MiR-125b Functions as a Tumor Suppressor and Enhances Chemosensitivity to Cisplatin in Osteosarcoma

Abstract

MicroRNAs are highly conserved noncoding RNA that negatively modulate protein expression at a posttranscriptional and/or translational level and are deeply involved in the pathogenesis of several types of cancers. To date, the potential microRNAs regulating the growth and progression of osteosarcoma are not fully identified yet. Previous reports have shown differentially expressed miR-125b in osteosarcoma. However, the role of miR-125b in human osteosarcoma has not been totally illuminated. In this study, we have shown that miR-125b was downregulated in human osteosarcoma tissues compared to the adjacent tissues and effects as a tumor suppressor in vitro. We found that stable overexpression of miR-125b in osteosarcoma cell lines U2OS and MG-63 inhibited cell proliferation, migration, and invasion. Our data also verified that Bcl-2 is the target of miR-125b. Meanwhile, we showed that Bcl-2 was inversely correlated with miR-125b in osteosarcoma tissues. More importantly, we proved that miR-125b increased the chemosensitivity of osteosarcoma cell lines to cisplatin by targeting Bcl-2. In conclusion, our data demonstrate that miR-125b is a tumor suppressor and support its potential application for the treatment of osteosarcoma in the future.

Introduction

Osteosarcoma (OS) is the most common bone tumor and a leading cause of cancer death among adolescents and young adults. ¹ Survival rates have improved considerably after the introduction of multiagent chemotherapy in the 1980s, with a 5-year survival rate of 60% to 65% for patients without evidence of metastasis. ² However, the survival rates have reached a bottleneck, and chemotherapy resistance has become one of the most intractable obstacles. Studying the molecular mechanism of tumor growth and chemoresistance is therefore urgent, if we are to restore the effective treatment of OS and to identify novel biology-based therapies that may be effective in resistant disease.

MicroRNAs (miRNAs) are short noncoding RNAs that posttranscriptionally modify as much as 60% of the human protein coding genes. ³ Expression of a single miRNA can silence a large number of genes, granting these molecules extensive control over many cellular functions, ⁴ such as developmental biology, cellular differentiation programs, and oncogenesis. ⁵ Recent studies have shown that miRNAs are frequently dysregulated expression in diverse cancer subtypes and function either as tumor suppressors or as oncogenes depending on the role of the messenger RNA (mRNA) targets. ⁶ A large number of progresses indicate that differential expressions of miRNAs were also existed both in OS tissues and human OS cell lines. ⁷

In recent years, accumulating studies have shown the possible connection between the abnormal miR-125b expression and several tumor types. Previous studies have shown that miR-125b was significantly downregulated in OS tissues and inhibited OS cells proliferation and migration by targeting STAT3. ⁸ Nevertheless, the regulation mechanism of miR-125b in OS is still not fully studied. In our study, we found the expression of miR-125b decreased in OS cell lines and 25 pairs of OS samples. We further validated the function of miR-125b in OS cells. We found that miR-125b may reduce the resistance of OS cells to the chemotherapy of cisplatin, which has been used universally in the treatment of OS. Additionally, we demonstrated that Bcl-2 is a downstream target of miR-125b and negatively regulated by miR-125b binding to its 3′ untranslated region (UTR). Meanwhile, we found that Bcl-2 was inversely correlated with miR-125b in OS tissues. In conclusion, overexpression of miR-125b inhibited proliferation, migration, and invasion of OS cells and reduced the chemotherapy resistance of OS cells to cisplatin by targeting Bcl-2. Our study suggests that aberrant expression of miR-125b is critical for the development of human OS and indicates its potential application in cancer therapy.

Materials and Methods

Clinical Tissues and Cell Culture

Human OS cell lines HOS, U2OS, Saos-2, and MG-63 and normal human osteoblast (NHOst) cell were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai,China) and maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 ng/mL streptomycin. All cells were incubated at 37°C in an atmosphere of 5% CO₂.

Osteosarcoma samples and the adjacent normal samples were collected from clinical patients undergoing OS resection. All these tissues were immediately snap-frozen in liquid nitrogen after surgery. This study was approved by the medical review board, and written informed consent was obtained from all patients.

