MicroRNA-33b Inhibits the Proliferation and Migration of Osteosarcoma Cells via Targeting Hypoxia-Inducible Factor-1α.

Recently, microRNA (miR)-33b has been demonstrated to act as a tumor suppressor in osteosarcoma. However, the regulatory mechanism of miR-33b in osteosarcoma cell proliferation and migration remains largely unknown. In this study, real-time PCR showed that miR-33b was significantly downregulated in osteosarcoma tissues compared to their matched adjacent nontumor tissues. Its expression was also decreased in several common osteosarcoma cell lines, including Saos-2, MG63, U2OS, and SW1353, when compared to normal osteoblast cell line hFOB. Overexpression of miR-33b suppressed U2OS cell proliferation and migration. HIF-1α was further identified as a target of miR-33b, and its protein levels were reduced after overexpression of miR-33b in U2OS cells. Moreover, overexpression of HIF-1α significantly reversed the suppressive effect of miR-33b on U2OS cell proliferation and migration. In addition, HIF-1α was found to be significantly upregulated in osteosarcoma tissues compared to adjacent nontumor tissues, and their expression levels were inversely correlated to the miR-33b levels in osteosarcoma tissues. According to these findings, miR-33b plays a suppressive role in the regulation of osteosarcoma cell proliferation and migration via directly targeting HIF-1α. Therefore, we suggest that the miR-33b/HIF-1α axis may become a promising therapeutic target for osteosarcoma.

INTRODUCTION

Osteosarcoma is the most common cancer of the bone, primarily present around regions with active bone growth and repair1,2. Although great attention has been paid to the diagnosis of and therapy for osteosarcoma, the 5-year survival rate of patients with this disease remains poor, mainly due to its recurrence and metastasis1. Therefore, exploring the molecular mechanisms involved in osteosarcoma tumorigenesis and progression is urgently needed.

MicroRNAs (miRs), a class of noncoding RNAs that are 18–25 nucleotides in length, have been demonstrated to act as gene regulators via directly binding to the 3′-untranslated region (3′-UTR) of their target mRNAs and causing mRNA degradation or translation inhibition3. Through negative mediation of the gene expression, miRs participate in various biological processes, such as embryonic development, cell proliferation, differentiation, survival, apoptosis, energy metabolism, and motility3–5. Recently, miRs have been found to provide promising diagnostic and therapeutic opportunities for human cancers6,7. Many miRs can regulate the expression of cancer-related genes including oncogenes and tumor suppressors8,9. In recent years, deregulation of miRs has been identified in osteosarcoma tissues, and their functions in osteosarcoma have been gradually revealed10,11. For instance, miR-199a-3p is downregulated in osteosarcoma tissues and can inhibit the proliferation and migration of osteosarcoma cells12. miR-218 was reported to inhibit the migration and invasion of osteosarcoma cells by inhibition of TIAM1, MMP2, and MMP913.

miR-33b, an important member of the miR-33 family, has been implicated in several human cancers14. For instance, Zhang et al. reported that miR-33b suppressed the epithelial-to-mesenchymal transition (EMT) and migration of malignant melanoma cells by inhibiting the protein expression of HMGA215. Qu et al. found that miR-33b inhibited growth, invasion, and EMT of lung adenocarcinoma cells by suppressing Wnt/β-catenin/ZEB1 signaling16. Recently, Xu et al. reported that miR-33b could inhibit the migration and invasion of osteosarcoma cells by targeting the c-Myc gene and thus act as a tumor suppressor17. However, the detailed molecular mechanism of miR-33b in regulating the proliferation and migration of osteosarcoma cells still remains largely unclear.

In the present study, we aimed to investigate the exact role of miR-33a in the regulation of osteosarcoma cell proliferation and migration. Furthermore, we explored the underlying mechanism.

MATERIALS AND METHODS

Tissue Sample Collection

This study was approved by the ethics committee of The Fourth Affiliated Hospital of Harbin Medical University, Harbin, P.R. China. A total of 15 primary osteosarcoma tissues and their matched adjacent nontumor tissues were collected at our hospital from April 2013 to March 2015. None of these osteosarcoma patients received any radiation therapy or chemotherapy before surgery. Tissues were immediately snap frozen in liquid nitrogen after surgical resection and stored in liquid nitrogen before use.

