miR-155 inhibitor reduces the proliferation and migration in osteosarcoma MG-63 cells

As the most common malignant primary bone tumor in childhood, osteosarcoma (OS) maintains a high recurrence, despite the significant improvements in the overall survival rate of high-grade OS patients during the recent decades. Therefore, a novel therapy strategy is required for OS treatment. Recently, various microRNAs (miRNAs or miRs) have been confirmed as deregulated in OS, and the miR-155 dysregulation in OS has been discovered by the microarray analysis. In the present study, the regulation of miR-155 on the OS cell proliferation, migration and invasion on the MG-63 cells was explored in vitro. The miR-155 mimics were found to promote cell proliferation, colony formation, migration and invasion significantly, compared to the control miRNA. An miR-155 inhibitor was also used to evaluate whether miR-155 served as a therapeutic target for OS. The results demonstrated that the miR-155 inhibitor significantly reduced the proliferation, colony formation, migration and invasion of the MG-63 OS cells. Thus, the study confirmed the oncogenic regulation on the OS progression of miR-155, which could serve as a therapeutic target with an miR-155 inhibitor.


Introduction
Osteosarcoma (OS) accounts for ~2.5% of all malignancies in pediatric patients and ~ 20% of all primary bone cancers (1), with a morphological and malignant heterogeneity (2). The majority of OS variant cells are extremely aggressive, with a capability of rapid growth and early metastasis. Currently, >30% of OS patients with localized disease eventually develop distant metastases, mostly to the lungs and bones (3), even following chemotherapy and surgical treatment. The outcome of OS patients has not significantly improved over the last 20 years, and there has been no significant advance in OS treatment, as the molecular mechanism underlying the highly efficient proliferation and migration of OS cells remains largely unknown. Thus, there is an urgency to identify the details regarding tumor progression and to develop novel therapy strategies for this disease. microRNAs (miRNAs or miRs) are endogenous non-coding RNAs with 18-24 nucleotides, which regulate gene expression (4) by binding the target mRNA's 3' untranslated region (5), in a wide range of organisms, and in a broad array of cell processes in mammals (5)(6)(7). It is well known that cancer is driven by the deregulation of a complexity of oncogenic and tumor suppressive genes, and emerging evidence shows that miRNAs are deregulated in various types of cancer (8)(9)(10), and play oncogenic and tumor suppressive roles, contributing to tumor formation and development (11)(12)(13). Recently, various miRNAs have been confirmed to be deregulated in OS (14,15). The oncogenic miRNA, miR-21, which is aberrantly overexpressed in numerous types of tumor and induces cancer cell growth, migration, invasion and metastasis (16,17), has also been indicated to be significantly overexpressed in OS tissues and induces invasion and migration of the OS cell line, MG-63, by negatively regulating the tumor suppressor gene, reversion-inducing-cysteine-rich protein with kazal motifs (18). The oncogenic miR-93 also induces proliferation and invasion in OS (19), whereas miR-20a promotes OS metastasis by regulating Fas expression (20). By contrast, the tumor suppressive miRNAs, including miR-199a-3p (21), miR-125b (22), miR-143 (23), miR-382 and miR-134 (24), are significantly downregulated in OS cells and attenuate proliferation and inhibition of migration, reduce cell viability and induce apoptosis. miR-155 is well identified as an oncogenic miRNA in leukemia (25,26) and breast cancer (14), contributing to tumorigenicity and progression.
Neoadjuvant chemotherapy has improved the cure rate of OS patients (27,28). However, patients that are not sensitive to these drugs have a poor prognosis. In addition, the frequent acquisition of drug-resistance is often associated with chemotherapy and is a significant obstacle to achieving favorable outcomes. Thus, exploring novel targets for therapy and developing more effective treatment strategies for this disease is miR-155 inhibitor reduces the proliferation and migration in osteosarcoma MG-63 cells required. Recently, Lauvrak et al (29) identified that miR-155 overexpression in OS cell lines was associated with aggressive cancer phenotypes. In the present study, the aim was to evaluate whether miR-155 is a sensitive target for therapy. The regulatory role of miR-155 was determined in the proliferation, invasion and migration of OS cells. Subsequently, the miR-155 inhibitor was evaluated for its inhibition on the OS cell proliferation and migration. The results demonstrated that the miR-155 mimic significantly increased, whereas the miR-155 inhibitor significantly reduced the proliferation and migration of OS MG-63 cells. Therefore, the study revealed miR-155 as a possible therapeutic target for OS.

