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. 2020 Dec 1;10(12):4357–4371.

Molecular mechanisms underlying the enhancement of carbon ion beam radiosensitivity of osteosarcoma cells by miR-29b

Eun Ho Kim 1,*, Jeong Yub Kim 2,*, Mi-Sook Kim 3, Guillaume Vares 4, Tatsuya Ohno 5, Akihisa Takahashi 5, Akiko Uzawa 6, Seung-Jun Seo 1, Sei Sai 6
PMCID: PMC7783744  PMID: 33415004

Abstract

Carbon ion radiotherapy (CIRT) is more effective than conventional photon beam radiotherapy in treating osteosarcoma (OSA); however, the outcomes of CIRT alone are still unsatisfactory. In this study, we aimed to investigate whether miR-29b acts as a radiosensitizer for CIRT. The OSA cell lines U2OS and KHOS were treated with carbon ion beam alone, γ-ray irradiation alone, or in combination with an miR-29b mimic. OSA cell death as well as invasive and migratory abilities were analyzed through viability, colony formation, Transwell, and apoptosis assays. miR-29 expression was downregulated in OSA tissues compared to that in normal tissues and was associated with metastasis and relapse in patients with OSA. Further, miR-29b was found to directly target the transcription factor Sp1 and suppress the activation of the phosphatase and tensin homolog (PTEN)-AKT pathway. Conversely, Sp1 was found to attenuate the inhibitory effects of miR-29b in OSA cells. When used in combination with miR-29b mimic, carbon ion beam markedly inhibited invasion, migration, and proliferation of OSA cells and promoted apoptosis by inhibiting AKT phosphorylation in a Sp1/PTEN-mediated manner. Taken together, miR-29b mimic improved the radiosensitivity of OSA cells via the PTEN-AKT-Sp1 signaling pathway, presenting a novel strategy for the development of carbon ion beam combination therapy.

Keywords: miR-29b, carbon ion, radiosensitivity, osteosarcoma cells, AKT, PTEN, Sp1

Introduction

Osteosarcoma (OSA) is the most common malignant primary neoplasm of the bone in children and young adults [1,2]. As OSA cells are not readily killed by photon beams, conventional radiation therapy does not play a major role in the treatment of OSA [3,4]. High linear transfer energy (LET) carbon ion radiotherapy (CIRT) is an innovative method in cancer treatment resulting in improved quality of life owing to its high radiocurability, which is attributable to its strong biological impact and more desirable dose distribution than that of proton beam and photon beam radiation therapies [5,6]. The physical and biological advantages of CIRT make it ideal for targeting radioresistant tumors or tumors located near sensitive organs [7,8]. Over the past 25 years, CIRT has been predominantly applied in the treatment of several cancers including OSA. According to our clinical data, CIRT is an effective and safe modality to treat unresectable OSA of the trunk; therefore, it offers appreciable long-term functional results and a decent survival advantage without accompanying morbidities [9-13]. We previously explored how and why CIRT alone or in combination with DNA-damaging drugs such as gemcitabine and cisplatin is more effective in killing radioresistant cancer stem cells (CSCs) than photon beam therapy [14-20]. However, limitations exist in the application of CIRT alone or in combination with existing anticancer drugs for the treatment of more advanced and aggressive cancers [21]. As the overall survival of patients with OSA is still poor, an effective novel molecular targeted combination therapy is warranted for expanding the therapeutic effects of CIRT.

MicroRNAs (miRNAs) are endogenously expressed on coding RNA molecules that regulate gene expression negatively at the post-transcriptional level by base-pairing with the untranslated region (UTR) of the target mRNAs [22,23]. A single miRNA binds and regulates nearly 100 different transcripts [24,25]. Therefore, miRNAs are deemed the master regulators of gene expression, influencing majority of cell events and activities, metabolism, infection, as well as cancer [26-28]. The anti-tumor effects of miRNAs have been demonstrated by targeting the miRNA network, indicating a great therapeutic potential of miRNAs in cancer treatment [29,30]. Thus, there is a need for developing combination therapy involving carbon ion beam and new potential molecular targeted drugs such as miRNA mimics/antagomirs. Recently, several studies have shown that miR-29b, a miR-29 family member, is altered in several human cancers and serves as a critical tumor suppressor [31]. The enforced expression of miR-29b in cancer cells impedes extracellular matrix remodeling [32], tumor-suppressor promoter methylation [30], and antiapoptotic signaling [33,34]. In addition, the expression of miR-29b and miR-29a is downregulated via activation of survival-promoting and multiple growth signaling pathways such as the ones that involve c-myc, Hedgehog, and NF-kB [35]. miR-29b overexpression induce apoptosis in vitro and anti-tumor effects in vivo in acute myeloid leukemia and rhabdomyosarcoma [36-46]. We previously reported that downregulation of miR-29c expression resulted in enhanced expression of KLF4, a transcription factor that maintains breast CSCs, leading to the inhibition of CSC production. This finding suggested that miR-29c negatively regulates breast CSCs [47]. Recently, we have demonstrated that zoledronic acid (ZOL), one of the bisphosphonates, is a drug used to treat osteoporosis and bone metastasis, effectively enhanced carbon ion beam radiosensitivity accompanied with upregulation of miR-29b expression in OSA cells [48].