Cell Culture

Human osteoblast cell line hFOB and osteosarcoma cell lines (Saos-2, MG63, U2OS, and SW1353) were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, P.R. China. All cell lines were cultured in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies) in a 37°C humidified atmosphere of 5% CO2.

Cell Transfection

Lipofectamine 2000 (Life Technologies) was used to perform cell transfection according to the manufacturer’s instruction. For miR-33b overexpression, miR-33b mimics (GenePharma, Shanghai, P.R. China) was used to transfect U2OS cells, and scramble miR mimic (miR-NC) was used as control. For restoration of hypoxia-inducible factor-1α (HIF-1α) expression, pcDNA3.1-HIF-1α plasmid (Amspring, Changsha, P.R. China) and miR-33b mimics were used to transfect the miR-33b-overexpressing U2OS cells. After transfection for 48 h, real-time RT-PCR or Western blot assay was conducted to examine the expression level of miR-33b or HIF-1α.

Real-Time RT-PCR Assay

Total RNA was extracted with TRIzol reagent (Life Technologies) according to the manufacturer’s instruction. RNA was then converted into cDNA using PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Tokyo, Japan). For miR-33b expression detection, real-time PCR was performed using miRNA Q-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) on the ABI 7500 thermocycler (Life Technologies). U6 gene was used as an internal control. For mRNA expression detection, SYBR Green I Real-Time PCR Kit (Biomics, Nantong, P.R. China) was used to perform real-time PCR. GAPDH was used as an internal control. The reaction condition was 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. The relative expression was analyzed by the 2−ΔΔCt method18.

MTT Assay

MTT assay was used to examine cell proliferation. U2OS cells (10,000 cells/per well) in each group were plated in a 96-well plate and then incubated at 37°C, 5% CO2 for 0, 24, 48, or 72 h, respectively. Then 10 μl of MTT (5 mg/ml) was added to each well and incubated at 37°C, 5% CO2 for 4 h. Then the supernatant was removed and 100 μl of DMSO was added. The absorbance was detected at 570 nm with a microplate reader (Model 680; Bio-Rad, Hercules, CA, USA).

Wound-Healing Assay

Wound-healing assay was used to examine cell migration. U2OS cells in each group were cultured to full confluence, and a wound of approximately 1-mm width was created with a plastic scriber. The U2OS cells were washed and then cultured in DMEM containing 10% FBS for 48 h. Then cells were observed and photographed under a microscope.

Western Blot

Cells were solubilized in cold RIPA lysis buffer (Beyotime, Shanghai, P.R. China). BCA Protein Assay Kit (Beyotime) was used to determine the protein concentration according to the manufacturer’s instruction. Proteins (50 μg) were separated with 12% SDS-PAGE and transferred onto a polyvinylidene difluoride PVDF membrane (Life Technologies), which was incubated with PBS containing 5% milk (Mengniu, Beijing, P.R. China) overnight at 4°C. After being washed with PBS (Life Technologies) three times, the PVDF membrane was then incubated with rabbit anti-human HIF-1α monoclonal antibody (1:100) and rabbit anti-human GAPDH monoclonal antibody (1:200) at room temperature for 3 h, respectively. After being washed with PBS three times, the PVDF membrane was incubated with mouse anti-rabbit secondary antibody (1:5,000) at room temperature for 40 min. The ECL Kit (Pierce Chemical, Rockford, IL, USA) was then used to perform chemiluminescence detection according to the manufacturer’s instruction. The relative protein expression of HIF-1α was represented as the density ratio versus GAPDH.

Bioinformatics Analysis

MiRanda software (http://www.microrna.org) was used to analyze the putative target genes of miR-33b.