Materials and methods
Reagents and cell culture. . miR-Con was used as a control.

RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR) miR-155 assay.
The mirVana miRNA Isolation kit (Ambion, Austin, TX, USA) was used to extract miRNAs from the MG-63 cells, and the mirVana RT-qPCR miRNA Detection kit (Ambion) was used to quantify the miR-155 expression, with the U6 small nuclear RNA as the internal control. ∆∆Ct method was used for relative quantification (30). The RT-qPCR was performed using SYBR Green with the LightCycle 2.0 (Roche Diagnostics GmbH, Mannheim, Germany).
Cell viability assay and cell colony formation assay. The MTT assay was adopted to determine the cell viability. MG-63 cells were seeded in 96-well plates and transfected with the miR-155 mimic, inhibitor or control, with ~85% confluence. The cells were washed with warm PBS 6 h post-tranfection and were replaced with RPMI-1640 medium containing 1% FBS, and were cultured for various time. Subsequently, the MTT assay was conducted. Briefly, the incubation medium in the cell wells was replaced with 50 µl 1x MTT solution, and the cells were incubated for 2 h at 37˚C. Post-incubation, the MTT solution was discarded and 150 µl DMSO was added to dissolve the precipitate completely at room temperature. The optical density was measured at 570 nm using a spectrophotometer, the cell viability was expressed as relative viable cells (%) to the control MG-63 cells. For the cell colony formation assay, 2x10 3 cells were incubated in 6-well plates at 37˚C containing 5% CO 2 . Ten days post-incubation, the cells were stained with crystal violet (0.005%) for 30 min and the colony numbers were recorded by Image J software (National Institutes of Health, Bethesda, MD, USA). For the proliferation assay, post-transfection with the miR-155 mimic, inhibitor or control, cells were incubated in cell counting kit 8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) for various times.
The 450 nm absorbance of each well was detected following visual color occurrence.
Cell migration and invasion assay. The cell migration was determined by the scratch assay. The cells were cultivated to 90% confluence on 12-well plates and were transfected with the miR-155 mimic, inhibitor or control. Subsequently, Cell Scrapers (Corning Inc., Corning, NY, USA) were utilized to scratch the confluent cells 24 h post-transfection. The procedures of cellular growth were observed at 0 and 96 h. All the experiments were repeated in triplicate. The Transwell migration chambers were used to evaluate the MG-63 cell invasion. The cells were first seeded at a density of 1x10 5 cells in serum-free media on the upper chamber with the non-coated membrane (8 µm pore size; Millipore, Zug, Switzerland). The lower chamber contained EMEM with 20% FBS as a chemoattractant. The cells in the upper chamber were discarded using cotton wool after 24 h and the migration cells in the lower chamber were counted using a microscope (Olympus, Tokyo, Japan). All the experiments were repeated in triplicate.
Statistical analysis. The results are expressed as mean ± standard error. Student's t-test was performed to compare the differences between two groups. Statistical analysis was conducted by SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference; and in particular, the results are shown as no significance, * P<0.05, ** P<0.01 or *** P<0.001.