In this study, we aimed to elucidate the molecular mechanisms underlying miR-29b-induced carbon ion beam radiosensitization of OSA cells.

Materials and methods

Cell culture

U2OS and KHOS/NP OSA cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle medium (DMEM) [before being supplemented with fetal bovine serum (FBS; WelGene), 1% (v/v) penicillin-streptomycin, and 10% FBS (Gibco®; Thermo Fisher Scientific, Waltham, MA)] in a humidified incubator at 37°C and 5% CO2. OSA tissues and matched non-tumor tissues were derived after obtaining informed consent from 14 patients who were operated at the Korea Institute of Radiological and Medical Sciences (Institutional Review Board Approval Number K-1603-001-001). Primary cell cultures were established from this tissue. Simply put, the tissue was finely chopped into a slurry with a blade, washed with phosphate buffered saline (PBS), and centrifuged at 1000 rpm for 3 minutes. The supernatant is then discarded and the pellet resuspended in serum-free Dulbecco-modified Eagle’s medium (DMEM, WelGene, Daegu, South Korea) containing 0.05-0.1% (w/v) Type I collagenase (Gibco®, Life Technologies). After 2 h, cells were washed clean with PBS and maintained in DMEM containing 20% (v/v) FBS.

Reagents

Anti-p21 (sc-397), anti-β-actin (sc-81178), anti-Slug (sc-166476), and anti-Snail (sc-10432) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-cleaved polyADP ribose polymerase (PARP) (#9541), CDK6 (#3136), MCL-1 (#4572), Sp1 (#9389), PTEN (#9559) p-AKT (Ser473) (#4060), total AKT (#9272), p-4EBP1 (S65) (#9451) and p-GSK-3b (Ser9) (#9336) were purchased from Cell Signaling Technology (Danvers, MA).

Irradiation

The cells were irradiated with carbon ion beams accelerated by the heavy ion medical accelerator in Chiba at the National Institute of Radiological Sciences. The details regarding the beam characteristics of carbon ion beams, biological irradiation procedures, and dosimetry are described elsewhere [30]. Briefly, we used 290 MeV/nucleon carbon ion beams with dose average LET of 50 KeV/µm at the center of spread-out Bragg peak. As a reference, we irradiated cells with Cs-137 γ-rays (Atomic Energy of Canada, Ltd., Ontario, Canada) or X-rays (Titan-320, GE Co., USA) at a dose rate of 2.45 or 3.81 Gy/min, respectively, at the Korea Institute of Radiological and Medical Sciences and/or NRIS. The cells were irradiated with γ-rays (2, 4, or 6 Gy) or carbon ion beams (1, 2, or 3 Gy).

miRNA and transient transfection

miR-29b mimic and control mimic were purchased from Bioneer (Daejeon, South Korea). The cells were transfected with 60 nM control or miR-29b mimic for 24 h by using G-fectin miRNA Transfection Reagent. Then, the cells were transfected with si-SP1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions.

Cell viability assay

The cells were seeded in 96-well plates at a density of 5000 cells/well and incubated for the time points indicated therein. To quantify cell viability, a culture medium containing an equal amount of EZ-Cytox reagent was added to the cells and incubated for 4 h. Then, the cells were treated with miR-29b mimic for 24 h and seeded and irradiated. Cell viability was measured after 48 h using a Multiskan EX instrument (Thermo Fisher Scientific) at 450 nm.