Luciferase Reporter Assay

QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to construct the mutant type (MT) of HIF-1α 3′-UTR lacking complementarity with miR-33b seed sequence, according to the manufacturer’s instruction. The wild type (WT) or MT of HIF-1α 3′-UTR was cloned into the downstream of the firefly luciferase coding region of pMIR-GLOTM Luciferase vector (Promega, Madison, WI, USA). U2OS cells were cotransfected with WT-HIF-1α-3′-UTR or MUT-HIF-1α-3′-UTR plasmid, and miR-33b mimic or miR-NC, respectively. After transfection for 48 h, Dual-Luciferase Reporter Assay System (Promega) was used to detect the luciferase activity, according to the manufacturer’s instruction.

Statistical Methods

The results are expressed as the mean ± SD of three independent experiments. SPSS.21 was used to perform statistical analysis. Student’s t-test was used to analyze the difference between two groups. One-way analysis of variance (ANOVA) was used to analyze the differences among more than two groups. Values of p < 0.05 were considered statistically significant.

RESULTS

Decreased Expression of miR-33b in Osteosarcoma

In the present study, we first examined the expression levels of miR-33b in osteosarcoma tissues and matched adjacent nontumor tissues using quantitative RT-PCR assay. Our data demonstrated that the miR-33b expression levels were lower in 83.3% (12/15) of osteosarcoma tissues compared to adjacent tissues (Fig. 1A). Moreover, the expression of miR-33b was significantly downregulated in osteosarcoma tissues compared to their matched adjacent normal tissues (Fig. 1B). We further detected miR-33b levels in several common human osteosarcoma cell lines, including Saos-2, MG63, U2OS, and SW1353. Human osteoblast cell line hFOB was used as the control. Our data indicated that the expression of miR-33b was also reduced in Saos-2, MG63, U2OS, and SW1353 cells, when compared with that in hFOB cells (Fig. 1C). Accordingly, miR-33b was downregulated in osteosarcoma and might play a suppressive role in this disease.

Figure 1

(A, B) Real-Time qPCR was used to examine the miR-33b expression in a total of 15 cases of osteosarcoma tissues and their matched adjacent nontumor tissues. **p < 0.01 versus Adjacent. (C) Real-Time qPCR was used to examine the miR-33b expression in human osteoblasts cell line hFOB and osteosarcoma cell lines including Saos-2, MG63, U2OS, and SW1353. **p < 0.01 versus hFOB.

miR-33b Has Suppressive Effects on U2OS Cell Proliferation and Migration

As U2OS cells showed the most significant decrease in the miR-33b level, we used U2OS cells to study the role of miR-33b in the regulation of cell proliferation and migration in vitro. U2OS cells were transfected with miR-NC or miR-33b mimics, respectively. After transfection for 48 h, the miR-33b levels were significantly increased in the miR-33b mimics group compared to the control group, while transfection with scramble miR (miR-NC) had no effect on the miR-33b expression (Fig. 2A). After that, MTT assay was performed to study cell proliferation. We found that overexpression of miR-33b led to a significant decrease in the proliferation of U2OS cells, when compared to the control group (Fig. 2B). These data indicate that miR-33b could inhibit U2OS cell proliferation. Wound-healing assay was then used to examine cell migration. Our data indicated that the migratory capacity of miR-33b-overexpressing cells was remarkably reduced compared to the control group (Fig. 2C). Accordingly, miR-33b can also suppress U2OS cell migration.

Figure 2

(A) Real-time RT-PCR was performed to determine the expression level of miR-33b in U2OS cells transfected with scramble miRNA (miR-NC) or miR-33b mimic, respectively. (B) MTT assay and (C) wound-healing assay were conducted to determine cell proliferation and migration. Nontransfected U2OS cells were used as the control group. *p < 0.05 versus Control. **p < 0.01 versus Control.

HIF-1α Is a Direct Target of miR-33b in U2OS Cells

As miRs function through regulating their target genes, we further performed bioinformatics prediction to analyze the putative target genes of miR-33b using the Miranda software. HIF-1α, a previously reported oncogene in osteosarcoma, was predicted to be a direct target of miR-33b. To clarify the targeting relationship between miR-33b and HIF-1α, the WT or MT of HIF-1α 3′-UTR luciferase reporter vector was generated (Fig. 3A and B). U2OS cells were then cotransfected with WT HIF-1α 3′-UTR or MT HIF-1α 3′-UTR luciferase reporter vector with miR-33b mimics or miR-NC, respectively. Dual-luciferase reporter assay data showed that the luciferase activity of miR-33b-overexpressing cells was significantly decreased after transfection with WT HIF-1α 3′-UTR luciferase reporter vector, which was abolished when transfected with the MT HIF-1α 3′-UTR luciferase reporter vector (Fig. 3C). These findings indicate that miR-33b can directly bind to the 3′-UTR of HIF-1α mRNA.

