miR-34a increases cisplatin sensitivity of osteosarcoma cells in vitro through up-regulation of c-Myc and Bim signal

Abstract

BACKGROUND AND OBJECTIVE:

Osteosarcoma is the most common primary malignancy in bone. Patients who respond poorly to induction chemotherapy are at higher risk of adverse prognosis. The molecular basis for such poor prognosis remains unclear. Recently, increasing evidence has suggested decreased expression of miR-34a is observed in a number of cancer types, including human osteosarcoma, and decreased miR-34a is involved in drug resistance. However, the underlying molecular mechanisms of decreased miR-34a on cisplatin chemoresistance in osteosarcoma has not been reported.

METHODS:

Osteosarcoma U2OS cells were transfected with miR-34a mimics for 48 h, then the cells were treated with 3.0 $\mu$m cisplatin for 24 h. Using siRNA targeting c-Myc and Bim to examine the relation between miR-34a, c-Myc and Bim expression exposure to cisplatin on cisplatin-induced apoptosis.

RESULTS:

Treatment of U2OS cells with cisplatin induced cell apoptosis by upregulation of c-Myc -dependent Bim expression; Osteosarcoma U2OS cells transfected with miR-34a mimics (miR-34a/U2OS) induced cell apoptosis and inhibited cell survival, and increased the sensitivity of U2OS cells to cisplatin. U2OS cells transfected with miR-34a mimics upregulated the protein expression of c-Myc and Bim. Targeting c-Myc downregulated the expression of Bim in the miR-34a/U2OS cells. In addition, Targeting Bim reversed the chemeresistance of miR-34a/U2OS cells to cisplatin.

CONCLUSIONS:

Our data indicated that miR-34a enhanced the sensitivity to cisplatin by upregulation of c-Myc and Bim pathway.

1. Introduction

Osteosarcoma (OS) remains the most common primary malignant bone cancer affecting children and adolescents [1]. The 5-year survival for patients with localized OS remains at 60–70% with multiagent chemotherapy treatment together with surgical techniques [2]. Up to 20–25% of patients present with intrinsic or acquired resistant to chemotherapy, leading to recurrence and metastasis. However, the underlying mechanisms of chemotherapy resistance are still unknown [3].

MicroRNAs (miRNAs, miRs) are a family of small noncoding RNAs that regulate gene expression by sequence-selective targeting of mRNAs, leading to a translational repression or mRNA degradation [4]. Experimental evidence demonstrates that dysregulation of specific miRNAs leads to drug resistance in different cancers and correction of these miRNAs using miRNA mimics or antagomiRs can normalize the gene regulatory network and signaling pathways and sensitize cancerous cells to chemotherapy [5, 6, 7, 8].

miR-34a is a member of an evolutionarily conserved miRNA family, miR-34s. It has been shown to repress several oncogenes directly or indirectly [9, 10]. Ectopic miR-34a expression resulted in cell cycle arrest and growth inhibition and attenuated chemoresistance to anticancer drug camptothecin by inducing apoptosis, suggesting a potential role of miR-34a for the treatment of prostate cancer [11]. Functional analysis of miR-34a in EWS cell lines indicated that restoration of miR-34a activity may be useful to decrease malignancy and increase tumour sensitivity to doxorubicin and vincristine, so sparing excessive long-term toxicity to EWS patients [12]. Zhao et al. [13] has recently reported that combination of doxorubicin chemotherapy and miR-34a replacement therapy synergistic antiproliferative effects and it is more effective than monotherapy in suppressing Ewing’s sarcoma xenograft tumor growth. However, the mechanisms of miR-34a sensitizes osteosarcoma cells to chemotherapy have not been totally elucidated. Therefore, it is of great significance to further study the function and mechanism of miR-34a in osteosarcoma.

c-Myc is a transcriptional activator implicated in the control of cell proliferation, differentiation and transformation, but is also involved in the regulation of programmed cell death, apoptosis [14]. Ectopic overexpression of c-Myc in both Rat1 fibroblasts and human osteosarcoma cells causes a dramatic increase of cell apoptosis [15]. Cerquetti et al. [16] has reported that silencing of c-Myc mRNA prevented paclitaxel induced apoptosis in SW13 cells, whereas in the H295R cells the overexpression of c-Myc rendered the cells more prone to growth inhibition after paclitaxel exposure.

