Real-time quantitative RT–PCR

Total RNA was extracted from the frozen tissue samples or cultured cells using the TRIzol kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. Stem-loop RT–PCR (P/N: 000449; TaqMan MicroRNA Assays; Applied Biosystems, Foster City, CA, USA) was used to quantitate miR-125b according to the manufacturer's instructions. TaqMan probes and primers for p38MAPK were obtained from Applied Biosystems (P/N: Hs01047706_m1; Applied Biosystems) and the PCR reaction condition followed the manufacturer's protocol.

All reactions were performed in duplicate and a negative-control lacking cDNA was included. U6 snRNA (P/N: 001973; Applied Biosystems) and GAPDH (P/N: Hs02758991_g1; Applied Biosystems) were used as internal controls, respectively, to analyse the expression of miR-125b and p38MAPK. The relative expression level of miR-125b and p38MAPK was calculated using the delta–delta Ct methods.

Synthesis of luciferase reporters and the p53 shRNA expression vector

The 3′-UTR of human p53 and p38MAPK was amplified using the total cDNA of U87 cells as the template and then inserted into the psiCheck2 vector to synthesise the luciferase reporter constructs, Luc+wt p53 3′-UTR and Luc+wt p38MAPK 3′-UTR, respectively. The sense and antisense sequences of the MRE were synthesised, annealed and linked into the psiCheck2 vector to construct miR-125b MRE luciferase reporter Luc+miR-125b MRE. The construct Luc+125b mutated MRE, which introduced some mutations in the seed region of miR-125b MRE, was synthesised by ligating the mutation MRE fragment of miR-125b into the psiCheck2 vector as described previously (Le et al, 2009b). pSilencer4.1-CMV/neo vector (Life Technologies, Carlsbad, CA, USA) was used to construct the p53 short hairpin RNA (shRNA) expression vector. Gene-specific shRNAs targeting p53 were ligated the downstream of the CMV promoter. The sequences of shRNA p53 are as follows: Forward: 5′-GACTCCAGTGGTAATCCTAC-3′reversal: 5′-GTAGGATTACCACTGGAGTCTT-3′.

The forward and reverse synthetic oligonucleotides were annealed and inserted into the HindIII and BamHI sites of the pSilencer4.1-CMV/neo vector to construct the p53 shRNA expression vectors, pSilencer-p53.

Transfection and drug treatments

The two miRNA mimics used in the experiments were purchased from Dharmacon (Dharmacon, Lafayette, CO, USA). One of the microRNA mimics is microRNA duplex (NC-DP, MIMAT0000039) as negative control and another is 125b-DP, which is a duplex of miR-125b (MIMAT0000423). The antisense of two microRNAs, negative antisense of negative control microRNA (NC-AS) and miR-125b antisense (125b-AS) were also purchased from Dharmacon. MiRNA duplexes and antisense oligonucleotides were transfected at a final concentration of 50 and 100nM, respectively. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent following the manufacturer's protocol. The expression level of miR-125b was analysed by real-time RT–PCR. Luciferase reporter constructs were transfected into HEK293 cells at a final concentration of 0.5mgml⁻¹. To construct p53 knock-down cell line U87(p53 KD), pSilencer-p53 plasmid was transfected into U87 cells with Lipofectamine 2000 (Invitrogen) and U87(p53 KD) cell line was established by continuous selection with G418 (Invitrogen). Temozolomide (Sigma-Aldrich, St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich). U87 Cells with or without miRNAs were treated with TMZ in certain concentrations. After being cultured for 6h, cells were collected and washed with drug-free medium and allowed to grow for another 24h. Then, cells were collected to determine the viability and apoptosis using morphological analysis or MTT assay.

Luciferase reporter assay

Luciferase reporter construct was co-transfected with 50nMl⁻¹ of miRNA duplexes or 100nMl⁻¹ miRNA antisenses into HEK-293T cells in a 24-well plate Lipofectamin-2000 (Invitrogen). After incubation for 48h, the cells were harvested by centrifugation at 1000g for 10min and lysed using lysis buffer. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase reporter system (Promega, Madison, WI, USA) following the manufacturer's protocol.

MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro-121 mide) assay

C6, U251, U87 and U87 (p53-) cells were plated onto 96-well plates with 5 × 10³ cells per well and allowed to adhere overnight. The cells were transfected with 125b-DP at the final concentration of 50nMl⁻¹ or 125b-AS at the final concentration of 100nMl⁻¹ using Lipofectamine 2000 reagent (Invitrogen). After incubation for 48h, MTT dye (30μl, 5mgml⁻¹, Sigma-Aldrich) was added into the culture medium. After incubation at 37°C for 4h, the MTT solution was removed and 150μl DMSO was added to dissolve the formazan crystals. The OD value at 490nm was measured by Multiskan EX microplate photometer (Thermo Fisher Scientific, Waltham, MS, USA).

Colony formation in Soft-agar

U87 cells were treated with olionucleodies for 24h and the cells were harvested by trypsinisation, and diluted to a density of 2000cells per ml. The cells were collected and suspended in 1ml of 0.3% melted agar in DMEM with 10% FBS and plated in 60-mm dishes overlayed with 0.6% agar in the same medium. Medium was changed every 3 days. After being cultured for 7 days, colonies containing at least 50 cells were counted and stained with 0.1% crystal violet (Sigma-Aldrich).

Apoptosis assay

Cell apoptosis was detected by Hoechst 33342 staining, terminal deoxynucleotidyl transferase–mediated nick-end labelling (TUNEL) assay and Annexin V-FITC assay. For Hoechst 33342 staining, cells were transfected with oligonucleotides or treated with drugs and washed with PBS twice, fixed with 70% ethanol for 30min at room temperature and then stained with Hoechst 33342 (1μgml⁻¹) for 5min. Chromatin fluorescence was observed under a UV-light microscope, and apoptotic cells were morphologically defined by cytoplasmic and nuclear shrinkage and chromatin condensation (Zhu et al, 2006). For TUNEL assay, cells were cultured on L-polylysine coated slides and oligonucleotides were transfected into cells. After treatment, the cells on the slides were fixed and analysed using the DeadEnd fluorometric TUNEL system (Promega) according to the manufacturer's protocol. Apoptotic cell death was quantified by flow cytometry with Annexin V-FITC and PI staining. Annexin V-FITC apoptosis detection kit (Invitrogen) was used according to the manufacturer's instructions. Briefly, both attached and floating cells were collected and resuspended in binding buffer before adding the Annexin V-FITC antibody and propidium iodide. Stained cells were analysed by flow cytometry (Beckman Coulter, Brea, CA, USA).

UV irradiation

The UV irradiation experiment was performed as previously described (Zhao et al, 2012). Briefly, cells were transfected with 125b-DP, NC-DP or 125b-AS at the final concentration of 100nMl⁻¹. After being cultured for 12h, cells were washed with PBS and irradiated with 15Jm⁻² UVC for 4h and then supplemented with fresh medium.

Western blot analysis

Cells were lysed in RIPA buffer (Solaibo, Beijing, China). Proteins were separated by a 10% polyacrylamide gel and transferred to a methanol-activated PVDF membrane (GE Healthcare, Little Chalfont, UK). The membrane was blocked in blocking solution (5% nonfat dry milk powder) for 2h at room temperature and subsequently probed with primary antibodies; including p53, p21, Bax, Bcl-2, Caspaese-3. Caspase-9, p38MAPK or β-actin; (used at a 1/1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three 10min washes with 0.1% Tween-20 in PBS buffer, membranes were incubated with rabbit anti mouse or goat anti rabbit HRP-conjugated secondary antibody (Santa Cruz) for 1h. After an additional three 10min washes with 0.1% Tween-20 in PBS buffer, the chemiluminescence method was employed to detect the signals using Super Signal West Dura (Thermo Scientific) and protein bands were visualised by autoradiography. Quantitation of signal intensities was performed using densitometry on a Hewlett-Packard ScanJet 5370C (Hewlett-Packard, Palo Alto, CA, USA) with NIH image 1.62 software (http://rsb.info.nih.gov/nih-image).