Figure 3

(A) The predicted miR-33b target sequence within the wild-type (WT) HIF-1α 3′-UTR and a mutant type (MT) lacking complementarity with miR-33b seed sequence are indicated. (B) The WT or MT of HIF-1α 3′-UTR luciferase reporter vector was generated. (C) Dual-luciferase reporter assay data showed that the luciferase activity of miR-33b-overexpressing U2OS cells was significantly decreased after transfection with WT HIF-1α 3′-UTR luciferase reporter vector, which was abolished when transfected with the MT HIF-1α 3′-UTR luciferase reporter vector. (D) Real-time PCR and (E) Western blot were used to examine the mRNA and protein expression of HIF-1α in U2OS cells transfected with scramble miRNA (miR-NC) or miR-33b mimic, respectively. Nontransfected U2OS cells were used as the control group. **p < 0.01 versus Control.

As miRs negatively regulate the expression of their target genes at the posttranscriptional level, we then detected the mRNA and protein levels of HIF-1α in U2OS cells in each group. Real-time RT-PCR data showed no difference in the HIF-1α mRNA level in U2OS cells with or without miR-33b overexpression (Fig. 3D). However, Western blot assay showed that the HIF-1α protein levels were significantly decreased after overexpression of miR-33b, when compared to the control group (Fig. 3E). On the basis of these findings, we demonstrate that HIF-1α is a target gene of miR-33b in U2OS cells.

Overexpression of HIF-1α Reversed the Inhibitory Effects of miR-33b on the Proliferation and Migration of U2OS Cells

We further studied whether the regulatory effects of miR-33b on U2OS cells was through mediation of HIF-1α expression. The miR-33b-overexpressing U2OS cells were transfected with pc-DNA3.1-HIF-1α plasmid. After transfection, the mRNA and protein levels of HIF-1α were significantly increased in the miR-33b + HIF-1α group, when compared to the miR-33b group (Fig. 4A and B). MTT assay was then conducted to examine cell proliferation. Cell proliferation was significantly increased in the miR-33b + HIF-1α group compared to the miR-33b group (Fig. 4C), indicating that overexpression of HIF-1α reversed the suppressive effects of miR-33b on U2OS cell proliferation. Besides, overexpression of miR-33b also reversed the inhibitory effect of miR-33b on the migration of U2OS cells (Fig. 4D). Therefore, our findings suggest that miR-33b plays a suppressive role in the regulation of osteosarcoma cell proliferation and migration by directly targeting HIF-1α.

Figure 4

(A) Real-time PCR and (B) Western blot were used to examine the mRNA and protein expression of HIF-1α in U2OS cell-transfected miR-33b mimic or cotransfected with miR-33b mimic and HIF-1α ORF plasmid, respectively. (C) MTT assay and (D) wound-healing assay were conducted to determine cell proliferation and migration. **p < 0.01 versus miR-33b.

HIF-1α Is Upregulated in Osteosarcoma and Inversely Correlated to miR-33b Levels

In addition, we examined the expression levels of HIF-1α in osteosarcoma tissues and adjacent nontumor tissues. Real-time RT-PCR assay showed that the mRNA levels of HIF-1α were frequently and significantly increased in osteosarcoma tissues compared to their matched adjacent tissues (Fig. 5A and B). Moreover, the HIF-1α levels were inversely correlated to the miR-33b levels in osteosarcoma tissues (Fig. 5C). These above findings suggest that the reduced expression of miR-33b may contribute to the upregulation of HIF-1α in osteosarcoma tissues.