A growing body of evidence supports a central role for the BCL2 homologous (BH) family of proteins in mediating MYC induced apoptosis. This family of proteins is subdivided into anti-apoptotic, pro-apoptotic BH3-only, and effector BH3 proteins, BAX and BAK. In response to pro-apoptotic stimuli, BAX and BAK oligomerize to form pores in the outer mitochondrial membrane (MOMP), triggering release of Cytochrome C, SMAC/DIABLO and consequent activation of effector Caspases [17, 18].

The recent identification of Bim as a transcriptional target of MYC suggests that this BH3-only protein may directly mediate MYC’s pro-apoptotic signal [19]. MYC induces Bim accumulation in Burkitt’s Lymphoma and MYC point mutants that fail to induce Bim also fail to induce apoptosis [20]. Muthalagu et al. has reported that

MYC induces apoptosis in postmitotic cells by upregulation of BIM [21]. Delbridge et al. has reported that MYC accelerated lymphomagenesis by downregulation of BIM [22]. In breast cancer cells, reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion [23]. Jiang et al. has reported that uberoylanilide hydroxamic acid (SAHA) induced apoptosis in rat fibroblast cells by selectively inducing the expression of Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA [24]. Egle et al. has reported that Bim is induced by Myc in B cells and mediates apoptosis. Inactivation of even a single allele of Bim accelerated Myc-induced development of tumors, particularly acute B cell leukemia [25]. Therefore, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of cancer cell death.

It has previously found that miR-34a functions as a potent tumor suppressor through the modulation of c-Myc in cellular senescence in HCC cells [26]. Yamamura et al. [27] reported that miR-34a was downregulated in prostate cancer tissues and silenced the expression of the c-Myc oncogene by targeting its 3’ UTR, inhibiting cell proliferation, cell invasion and promoting apoptosis. However, whether miR-34a induces apoptosis by c-myc/Bim signals remain still elusive.

In the present study, the antitumor effects of miR-34a were analyzed in osteosarcoma cells in vitro. The results show that overexpression of miR-34a induced osteosarcoma cells apoptosis and sensitized osteosarcoma cells to cisplatin by upregulation of c-Myc/Bim signal. These observations provide a promising gene therapeutic agent that miR-34a functions as a tumor suppressor gene, which could increase the susceptibility of osteosarcoma cell lines to cisplatin through up-regulating c-Myc/Bim signal in osteosarcoma.

2. Materials and methods

2.1. Cell culture

Human osteosarcoma U2OS cell line was purchased from the American Type Culture Collection (ATCC, Shanghai, China). The cells were cultured in F-12K or DMEM (Gibco, NY, USA) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO${}_2$ at 37${}^{\circ }$C.

2.2. miR-34 a transfection

U2OS cells were plated a day prior to transfection. When cells reached 80% confluency, cultures were transfected overnight with miR-34a mimics and negative controls (scrambled oligos) (Life Technologies) at a final concentration of 100 nmol/l using Lipofectamine 2000. The transfection media were replaced with fresh media and cells harvested 48 h after transfection for functional analysis. To acquire the stable clones, 48 h after transduction, U2OS cells were selected with 400 $\mu$g/ml neomycin (Gibco, Life Technologies, Thermo Fisher Scientific) for 2 weeks.

2.3. siRNA transfection

U2OS cells, miR-34a overexpressing U2OS cells and scrambled oligos overexpressing U2OS cells were seeded on 60 mm dishes at 2 $\times$ 10${}^6$ cells/dish. Twenty-four hours later, cells were transfected with 100 nM of c-Myc siRNA or Bim siRNA and a nonsilencing siRNA control using Lipofectamine 2000 according to the manufacturer’s instructions (all from Invitrogen, Life Technologies, Thermo Fisher Scientific). Forty-eight hours after transfection, cells were detached and reseeded in 6-well plates at 3 $\times$ 10${}^5$ cells/well, and in 96-well plate or 96-well E-plate at 5,000 cells/well, respectively for the evaluation of protein expression, cell viability, and death.