Oligonucleotides and Cell Transfection

All cells were seeded into 6-well, 12-well, 24-well, or 96-well plates and kept in an incubator at 37°C and 5% CO₂ overnight. MiR-125b mimics and miR negative control (NC) were chemically synthesized by GenePharma (Shanghai, China). Cells at 50% to 70% confluence were transfected with miR-125b or miR-NC using lipofectamine reagent (Invitrogen, California) according to the manufacturer’s instructions. Cells were harvested for further experiments 24 or 48 hours after transfection.

RNA Extraction and Real-Time Reverse Transcription–Polymerase Chain Reaction

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions and stored at −80°C until use. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis for mature miR-125b was carried out in triplicate using the PrimeScript RT Reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. Total RNA (500 ng) was converted to complementary DNA for qRT-PCR and was performed using SYBR Premix DimerEraser (Takara) on a 7900HT system. The expression of miR-125b in each group was calculated relative to that of U6, and fold changes were calculated by relative quantification (2^−DDCt).

Cell Proliferation Assay

Cell Counting Kit-8 (CCK8; Dojindo Laboratories, Kumamoto, Japan) assay was used for cell viability. Cells were seeded at 2000 per well in 96-well plates and cultured as described previously for 48 hours after transfection. After 24-, 48-, 72-, and 96-hour incubation, CCK-8 was added into each well, followed by 2-hour incubation. Absorbance value at 450 nm was then measured. Experiments were carried out in triplicate.

Migration and Invasion Assay

We determined the migration and invasion assay of cells using 24-well BD Matrigel invasion chambers (BD Biosciences, Cowley, United Kingdom) in accordance with the manufacturer’s instructions. Cells (5 × 10⁴) were seeded per well in the upper well of the migration (invasion with growth factor-reduced Matrigel) chamber in DMEM without serum, and the lower chamber well contained DMEM supplemented with 10% FBS to stimulate cell migration and invasion. After 24 hours, any noninvading cells on the top well were removed with a cotton swab, and the bottom cells were fixed with 3% paraformaldehyde stained with 0.1% crystal violet. Images were captured in 3 independent 10× magnification fields. The membrane was then air-dried, soaked for 15 minutes with 33% acetic acid (200 µL/well) at room temperature, and transferred to a 96-well plate. The absorbance value was read at optical density 570. Results were obtained from 3 independent experiments.

Western Blot

Cells were treated as described previously, and 24 hours later, cells were harvested and lysed on ice for 30 minutes in radioimmunoprecipitation assay buffer supplemented with protease inhibitors (100 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, 1% deoxycholate acid, 0.1% sodium dodecyl sulfate [SDS], 2 mmol/L dithiothreitol, 1 mmol/L sodium orthovanadate, 2 mmol/L leupeptin, and 2 mmol/L pepstatin). After a centrifugation, protein concentrations were determined by the bicinchoninic acid method (Beyotime, China) and separated by 10% SDS–polyacrylamide gel electrophoresis. Then, protein was transferred to a nitrocellulose membrane (Whatman, Germany). The membrane was incubated into Bcl-2 antibody (1:1000; Cell Signaling Technology) and β-actin (1:5000; Bioworld Technology [Nanjing, China]) at 4°C overnight.

Luciferase Reporter Assay

Prediction of miR-125b binding sites was performed using TargetScan software (http://www.targetscan.org). A fragment of 3′-UTR of Bcl-2 containing the putative miR-125b binding site was amplified by polymerase chain reaction (PCR). To generate a construct containing the miR-125b binding site mutant, we substituted 3 nucleotides corresponding to the 5′-seeding region of the miR-125b binding site on the wild-type (WT) fragment. Its complementary sequence in the 3′-UTR of Bcl-2 (UCAGGGA) was replaced by UGACCGA. The PCR products were digested using SacI (Takara, Shanghai, China) and HindIII (Takara, Shanghai, China), inserted into pMIR-REPORTER (GenePharma, Suzhou, China), and validated by DNA sequencing. Constructs were transfected into MG-63 cells in 24-well plates and cotransfected with miR-125b or miR-NC. Twenty-four hours after transfection, luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Wisconsin).