Colony formation assay

At 24 h post-transfection with miR-29b mimic, the cells were reseeded and incubated for 7-9 days. The colonies were then fixed with methanol, stained with 0.4% crystal violet (Sigma-Aldrich, St. Louis, MO), and counted.

Western blotting

OSA cells were treated with miR-29b or irradiation and incubated for 24 or 48 h. The cells were then lysed with RIPA buffer. Next, the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% (v/v) skim milk in PBS and 0.1% Tween 20 and then incubated with the indicated antibodies (1:1,000), followed by secondary antibodies (1:1,000). Finally, the blots were developed using the Pierce Enhanced Chemiluminescence Western Blotting Substrate (Thermo Fisher Scientific) and scanned.

Detection of apoptotic cells by Annexin V staining

The cells were treated with miR-29b and incubated for 48 h. Then, the cells were washed with ice-cold PBS, trypsinized, and suspended in 1 × binding buffer at a density of 1 × 106 cells/mL. Cell suspension aliquots (100 μL) were mixed with 5 μL annexin V FITC and 10 μL propidium iodide stock solution (50 μg/mL in PBS) through gentle vortexing and incubated for 15 min at room temperature. To each sample, 1 × buffer (400 μL) was added and analyzed using FACScan flow cytometer. At least 10000 cells were counted for all samples, and data analysis was performed using CellQuest software.

Transwell assays

The invasive and migratory abilities of OSA cells were determined using Transwell chambers (Millipore) according to the manufacturer’s instructions. The cells were placed on the upper chamber of Transwell containing 150 μL of medium that was either untreated or treated with irradiation, miR-29b, or miR-29b along with irradiation for 24 h. The cells that migrated through the Matrigel/gelatin-coated membrane were stained using a cell stain solution provided with the Transwell chamber assay kit (Chemicon, Millipore).

DNA constructs

A DNA fragment of the Sp1 3’-UTR that contained the putative binding site of miR-29b was amplified with PCR by using the following primers: 5’-GTGTCTAGAGATTAGGGGAGGGTTGGAG-3’ (forward) and 5’-GTGGAATTCGTCCAAAAGGCATCAGGG-3’ (reverse), before cloning into the pGL3luc vector. For creating the mutant reporter, four nucleotide mutations were induced in the putative miR-29b binding site using EZchange™ Multi Site-directed Mutagenesis Core Kit (Enzynomics, Daejeon, South Korea). The primer sequences used were 5’-GGTTCTTCTGGGGacgaAATCAGGCCCCTG-3’ (forward) and 5’-CAGGGGCCTGATTtcgtCCCCAGAAGAACC-3’ (reverse). The vectors were confirmed by sequencing (Bioneer).

Luciferase assay

miR target prediction sites were used to predict Sp1 as a direct target gene of miR-29b. According to these prediction sites, Sp1 was included as the potential target. The cells were seeded in a 24-well plate. Then, Sp1 3’-UTR mutant plasmids or reporter constructs were co-transfected with miR-29b as well as pRL-CMV-Renilla internal control plasmid using Lipofectamine 2000. The Dual-GloTM Luciferase Assay System was used to determine the luciferase activity. The relative activities were normalized to Renilla luciferase activity.

Plasimd transient transfection

Prior to transfection, cells were plated on 6-well plates. After reaching 80% confluence, it was transfected with 2 μg of plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s procedure.

RNA extraction and quantitative reverse-transcription PCR

Total RNA was isolated using TRIzol reagent (Invitrogen). Quantitative reverse-transcription PCR was performed using KAPA Biosystems’ KAPA SYBR FAST qPCR kit according to the manufacturer’s instructions.

Caspase activity assay

Caspase activity was measured using the caspase family activity assay kits, according to the manufacturer’s instructions.

Statistical analysis

Experiments involving cell culture were performed in triplicate. The data were expressed as values of mean ± standard deviation. The statistical differences between different groups were analyzed using ANOVA and Student’s t-test (two-tailed). The following P-values were considered significant: *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

miR-29b expression is downregulated in OS specimens and cell lines

To understand the signatures of differentially expressed genes (DEGs), we performed miR-29b gene ontology analysis. Accordingly, these DEGs were significantly enriched in the processes of cell death and negative regulation of cell proliferation (Figure 1A). qRT-PCR analysis revealed that miR-29b expression in primary tumor tissues was significantly enhanced when compared to that in matched non-tumor tissues (IRB: No. K-1603-001-001) (Figure 1B).

Figure 1.