2.4. Quantitative RT PCR analysis of cell RNA

RNA was isolated from cells using TRIzol reagent and DNA prepared using TaqMan MicroRNA Reverse Transcription Kit (Invitrogen) according to the manufacturer’s protocol. Expression of miR-34a was assessed by quantitative reverse transcription PCR (RT-qPCR), using $U6$ snRNA (Invitrogen) as the control. Single tube TaqMan assays (Invitrogen) were obtained for miR-34a and qPCR was carried out as follows: 95${}^{\circ }$C for 10 min, then 40 cycles of 95${}^{\circ }$C for 15 s and 60${}^{\circ }$C for 60 s. All analyses were performed in triplicate and the data were normalized to $U6$ snRNA.

2.5. Western blot analysis

Cells were lysed using radioimmunoprecipitation (RIPA) assay lysis buffer (PBS containing 1% NP40, 0.5% Na-deoxycholate, and 0.1% SDS) supplemented with 1 $\mu$mol/L phenylmethylsulfonyl fluoride and 1 protease inhibitor cocktail tablet per 10 mL on ice for 20 min, and lysates protein concentration were determined using the Bio-Rad protein assay kit according to the manufacturer’s instructions. Proteins (40 $\mu$g) were electrophoresed onto 10% SDS-PAGE gels (Invitrogen, Hangzhou, China) and transferred onto polyvinylidene difluoride membranes. Following blocking in 5% milk, the membranes were incubated with a specific primary antibody, washed, and incubated with horseradish peroxidase-linked secondary antibody (GE Healthcare, Beijing, China). Signals were visualized with chemiluminescent horseradish peroxidase antibody detection reagent (Denville Scientific, Guangzhou, China). The primary antibodies were the following: anti-c-Myc and anti-Bim (1:1000, Cell Signaling Technology, Shanghai, China) and anti-$\beta$-actin (1:1000, Santa Cruz Biotechnology, Shanghai, China).

2.6. MTT assay

U2-OS cells (2 $\times$ 10${}^3$ per well) were plated in 96-well plates in a final volume of 100 $\mu$l. Twenty-four hours after plating, miR-34a mimics (100 nM) or/and siRNAs (100 nM) or/and scrambled oligos (100 nM) were transfected into the cells with Lipofectamine 2000 (Qiagen) for 24 h; then the U2-OS cells were treated with 3.0 $\mu$M cisplatin for 24 h. Assays were done by incubating each plate with 20 $\mu$l of MTT substrate for 2 h followed by removal of medium and addition of 200 $\mu$l of dimethylsulfoxide. Absorbance (550 nm) was measured using a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA, USA). Triplicate wells were used for each treatment.

2.7. AnnexinV fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining assay

Flow cytometry was used to discriminate between intact and apoptotic cells. U2-OS cells were stained with fluorescein isothiocyanate (FITC) labeled annexinV that binds to membrane phosphatidylserine and with propidium iodide (PI) that binds to cellular DNA according to the manufacturer’s instructions (BD bioscience, USA). Briefly, the U2-OS cells (following siRNA transfection or/and miR-34a transfection for 48 h and treated with 3.0 $\mu$M cisplatin for 24 h) were plated at 1 $\times$ 10${}^5$ cells/well in 24-well plates.After that, the cells were trypsinized, washed with cold PBS, and resuspended in 100 $\mu$l of binding buffer containing 5 $\mu$l of FITC Annexin V and 5 $\mu$l of PI. Then cells were gently vortexed and incubated for 20 min at room temperature in the dark. Four hundred microliters of binding buffer was added to each tube. Cells were then collected using a FACSCalibur flow cytometer (Becton Dickinson, USA) and analyzed with CellQuest Software (BD bioscience, USA).

2.8. Statistical analysis

All data were expressed as mean $\pm$ standard error of mean (SEM) from at least three independent experiments. Statistical significance was evaluated using the Student’s $t$ test. Values of 0.05 were considered significant.