Chemosensitivity Array

Cells were seeded at a density of 4000 cells/well in a 96-well plate overnight. Freshly prepared cisplatin (Sigma-Aldrich, St Louis, Missouri) was added with the final concentration ranging from 1.25 to 80 μmol/L. Forty-eight hours later, cell viability was assayed by CCK8. Results were obtained from 3 independent experiments.

Flow Cytometry Assay

Apoptosis was measured by flow cytometry. For annexinV staining, 5 μL phycoerythrin-annexinV, 5 μL propidium iodide (BD Biosciences, California), and 400 μL 1× binding buffer were added to the samples, which were incubated for 15 minutes at room temperature in the dark. Then, the samples were analyzed by flow cytometry (FACSCanto II; BD Biosciences) within 1 hour. The data were analyzed using FlowJo software (http://www.flowjo.com). Three experiments were performed in triplicate.

Statistical Analysis

All experiments were performed 3 times, and data were analyzed with GraphPad Prism5 (La Jolla, California). The correlation between miR-125b expression and Bcl-2 levels in OS samples was analyzed using Spearman rank test. Statistical evaluation for data analysis was determined by t test. The differences were considered to be statistically significant at P < .05.

Results

miR-125b Was Frequently Downregulated in OS Tissues and Cell Lines

To investigate the role of miR-125b in OS, 25 pairs of adjacent normal and OS samples were evaluated for the expression of miR-125b by qRT-PCR (Figure 1A). The results showed that the expression level of miR-125b was significantly lower in the OS tissues compared with normal tissues. We next sought to determine whether miR-125b downregulation also occurred in other cell lines. As shown in Figure 1B, expression of miR-125b in 4 OS cell lines, HOS, Saos-2, MG-63, and U2OS, was significantly decreased compared with NHOst. These results indicated that miR-125b was downregulated in OS tissues and cell lines.

Figure 1.

miR-125b was frequently downregulated in osteosarcoma tissues and cell lines. A, The expression of miR-125b was analyzed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) in 25 pairs of osteosarcoma samples (OS) and adjacent normal samples (NS). U6 was used as an internal control. B, Relative miR-125b expression was determined in normal human osteoblast cells (NHOst) and 4 osteosarcoma cell lines, HOS, Saos-2, MG-63, and U2OS. Data represent mean ± standard deviation (SD) of 3 replicates. *Significant difference at P < .05; **Significant difference at P < .01.

miR-125b Negatively Regulates Proliferation, Migration, and Invasion of OS Cell

To explore the potential biological significance of miR-125b in tumorigenesis, miR-125b–overexpressing OS cells were used to analyze cell growth. The results showed that cell growth was attenuated in miR-125b–overexpressing OS cells when compared with OS cells overexpressing miR-NC (Figure 2A).

Figure 2.

miR-125b negatively regulates the proliferation, migration, and invasion of osteosarcoma cell. A, The Cell Counting Kit-8 (CCK-8) assay showed that MG-63 and U2OS cells stably expressing miR-125b grew slower than cells stably expressing miR-negative control (NC). B and C, Transwell migration and invasion assays of MG-63 and U2OS cells stably expressing miR-NC or miR-125b were performed. MiR-125b overexpression decreased the activity of cell migration and invasion in MG-63 and U2OS cells. Data represent mean ± standard deviation (SD) of 3 replicates. *Significant difference at P < .05; **significant difference at P < .01.

Since migration and invasion are key characteristics of malignant tumor, we next assessed the effects of miR-125b on the migration and invasion of OS cell. Restoration of miR-125b dramatically inhibited the normally strong migration and invasive capacity of OS cells (Figure 2B and C). Thus, our results suggest that overexpression of miR-125b suppresses the proliferation, migration, and invasion of OS cell.