Figure 5

(A, B) Real-time qPCR was used to examine the mRNA expression of HIF-1α in a total of 15 cases of osteosarcoma tissues and their matched adjacent nontumor tissues. **p < 0.01 versus Adjacent. (C) The HIF-1α mRNA levels were inversely correlated to the miR-33b levels in osteosarcoma tissues.

DISCUSSION

miRs have been found to play promoting or suppressive roles in the regulation of the malignant phenotypes of osteosarcoma cells. However, the underlying mechanism of miR-33b in mediating osteosarcoma cell proliferation and migration still remains largely unclear. In this study, we found that the expression of miR-33b was significantly downregulated in osteosarcoma tissues compared to matched adjacent nontumor tissues. miR-33b was also downregulated in osteosarcoma cell lines compared to normal human osteoblast cells. Overexpression of miR-33b suppressed the proliferation and migration of osteosarcoma U2OS cells. HIF-1α was further identified as a target gene of miR-33b in U2OS cells, and overexpression of HIF-1α reversed the suppressive effects of miR-33b on the proliferation and migration of U2OS cells. These findings suggest that miR-33b can inhibit osteosarcoma cell proliferation and migration via directly targeting HIF-1α.

Multiple miRs have been implicated in the development and malignant progression of osteosarcoma. For instance, miR-145 inhibits invasion and metastasis of osteosarcoma cells via inhibiting the protein levels of VEGF19. miR-101 inhibits proliferation and induces apoptosis of osteosarcoma cells by targeting mTOR20. Recently, miR-33a was found to be downregulated in osteosarcoma tissues compared with adjacent nontumor bone tissues, and overexpression of miR-33a significantly reduced osteosarcoma cell growth21. Moreover, miR-33b was also reported to be significantly downregulated in osteosarcoma tissues and can inhibit the migration and invasion of osteosarcoma cells by targeting the c-Myc gene17. These findings suggest that the miR-33 family plays a suppressive role in osteosarcoma. In this study, we also found that the miR-33b levels were significantly decreased in osteosarcoma tissues and cell lines. Moreover, we showed that overexpression of miR-33b markedly inhibited the proliferation and migration of osteosarcoma U2OS cells. Therefore, miR-33b may have a suppressive effect on tumor growth and metastasis in vivo, which should be clarified in future studies.

Low levels of oxygen in the tumor microenvironment can induce the expression of HIF during tumor progression, which further enhance the glycolytic metabolism in cancer cells22,23. HIF-1α is the α subunit of transcription factor HIF-1 and functions as a key regulator of cellular and systemic homeostatic response to hypoxia24. By activating the transcription of those genes involved in energy metabolism, angiogenesis, and apoptosis, HIF-1α plays an important role in embryonic vascularization, tumor angiogenesis, and pathophysiology of ischemic disease25,26. Moreover, inhibition of HIF-1α-mediated signaling can drive tumor cells toward mitochondrial oxidative metabolism, which potentiates the therapeutic activity of pro-oxidants27. Recently, HIF-1α was found to play an oncogenic role in osteosarcoma28. Liang et al. reported that HIF-1α could protect osteosarcoma cells from undergoing apoptosis29. In addition, HIF-1α contributes to the proliferation, migration, EMT, and chemoresistance in osteosarcoma30,31. In the present study, bioinformatics analysis and luciferase reporter assay data indicated that HIF-1α was a direct target gene of miR-33b, and the expression of HIF-1α was negatively mediated by miR-33b in U2OS cells. Therefore, we speculated that HIF-1α might be involved in the suppressive effect of miR-33b on U2OS cell proliferation and migration. To further verify this speculation, we performed reverse experiments and found that overexpression of HIF-1α significantly reversed the suppressive effects of miR-33b on osteosarcoma cell proliferation and migration. Accordingly, we suggest that miR-33b inhibits osteosarcoma cell proliferation and migration by inhibition of HIF-1α expression.

To our knowledge, this is the first study that revealed a suppressive role of miR-33b in the regulation of osteosarcoma cell proliferation and migration via directly targeting HIF-1α. Our findings expand the understanding of the miRs’ function in the malignant progression of osteosarcoma. Therefore, we suggest that the miR-33b/HIF-1α axis may become a potential target for osteosarcoma treatment.