3. Results

3.1. Cisplatin treatment upregulats c-Myc-dependent Bim expression

Osteosarcoma U2OS cells were treated with 30 $\mu$M cisplatin for 24 h, and the levels of c-Myc and Bim protein were assessed by Western blot analysis. As shown in Fig. 1A, cisplatin treatment caused significant increase of c-Myc and Bim expression. When c-Myc expression is knocked down by siRNA, cisplatin-induced Bim expression is significantly inhibited (Fig. 1A). However, knockdown of Bim has no effect on cisplatin-induced c-Myc expression (Fig. 1A). Our results demonstrate that cisplatin activates c-Myc/Bim signal.

Figure 1.

Activation of c-Myc/Bim by cisplatin confers cisplatin sensitivity in U2OS cells. A, Western blot analyses of c-Myc and Bim expression in U2OS cells treated with 3.0 $\mu$M cisplatin for 24 h in the presence or absence of c-Myc siRNA transfection for 48 h, $\beta$-actin was used as a loading control. B, Flow cytometric analysis of cell apoptosis after 24 h of cisplatin treatment in the presence or absence of Bim siRNA transfection for 48 h. C, MTT analyses of cell growth inhibition after 24 h of cisplatin treatment in the presence or absence of Bim siRNA transfection for 48 h.

3.2. Activation of Bim is essential for cisplatin-induced apoptosis

U2OS cells were treated with 3.0 $\mu$M cisplatin for 24 h, about 32% of the cells underwent apoptosis as measured by FCM (Fig. 1B). And the cell survival rate was 56% as measured by MTT assay (Fig. 1C). Transient transfection with Bim siRNA alone has no effect on the viability and apoptosis of U2OS cells (data not shown). However, when Bim expression is knocked down, cisplatin-induced cell apoptosis (early apoptosis+late apoptosis) is significantly inhibited since only 8% cells underwent apoptosis (early apoptosis $+$ late apoptosis) (Fig. 1B), and cell survival rate increased to 92% (Fig. 1C) ,which suggests that Bim is responsible for cisplatin-induced apoptosis.

3.3. c-Myc/Bim activation is essential for miR34a-induced apoptosis

To investigate the function of miR-34a in U2OS cells tumorigenesis, we transfected miR-34a mimics or miR-Ctrl in U2OS cells and determined their miR-34a levels 48 h after transfection. Results showed increased miR-34a levels in miR-34a transfected U2OS cell compared to miR-Ctrl transfected cells (Fig. 2A).

Figure 2.

The role of miR-34a in the regulation of proliferation and apoptosis in U2-OS cells. A, U2OS cells were transfected with miR-34a mimics or miR-Ctrl for 48 h. miR-34a level was determined by qRT-PCR assay. B, Western blot analyses of c-Myc and Bim expression in U2OS cells transfected with miR-34a in the presence or absence of c-Myc siRNA transfection for 48 h, $\beta$-actin was used as a loading control. C, MTT analyses of cell growth inhibition after miR-34a transfection in the presence or absence of Bim siRNA or c-Myc siRNA transfection for 48 h. D, Flow cytometric analysis of cell apoptosis after miR-34a transfection in the presence or absence of Bim siRNA or c-Myc siRNA transfection for 48 h.

c-Myc and Bim protein levels were analyzed by western blot to investigate the mechanism by which miR-34a induced apoptosis in U2OS cells. c-Myc and Bim protein were significantly increased in U2OS cells, which express lower levels of endogenous c-Myc and Bim after transfection with miR-34a mimics (Fig. 2B).

We then investigated the effect of miR-34a on U2OS cell viability. As shown in Fig. 2C, miR-34a significantly inhibited U2OS proliferation by MTT assay. To investigate whether miR-34a affects U2OS cell apoptosis, U2OS cells were transfected with miR-34a and apoptosis assessed 24 h later. As shown in Fig. 2D, miR-34a transfected U2OS cells had a higher incidence of apoptosis than miR-Ctrl transfected U2OS cells by Flow cytometric analysis (FCM). These results suggest that miR-34a inhibits U2OS cell proliferation and induces apoptosis.