MiR-125b promotes the cell proliferation and survival of GMB cells

We next investigated the effect of miR-125b on the proliferation of GMB cells. MiR-125b duplex (125b-DP) or 2′-omethoxyethy (2′-O-MOE) modified miR-125b anti-sense (125b-AS) were transfected into rat GMB C6 cells and human GMB U87 and U251 cells to upregulate or downregulate the expression of miR-125b, respectively. The miR-125b expression level was detected by quantitative RT–PCR (qRT–PCR) in each cell line. The level of miR-125b was increased about fourfold with transfection of 125b-DP, while it was decreased more than fivefold with transfection of 125b-AS compared with the endogenous miR-125b level (Supplementary Figure 1). These results suggest that both 125b-DP and 125b-AS are effective in modulating the level of miR-125b. We then determined the cell proliferation by morphological changes and MTT assay. As shown in Figure 2A, cell proliferation was promoted by overexpression of miR-125b in all of the three cell lines, including C6, U87 and U251 cells. In contrast, cell proliferation was reduced by knockdown of endogenous miR-125b in C6, U87 and U251 GMB cells (Figure 2A). MTT assay revealed that after treating the cells with 125b-DP, the cell proliferation rate was increased by 31.0%, 35.0% and 28.5% in C6, U87 and U251 cells, respectively (Figure 2C). However, the cell growth was inhibited by 30.6%, 33.4% and 28.7% in C6, U87 and U251 cells, respectively, when the cells were treated with 125b-AS (Figure 2B). As the effect of miR-125b is most significantly on the proliferation of U87 cells, we selected U87 cells for our future studies. Cell-cycle analysis showed that overexpression of miR-125b in U87 cells increased the cell cycle on S phase. In contrast, downregulation of miR-125b caused cell-cycle arrest at G1 phase (Figure 2C). Further study showed that overexpression of miR-125b increased colony formation of U87 cells in soft-agar assay. There were 28.5% increase of colony numbers in cells treated with 125b-DP compared with the cells transfected with NC-DP (Figure 2D). When the endogenous miR-125b was inhibited by 125b-AS, the cell colony number was significantly decreased up to 50% compared with the cells transfected with negative control miRNAs (NC-DP, Figure 2D). To further confirm the effects of miR-125b on GMB cell proliferation, we established a miR-125b stable knock-down U87 cell line as described in Supplementary Method 1. The results showed that the cell proliferation was significantly increased in the miR-125b knock-down U87 cells treated with 125b-DP (Supplementary Figure 2). These results suggest that miR-125b has a key role in the proliferation of GMB cells and it may function as an oncogene in GMB cells.

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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 48h, 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 48h, 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 48h, 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.

MiR-125b has been proposed to regulate both apoptosis and proliferation (Le et al, 2011). We further investigated the role of miR-125b in the apoptosis of GMB cells. NC-DP, 125b-DP or 125b-AS were transfected into U87 cells, respectively. After being cultured for 48h, cells were collected and Hoechst 33342 staining was performed to detect the apoptosis cells. Cell nuclear pyknosis, chromosome condensation and formation of apoptotic bodies were observed in U87 cells treated with 125b-AS as detected by Hoechst 33342 staining. However, no apoptosis was found in cells transfected with NC-DP or 125b-DP (Figure 3A). TUNEL assay also confirmed the results (Figure 3B). AnnexinV-FITC and PI staining assays were preformed to quantitate the number of apoptotic cells. As showing in Figure 3C, the percentage of early apoptotic cells (AnnexinV-FITC-positive/PI-negative) was significantly increased in cells transfected with 125b-AS. However, overexpression of miR-125b didn't affect the apoptosis of U87 cells (Figure 3B). The results indicate that miR-125b is critical for the survival of GMB cells, in which knockdown of endogenous miR-125b by 125b-AS induces cells apoptosis.

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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 72h, 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.

Downregulation of miR-125b increases the sensitivity of GMB cell to temozolomide

Temozolomide (TMZ), a DNA methylating agent with pro-apoptotic activity, is widely used in GMB therapy (Das et al, 2004). We examined whether downregulation of endogenous miR-125b was capable of promoting TMZ-induced apoptosis. U87 cells were transfected with 125b-AS and then treated with TMZ. Cell morphological observation revealed that there were few pieces of debris and fewer dead cells in miR-125b-transfected cells. In contrast, more pieces of debris and more detached cells were observed in the cells treated with 125b-AS (Figure 4A). MTT assay also confirmed that overexpression of miR-125b in U87 cells resulted in cells resistance to TMZ, but downregulation of miR-125b with 125b-AS increased the sensitivity of U87 cells to TMZ (Figure 4B). The inhibitory rate of 125b-AS transfected cells treated with TMZ for 72h was up to 58.7% that was significantly higher compared with that of TMZ plus NC-DP (41.2%) and TMZ plus 125b-DP (30.8%) treated groups. In addition, the cell numbers of nuclear shrinkage and chromatin condensation were also increased when the cells treated with 125b-AS as determined by Hoechst 33342 staining analysis (Figure 4C). Moreover, Annexin V-FITC and PI staining assay revealed that the apoptotic rate was increased from 18.81 to 25.43% in 125b-AS transfected cells compared with the control (Figure 4D). These results suggest that knockdown of endogenous miR-125b enhances TMZ-induced apoptosis in U87 cells, and miR-125b inhibitor (125b-AS) possesses potential to be developed as a therapeutic agent in the treatment of GMB.