Bcl-2 Is a Direct Target of miR-125b in OS Cells

To elucidate the underlying mechanisms of miR-125b in OS, we analyzed databases TargetScan, miRanda, and PicTar. Among the candidates, we found that seed sequence of miR-125b matched 3′-UTR of Bcl-2 (Figure 3A). To verify whether miR-125b directly targets Bcl-2, 3′-UTR sequences containing putative binding sites of WT or mutated (mut) were cloned into the pMIR-REPORT vector. Osteosarcoma cells were cotransfected with the WT or mut Bcl-2 luciferase reporter vector together with miR-125b or miR-NC for 24 hours, and luciferase activities in those cells were measured. As shown in Figure 3B, luciferase activities were significantly reduced in those cells transfected with the wild sequence and miR-125b but not in the cells with the mutant sequence and miR-125b. Then, Western blotting analysis was conducted to measure the levels of Bcl-2 protein, and we found that the expression of Bcl-2 protein was downregulated in miR-125b–treated cells and forced expression of Bcl-2 reversed miR-125b–mediated suppression of Bcl-2 protein (Figure 3C). These results suggest that miR-125b directly targets Bcl-2 by binding its seed region to their 3′-UTR in OS cells. To further determine the correlation between miR-125b and Bcl-2 levels, we measured the protein levels of Bcl-2 in OS tissues. Spearman correlation analysis demonstrated that Bcl-2 levels in OS samples were inversely correlated with miR-125b expression levels (Spearman correlation r = −.4015; Figure 3D).

Figure 3.

Bcl-2 (B-cell lymphoma-2) is a direct target of miR-125b in osteosarcoma cells. A, Sequence of the miR-125b–binding site within the human Bcl-2 3′-untranslated region (UTR) and a schematic diagram of the reporter construct showing the entire Bcl-2 3′-UTR sequence and the mutated Bcl-2 3′-UTR sequence. The mutated nucleotides of the Bcl-2 3′-UTR are labeled in red. B, Luciferase assay on MG-63 cells which were cotransfected with miR-negative control (NC) or miR-125b and a luciferase reporter containing the full length of Bcl-2 3′-UTR (wild type [WT] or a mutant [Mut]) in which 3 nucleotides of the miR-125b-binding site were mutated. Luciferase activities were measured 24 hours after transfection. MiR-125b markedly suppressed luciferase activity in Bcl-2 3′-UTR (WT) reporter constructs. The data are means ± standard deviation (SD) for separate transfections (n = 4). C, The expression levels of Bcl-2 were decreased in cells with miR-125b overexpression by Western blotting, Overexpression of Bcl-2 restored the inhibition of miR-125b at protein level. D, Spearman correlation analysis was used to determine the correlation between the expression levels of Bcl-2 and miR-125b in 25 pairs of adjacent normal and osteosarcoma samples (Spearman correlation r = −.4015, P < .05).

Elevated Expression of miR-125b Enhances the Chemosensitivity of OS Cells to Cisplatin by its Target Bcl-2

Resistance to cisplatin treatment is one of the major causes for the failure of chemotherapy in treating OS. Therefore, it is critical to discover new strategies to increase the effectiveness of cisplatin for therapeutic purposes. To explore the potential role of miR-125b in chemotherapy, we treated MG-63 cells stably expressing miR-125b or miR-NC with different concentrations of cisplatin. Our results showed that miR-125b–overexpressing MG-63 cells significantly increased chemosensitivity to treatment of cisplatin (Figure 4A). Furthermore, cell growth rate in the presence of cisplatin (5 μmol/L) was assayed by CCK-8 assay at different time points; interestingly, forced expression of Bcl-2 reversed miR-125b–induced OS cells chemosensitivity to cisplatin (Figure 4B). To further study whether miR-125b and its target Bcl-2 play a role in cell apoptosis in the presence of cisplatin treatment, fluorescence-activated cell sorting analysis was performed to detect cell apoptosis rates. The combination of miR-125b and cisplatin treatment significantly induced cell apoptosis, whereas Bcl-2 overexpression partially abolished the effect induced by miR-125b plus cisplatin treatment (Figure 4C). Moreover, we found that the activity of caspase 3, a key executor of cell apoptosis, was significantly upregulated upon treatment by miR-125b plus cisplatin compared with miR-125b or cisplatin treatment alone, whereas forced expression of Bcl-2 attenuated the activation of caspase 3 induced by miR-125b plus cisplatin treatment (Figure 4D). These results indicated that miR-125b renders OS cells more sensitive to cisplatin treatment, and miR-125b and cisplatin combination induced apoptotic effect through targeting Bcl-2 in OS cells.

Figure 4.