To further verify that the effects of miR-34a on proliferation and apoptosis in U2OS cells were mediated by regulation of c-Myc/Bim signal, we silenced Bim or c-Myc in U2OS cells by using siRNA, respectively. As shown in Fig. 2, knockdown of Bim reversed miR-34a-induced U2OS cell proliferation (Fig. 2C) and inhibited cell apoptosis (Fig. 2C), which was consistent with the effects of targeting c-Myc in miR-34a transfected U2OS cells (Fig. 2C–D).

To test whether c-Myc is involved in activating Bim during miR-34a-induced apoptosis, an siRNA strategy was utilized to knock down the expression of c-Myc in human osteosarcoma U2-OS cells. After 48 h of transient c-Myc siRNA transfection, c-Myc and Bim levels were reduced 80–90% (Fig. 2B). These data indicate that miR-34a upregulated c-Myc/Bim signal under these experimental conditions. We also showed that cisplatin treatment did not induced miR-34a expression in U2OS cells, indicating that miR-34a is not involved in cisplatin-induced apoptosis (data not shown).

3.4. miR-34a enhances cisplatin sensitivity of U2-OS cells

We next determined the effects of miR-34a overexpression on cisplatin sensitivity in U2OS cells. Briefly, U2OS cells were transfected with a miR-34a mimic or a miR-Ctrl for 48 h, then exposed to 3.0 $\mu$M of cisplatin for 24 h. Cell viability was determined using an MTT assay. Cell apoptosis was detected by Flow cytometric analysis. As is shown in Fig. 3A–B, transfection with miR-34a mimic gave rise to a marked increase in sensitivity after treatment with 3.0 $\mu$M cisplatin (both 0.05) in the U2OS cells.

Figure 3.

miR-34a sensitizes U2-OS cells to cisplatin by activation of c-Myc/Bim pathway. A, MTT analyses of cell growth inhibition after miR-34a transfection and cisplatin treatment in the presence or absence of Bim siRNA or c-Myc siRNA transfection for 48 h. B, Flow cytometric analysis of cell apoptosis after miR-34a transfection and cisplatin treatment in the presence or absence of Bim siRNA or c-Myc siRNA transfection for 48 h. C, Western blot analyses of c-Myc and Bim expression in U2OS cells. $\beta$-actin was used as a loading control.

3.5. miR-34a sensitizes U2-OS cells to cisplatin by activation of c-Myc/Bim pathway

miR-34a or a miR-Ctrl transfected U2OS cells were transfected with c-Myc siRNA or Bim siRNA or control siRNA for 48 h, then exposed to 3.0 $\mu$M of cisplatin for 24 h.Western blot analysis of lysates harvested 24 h following cisplatin exposure revealed a marked decrease in c-Myc and Bim protein levels in cells transfected with c-Myc siRNA compared to control (Fig. 3C). miR-Ctrl transfection had no effect of cisplatin-induced Bim and c-Myc expression in the U2OS cells (data not shown).

In order to determine whether the observed enhancement in sensitivity to cisplatin following overexpression of miR-34a was due to its induction of c-Myc-dependent Bim, the miR-34a or a miR-Ctrl transfected U2OS cells were transfected with Bim siRNA or control siRNA for 48 h, then exposed to 3.0 $\mu$M of cisplatin for 24 h. Figure 3A–B shows that U2OS/ miR-34a cells exposed to 3.0 $\mu$M of cisplatin for 24 h caused 85% growth inhibition and 70% cell apoptosis, whereas 3.0 $\mu$M cisplatin had a minimal effect in cells transfected with Bim siRNA as compared with cells transfected with control siRNA. Thus, these data suggest that Cisplatin and miR34a induced cell apoptosis by activation of c-Myc and Bim. Combined cisplatin and miR34a sensitized U2OS cells to cisplatin via activation c-Myc and Bim.

