Downregulation of miR-125b enhances the sensitivity of U87 cells to TMZ. U87 cells were transfected with NC-DP, 125b-DP or 125b-AS, respectively. After incubation for 48 h, the cells were treated with TMZ. (A) Observation of cell morphology. After being treated with TMZ, the cells were incubated for another 24 h and cell morphology was examined under a microscrope. (B) Determination of cell proliferation rate using MTT assay. U87 cells were transfected with oligonucleotides and TMZ were added into the cell culture medium. After being cultured for 24 h, the cell proliferation rates were evaluated by MTT assay. The inhibitory rates of cells were calculated using the formula: (OD value in 0.1% DMSO-treated group−OD value in TMZ and oligonucleotide treated group/OD value in 0.1% DMSO-treated group × 100%. Statistically significant differences between the groups of 125b-DP+TMZ and NC-DP+TMZ or between 125b-AS+TMZ and NC-DP+TMZ group were observed: *P<0.05 (P=0.0216) and **P<0.01 (P=0.0043). (C) Observation of nuclear morphology. Cells were stained with Hoechst 33342 and nuclear morphology was observed under a fluorescence microscope. The white arrows show the apoptotic cells with condensed, fragmented nuclei. (D) Quantities of the apoptosis cells with Annexin V-FITC/PI stain and Flow cytometry analysis. Early apoptotic cells (Annexin V+/PI−) are in the right lower quadrant.

Figure 5

The expression of p53 is regulated by miR-125b in glioma cells. (A) miR-125 downregulates p53 expression in U87 cells. U87 cells were transfected with NC-DP, 125b-DP, NC-AS or 125b-AS, respectively. After incubation for 72 h, the expression level of p53 was analysed by western blot as described in Materials and Methods. P53-specific siRNA was used as a positive control. (B) Quantitative results of p53 expression. Quantitation of signal intensities was performed using densitometry on a Hewlett-Packard ScanJet 5370 C. The percentage of p53 is calculated using the formula: the value of densitometry of p53/the value of densitometry β-actin expression (n⩾3) × 100%. The relative p53 expression level in nontransfected U87 cells was defined as 1.0. The relative expression level of p53 siRNA-treated group was used as a positive control. The two-tail Student t-test was employed to statistically analyse the results: **P<0.01. (C) Downregulation of miR-125b activates the p53 and p53 related gene expression dominant cell apoptosis. U87 cells were transfected with NC-DP, 125b-DP or 125b-AS. After incubation for 72 h, cells were collected by centrifugation at 1000 r.p.m. for 5 min and cell protein was isolated. The expression level of p53, p21, Bax, Bcl-2, caspase-3 and capsase-9 were detected by western blot. The expression of β-actin was used as a loading control.

Figure 6

Knockdown of miR-125b induces cell apoptosis independent on p53 expression. (A) Effect of UV radiation of p53 expression in U251 (p53 mutation) and U87 with p53 wild-type and p53 knock-down [U87(p53KD)] cells. (B) Downregulation of miR-125b activates p53 expression in p53 wild-type U87 cells but not in U251 and U87(p53 KD) cells. (C) Effect of 125b-AS on the proliferation of glioblastoma cell lines. 125b-AS was transfected into cells and the cell proliferation inhibitory rates were evaluated by MTT assay at 12, 24, 36, 48 and 72 h, respectively. The inhibitory rates of cells were calculated using the formula: the OD value in cells transfected with 125b-AS /to the OD value in cells transfected with NC-AS × 100%. (D) Observation of nuclear morphology. Cells were stained with Hoechst 33342 and nuclear morphology was observed under a fluorescence microscope. The white arrows showed the apoptosis cells with condensed, fragmented nuclei. (E) Quantities for the apoptosis cells with Annexin V-FITC/PI stain and Flow cytometry analysis. U251, U87 and U87(p53 KD) cells were transfected with NC-AS or 125b-AS. After incubation for 72 h, the cell apoptosis rates were detected by Annexin V-FITC/ PI stain and Flow cytometer analysis. The two-tail Student's t-test was employed to statistically analyse the results: **P<0.01. (F) Effect of 12b-AS on caspases expression in U251, U87 and U87(p53 KD) cells. Cells were treated with 125b-AS. After incubation for 72 h, cells were collected and the expression of caspase-9, cleaved caspase-9, caspase-3 and cleaved caspase-3 were detected by western blot. The expression of β-actin was used as a loading control.

Figure 7

Knockdown of miR-125b induces apoptosis in glioblastoma cells independent of p38MAPK. (A) The predicted miR-125b-binding site in the 3′-UTR of p38MAPK. The miR-125b seed sequences and their predicted binding sites in the 3′-UTR of p38MAPK 3′-UTR shown in frame. The underlined sequences represented the mutation seed sequence of miR-125b in 3′-UTR of p38MAPK. (B) miR-125b targets the 3′-UTR of p38MAPK in luciferase reporter assay. HEK-293 cells were co-transfected with 125b-DP and p38MAPK full-length 3′-UTR-luciferase reporter or p38MAPK 3′-UTR mutation luciferase reporter, respectively. After incubation for 48 h, luciferase assay was performed as described in Materials and Methods to determine the expression of p38MAPK. (C) Overexpression of miR-125b downregulates p38MAPK expression in U251, U87 and C6 cells. NC-DP and 125b-DP were transfected into U87, U251 and C6 cells and the level of p38MAPK was analysed by western blot. (D) p38MAPK is activated by knock-down of miR-125b in U87 cells. 125b-AS or 125b-DP was transfected into U87(p53 KD) cells, and treated with or without UV radiation. After being cultured for 48 h, the cells were collected and total cell proteins were isolated. The total expression of p38MAPK and phosphorylation p38MAPK (P-p38MAPK) were detected by western blot. β-Actin was used as a loading control. (E) Cell apoptotic determination with Hoechst 33342 stain in U87(p53 KD) cells transfected with 125b-AS treated with or without SB203580. U87(p53 KD) cells were transfected with 125b-AS, after incubation for 24 h, p38MAPK-specific inhibitor was added. After incubation for another 48 h, the apoptotic cells were stained by Hoechst 33342 as showed by arrows. (F) The expression of p-p38MAPK and p-p53 in U87(p53 KD) cells transfected with 125b-AS treated with or without SB203580 was determined by western blot. After incubation for 24 h, cells were treated with or without p38MAPK-specific inhibitor, SB203580. After being cultured for another 24 h, cells were collected and nuclear protein was isolated. Western blot was performed to detect the expression of p-p38MAPK and p-p53. (G) Caspases 9 and 3 expression in U87(p53 KD) cells after being transfected with 125b-AS and treated with or without SB203580. Cell treatment was described as above and the expression of caspase-9, cleaved caspase-9, caspase-3 and cleaved caspase-3 were detected by western blot. The expression of β-actin was used as a loading control.