Elevated expression of miR-125b enhances the chemosensitivity of osteosarcoma cells to cisplatin by its target Bcl-2. A, MG-63 cells stably expressing miR-negative control (NC) or miR-125b were pretreated with various concentration of cisplatin for 48 hours and subjected to CCK-8 assay. B, MG-63 cells stably expressing miR-NC, miR-125b, or miR-125b–forced expression of Bcl-2 were pretreated with 5 μmol/L of cisplatin for definite time points and subjected to CCK-8 assay. C, Apoptosis analysis by flow cytometry. D, MG-63 cells stably expressing miR-NC or miR-125b were transfected with 1 μg pCMV6 vector or pCMV6–Bcl-2 plasmid and cultured with or without cisplatin. After 72 hours, the relative caspase 3 activities were determined. Data represent mean ± standard deviation (SD) of 3 replicates. *or ^# P < .05. **P < .01. *Significant difference compared to control. ^#Significant difference compared to miR-125b treatment alone.

Discussion

Increasing evidence indicate that miRNAs may exert functions as oncogenes or tumor suppressors in human cancers depending on the role of their targets. ^{9 –16} To date, different expression profiles of miRNAs in OS cells are identified. ^{17 –21} Jones et al identified 34 differentially expressed miRNAs from 18 human OS tissue samples compared with NHOst. ⁷ Previous studies shown miR-125b was downregulated in both OS tissue samples and OS cell lines. ⁸ In this study, we further confirmed that miR-125b may act as a tumor suppressor in OS carcinogenesis and enhanced the chemosensitivity of OS cells to cisplatin. We found that Bcl-2 was a target of miR-125b, consistent with previous studies. More importantly, we showed that miR-125b increased the chemosensitivity of OS cell lines to cisplatin by targeting Bcl-2. Moreover, the expression levels of Bcl-2 in OS samples were inversely correlated with miR-125b levels. Thus, this study may provide novel therapeutic strategies for OS treatment.

Previous studies have shown that miR-125b is frequently deleted in breast cancer, ²² endometrial cancer, and lung cancer, ^23,24 indicating that miR-125b may function as a tumor suppressor. For instance, miR-125b inhibited cell growth and reduced migration and invasion in breast cancer cells through suppression of ERBB2 and ERBB3 in breast cancer cells. ²⁵ Zhang et al reported that miR-125b is methylated and functions as a tumor suppressor through targeting the ETS1 proto-oncogene in human invasive breast cancer. ²⁶ However, dysregulation of miR-125b has been identified to play opposite role in different tumors. MiR-125b has various regulating pathways in the process of cell proliferation, differentiation, and apoptosis, such as inhibiting p53-induced apoptosis during development and the stress response ²⁷ and inhibiting cell apoptosis through p53 and p38-MAPK-independent pathways in glioblastoma cells. ²⁸ . In combination with previous reports revealing the roles of miR-125b in OS cells, we confirmed that miR-125b functions as a tumor suppressor in OS carcinogenesis and enhanced the chemosensitivity of OS cells to cisplatin. However, pluripotency of miR-125b still needs further study in variant cancers.

The apoptotic process can be divided into 3 interdependent phases: induction, decision, and execution. The decision phase is largely regulated by the Bcl-2 family of apoptotic regulators. In tumor cells, abnormal expression of Bcl-2 can suppress cell death induced by a variety of stress applications including chemotherapy, irradiation, and viral infection. ^29,30 Bcl-2 is a therapeutic target, and small molecule inhibitors have been produced and are entering clinical trials. These agents may have clinical toxicities due to antagonism of other Bcl-2 family proteins or effects on other lineages. ³¹ Our study found that miR-125b negatively regulated Bcl-2 and directly binds to the 3′-UTR region of its mRNA. Thus, miR-125b affected the function of Bcl-2 and suppressed tumor progression. It would be advantageous to exploit mechanisms of Bcl-2 regulation to produce therapies that are highly lineage and gene specific.

In conclusion, our investigation shows that miR-125b acts as a tumor suppressor in OS cells by targeting Bcl-2. In 25 cases of OS tissues, miR-125b levels are inversely related to the expression levels of Bcl-2. MiR-125b inhibits the proliferation, migration, and invasion of OS cells and enhanced the chemosensitivity of OS cells to cisplatin by targeting Bcl-2. Our study would provide new insights into the molecular mechanism of OS development as well as provide new therapeutic strategy for OS treatment in the future.