4. Discussion

miR-34a was reported as a tumor suppressor. Furthermore, miR-34a was found to inhibit cancer stem cells (CSCs) self-renewal [28] and invasion [29] promoting their sensitivity to chemo- and radioth-erapy [28], providing evidence that miR-34a may function as an anti-oncogene. In the present study, we found that U2OS cells over-expressing miR-34a inhibited cell proliferation, decreased colony formation rate and induced apoptosis. The findings indicated that identification of miR-34a as a potent tumor suppressor is a highly significant finding with respect to the development of potential therapeutics for cancer. Although it is well accepted that miR-34a is a tumor-suppressor gene, the miR-34a KO mice are not tumor prone [30]. The tumor-suppressive function of miR-34a might be restricted to specific tissues and loss of miR-34a might cooperate with specific oncogenic lesions.

Cisplatin, which is widely used in chemotherapeutic therapy, is effective for treating patients with osteosarcoma [31], markedly increasing the survival rate. However, via various mechanisms, chemoresistance is one of the main obstacles preventing this increase [32]. It is well known that miRNA dysregulation influences tumor malignant progression. It has been demonstrated that miR-34a also play important roles in osteosarcoma chemoresistance [33, 34]. Consequently, our purpose was to elucidate the mechanism of cisplatin resistance and to discover possible means of improving osteosarcoma treatment. We demonstrated in our study that miR-34a overexpression enhanced the sensitivity of U2OS cells to cisplatin in vitro. Additionally, we showed that miR-34a overexpression enhanced cisplatin induced apoptosis in U2OS cells. These results suggest that miR-34a overexpressing therapies could be effective for the treatment of cisplatin-resistant osteosarcoma.

The c-Myc transcription factor, which regulates 15% of all human genes, plays an important role in a myriad of biological processes including cell growth and proliferation, cell cycle progression and apoptosis [35, 36, 37]. In other tumor types, c-Myc expression levels have been associated with drug resistance [38, 39, 40]. However, in other tumor types, c-Myc expression levels have been associated with drug sensitivity [41, 42, 43]. We demonstrated in our study that miR-34a overexpression upregulated c-Myc expression in U2OS cells, and accelerated cisplatin-induced apoptosis. However, targeting c-Myc decreased miR-34a induced apoptosis in U2OS cells, indicating that c-Myc is required for the synthetic induction of apoptosis driven by the combination of miR-34a and cisplatin.

Proapoptotic BH3-only proteins such as Bim bind to antiapoptotic proteins and thus allow the proapoptotic multidomain proteins, Bax and Bak, to form channels on the mitochondrial membrane leading to cytochrome $c$ release and apoptosis [44]. In hepatocarcinoma cells, inhibition of Bim or Bik up-regulation using siRNA strategies significantly attenuated bortezomib-induced apoptosis [45]. Here, we have shown that miR-34a overexpression up-regulate c-Myc-dependent Bim expression in the U2OS cells. In addition, our data indicate that targeting Bim inhibited miR-34a induced apoptosis and sensitivity to cisplatin in U2OS cells.

In ovarian cancer, degradation of Bim plays an important role in cisplatin resistance, and treatment of ovarian cancer cells with cisplatin caused Bim phosphorylation and subsequent degradation and that its degradation is associated with cisplatin resistance [46]. We demonstrated in our study that treatment of U2OS cells with cisplatin did not activate miR-34a, but caused the activation of c-Myc and Bim. By inhibiting c-Myc expression with siRNA, we show that Bim expression were blocked, which suggests that Bim is positively regulated by c-Myc. It has been shown that Bim transcriptional repression may mediate tumor chemoresistance [47]. Our data reveal that treatment of U2OS cells with cisplatin induced apoptosis that was suppressible by siRNA to Bim, suggesting that Bax is the primary mediator of cisplatin -induced apoptosis in U2OS cells.

In conclusion, the results show that miR-34a suppressed osteosarcoma cells growth and induced apoptosis by up-regulation of c-Myc-dependent Bim expression. Treatment of U2OS cells with cisplatin induced apoptosis by c-Myc-dependent Bim activation. miR-34a overexpression resulted in sensitivity to cisplatin-induced apoptosis in U2OS cells by activation of c-Myc-dependent Bim pathway. Taken together, these results suggest miR-34a induction as an indicator of the therapeutic efficacy. They also provide an anticancer mechanism of miR-34a overexpression, and imply one of the potential strategies contributing to chemotherapeutic resistance in tumors.

Conflict of interest

The authors have no conflict of interest to report.