miR-155 inhibitor reduces the viability and proliferation of MG-63 cells.
To confirm the promotion of miR-155 to the OS cell proliferation, the miR-155 expression level was manipulated in MG-63 cells, via transfection with the miR-155 mimic or inhibitor. The miR-155 in mimic-transfected cells was significantly higher than that of the control cells (P<0.001) 48 h post transfection, whereas the miR-155 level in the miR-155 inhibitor-transfected cells was significantly lower than in the control cells (P<0.05) (Fig. 1A). Subsequently, the influence of the miR-155 mimic, inhibitor or control on the cell viability was examined. The MTT assay results (Fig. 1B) Fig. 1C shows that in either concentration, the miR-155 mimic group exhibited a higher proliferation than miR-155 control, whereas the miR-155 inhibitor group reduced proliferation (P<0.05). In addition, the time-dependent promoting or reducing effect in cell proliferation of the miR-155 mimic or inhibitor was indicated under the condition of enhanced or reduced miR-155 levels in the MG-63 cells (P<0.05) (Fig. 1D).   (Fig. 3B). In addition, the miR-155 inhibitor reduced the migration of MG-63 cells significantly, as less cells crossed the base line in this group than in the control group (P<0.01) (Fig. 3B). The miR-155 inhibitor clearly reduced the MG-63 cell migration. The blockage of the miR-155 inhibitor to the cell invasion was also demonstrated.
The Transwell invasion chamber assay demonstrated clearly that there was a significant difference in the cell invasion between the miR-155 mimic and control groups, or between the miR-155 inhibitor and control groups. The number of invasive cells was 50±10 cells in the control group, whereas the invasive cell number in the miR-155 mimic or inhibitor group was 88±12 and 25±4 cells, respectively (Fig. 3C) (P<0.05, respectively). All the results indicated that overexpression of miR-155 stimulated the migration and invasion of OS cells, and the miR-155 inhibitor reduced the migration and invasion of the MG-63 cells.

Discussion
As the most common malignant primary bone tumor in childhood (32), OS maintains a high recurrence of 30-40%, and 80% of OS patients with metastatic disease at diagnosis will relapse (27,33,34), regardless of the significant improvements in the overall survival rate of high-grade OS patients during the past decades. Failure of standard multimodal therapy for the disease is associated with an extremely poor prognosis, and therefore, novel drugs or combination therapies are required for patients with recurrent or refractory high-grade OS. Several clinical studies have been conducted to evaluate the efficiency of a combined therapy with gemcitabine and docetaxel in recurrent or refractory OS, and the effect of the gemcitabine-docetaxel combination regimen in recurrent or refractory OS patients remains controversial (35)(36)(37). Extensive studies have been conducted to identify the oncogenes that are suitable to become targets of monoclonal antibodies and small inhibitors. Antibodies or inhibitors were used to knockdown the tyrosine kinase receptors, KIT, platelet-derived growth factor receptors and

C B
A vascular endothelial growth factor receptors (38)(39)(40)(41), however, their inhibition lacked antitumor activity. The monoclonal antibody anti-insulin-like growth factor receptor-I was also promising preclinically, but was not confirmed to be effective in the clinical setting (42). Recently, several studies have focused on the signal transduction pathways of phosphatidylinositol 3'-kinase/mammalian target of rapamycin (43) and mitogen-activated protein kinases. Their inhibition proved highly effective in OS preclinical models (44).
Previously, various miRNAs have been confirmed to be deregulated in OS (14,15). Several oncogenic miRNAs, including miR-21, miR-93 and miR-29, have been indicated to be overexpressed and to induce cancer cell growth, migration, invasion and metastasis (16)(17)(18)(19)45). Recently, the miR-155 dysregulation in OS was discovered by microarray analysis (29). In the present study, the regulation of miR-155 was explored on the OS cell proliferation, migration and invasion on the MG-63 cell in vitro. The miR-155 mimic was shown to promote the cell proliferation, colony formation, migration and invasion significantly, compared to the control miRNA. An miR-155 inhibitor was also used to evaluate whether miR-155 could serve as a therapeutic target for OS. The results demonstrated that the miR-155 inhibitor significantly reduced the proliferation, colony formation, migration and invasion of MG-63 OS cells.
In conclusion, the present study confirmed that the oncogenic regulation on the OS progression of miR-155 could serve as a therapeutic target with an miR-155 inhibitor.