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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 48h, the cells were treated with TMZ. (A) Observation of cell morphology. After being treated with TMZ, the cells were incubated for another 24h 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 24h, 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.

Downregulation of miR-125b activates p53 and p53 pathway

Previous studies have shown that p53 is involved in miR-125b-mediated cell apoptosis and proliferation; miR-125b could regulate both human and zebrafish p53 and p53 network related genes (Hyun et al, 2009). During zebrafish embryogenesis, loss of miR-125b leads to widespread apoptosis in a p53-dependent manner (Le et al, 2009b). To examine the possibility of p53 regulation by miR-125b in GMB cells, we overexpressed miR-125b by transfecting 125b-DP into U87 cells. As shown in Figure 5A and B, the p53 expression level was significantly inhibited. In contrast, silencing miR-125b by 125b-AS increased the p53 expression level. NC-DP and NC-AS was transfected as negative control and p53-specific siRNA was used as positive control. Quantitive analysis further confirmed that ectopic expression of miR-125b reduced the level of p53 protein by 40% (P<0.01) in U87 cells (Figure 5B). In addition, the expression of p21 and Bax, the two main targets of p53, was also decreased significantly in cells transfected with 125b-DP (Figure 5C). In contrast, knockdown of miR-125b increased the endogenous p53 protein expression by 35% (Figure 5B) and downregulation of miR-125b also led to activation of the p53-dominant apoptosis pathway. As shown in Figure 5C, treatment of U87 cells with 125b-AS resulted in the reduction of Bcl-2 expression and enhancement of Bax expression. Moreover, the cleavage of procaspase-9 and procaspase-3 was also elevated. These data suggest that p53 is an important target gene of miR-125b, and miR-125b functions as a regulator in the p53-dominant apoptosis pathway.

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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 72h, 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 (n3) × 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 72h, cells were collected by centrifugation at 1000r.p.m. for 5min 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.

MiR-125b regulates cell apoptosis independent of p53 in GMB cells

To further elucidate if the proliferation and apoptosis of GMB cells mediated by miR-125b is in a p53-dependent manner or not, we analysed the miR-125b-mediated apoptosis in cells with different p53 levels. We selected three kinds of cells including U251 cells with a mutation on the p53 gene (Godbout et al, 1992) and U87 cells expressing the wild-type p53 (Van Meir et al, 1994) and p53 knock-down with the p53 gene stable knockdown by a p53-specific shRNA expression vector. As shown in Figure 6A, the p53 expression was almost abolished in the U87(p53 KD) cells either with or without UV radiation. UV radiation only increased the phosphorylation of p53 protein in U87 cells with wild-type p53 but not in U251 and U87(p53 KD) cells. In addition, downregulation of miR-125b increased p53 expression levels of total p53 and p53 phosphorylation in U87 cells, but not in the cells with p53 mutation (U251 cells) or p53 silencing U87(p53 KD) cells. Next, we investigated the effects of downregulation of miR-125b on proliferation and apoptosis of the three cells. 125b-AS was transfected into the cells and cells proliferation rates were detected by MTT assay. As shown in Figure 6C, transfection of 125b-AS resulted in inhibition of the cell proliferation in all of the three cells without significant difference (Figure 6C). Cell apoptosis were also observed in all three cells by Hoechst 33342 staining (Figure 6D) and Annexin V-FITC/PI staining and flow cytometry assay (Figure 6E). In addition, the caspase-9 and caspase-3 still be activated by downregulation of miR-125b in U87(p53 KD) cells (Figure 6F). These results reveal that under lacking p53 condition, the mitochondrial apoptotic pathway still can be triggered by reducing miR-125b expression and p53 is not necessary for miR-125b-induced cell apoptosis although it is involved in miR-125b-induced cell apoptotic pathway.

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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 72h, 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 72h, 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 72h, 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.

We thank Dr Weicheng Yao and Jianpeng Wang for clinic tissues collection. This work was supported by National Natural Science Foundation of China (Nos. 81072065; 81273550 and 41306157) and Basic Research Projects of Qingdao Science and Technology plan (12-1-4-8-(4)-jch).