Figure 1

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A. DAVID function analysis for the miR-29b targeted genes of pathways in OSA. The horizontal axis represented p-value transformed by -log2 and the gene number of each cluster respectively. The vertical axis represented the functions of the target genes. Only the most significantly enriched clusters were shown. B. Relative expression of miR-29 in matched primary OSA tissues and non-tumor tissues; *P < 0.05.

miR-29b inhibits OSA cell proliferation, survival, migration, and invasion via Sp1 suppression

miR-29b was transfected into KHOS and U2OS cells, which was confirmed by qRT-PCR analysis after 24 h (Figure 2A). To determine whether miR-29b suppresses the oncogenic phenotype of OSA cells, we performed gain-of-function assays in OSA cells using miR-29b mimics. As shown in Figure 2B, miR-29 mimic significantly inhibited OSA cell growth from 48 h after transfection. We also found that miR-29b mimic significantly inhibited colony formation and cell proliferation in KHOS and U2OS cells (Figure 2C, 2D). Interestingly, growth inhibition was associated to upregulation of the cell-cycle inhibitor p21 and downregulation of pro-survival factors CDK-6 and MCL-1, both observed 24 h after cell transfection with miR-29b. Importantly, miR-29b-induced apoptosis was found to be associated with PARP activation, as evidenced by increased cleavage using Western blotting (Figure 2E). And transfection after 48 h resulted in a substantial accumulation of cell populations in G1 and a decrease in cell cycle S phase populations (Figure 2F). Furthermore, we found that miR-29b mimic suppressed the migratory and invasive ability of KHOS and U2OS cells (Figure 2G). Data regarding the circRNA/miRNA/mRNA interaction network of miR-29b, obtained using Cytoscape, were integrated to construct a cancer-related signaling network that includes miR-29b target genes. Most target genes in the network were considered closely associated with cell proliferation and tumor growth (Figure 2H). Thus, these findings indicate that miR-29b significantly inhibits proliferation, survival, migration, and invasion of OSA cells.

Figure 2.

Figure 2

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miR-29b inhibits OSA cell proliferation, survival, migration, and invasion. (A) qPCR analysis showing relative miR29b expression; ***P < 0.001. (B) Trypan blue assay of two OSA cell lines transfected with miR-29b or scrambled oligonucleotides (Scr); *P < 0.05, **P < 0.01, ***P < 0.001. (C, D) Colony formation assay (upper) and MTT assay (lower) using the two OSA cell lines after transfection with synthetic miR-29b or Scr; *P < 0.05, **P < 0.01. (E) Immunoblotting using the indicated antibodies 48 h after transfecting the two OSA cell lines with synthetic miR-29b or Scr. (F) Inhibition of cell cycle progression by overexpression of miR-29b. Two OSA cell lines were transfected as in (A). Cells were stained with propidium iodide (PI) at 48 h post-transfection and analyzed with FACS. *P < 0.05, (G) Migration and invasion of OSA cells decreased with miR-29b overexpression. Photo micrographs of Transwell migration and invasion of OSA cells transfected for 24 h; *P < 0.05. (H) Establishment of a circRNA/miRNA/mRNA interaction network of pathways in cancer.

Sp1 is a direct target of miR-29 and miR-29 suppresses OSA growth and invasion through Sp1

The predicted binding site of miR-29b was identified in the 3’-UTR of Sp1 mRNA. Therefore, we first constructed luciferase reporter plasmids containing the wild-type or mutant miR-29b target site in the Sp1 3’-UTR sequence (Figure 3A). KHOS and U2OS cells were co-transfected with miR-29b expression and reporter plasmids. miR-29b expression significantly decreased firefly luciferase activity but not mutant reporter activity, in the presence of wild-type 3’-UTR (Figure 3B). Furthermore, miR-29b overexpression for 48 h significantly decreased mRNA and protein levels of Sp1 compared with those in the control group (Figure 3C, 3D), indicating that miR-29b suppressed Sp1 expression by binding to its target site at the 3’-UTR of Sp1. We also found that Sp1 overexpression was associated with reduced miR-29b levels (Figure 3E). And Sp1 silencing led to inhibition of OSA cell growth and cell proliferation, and Sp1 overexpression showed the opposite result (Figure 3F-H). These results demonstrate that miR-29 suppressed the growth of OSA cells by directly downregulating Sp1. Thus, our findings show that miR-29 forms an auto-regulatory loop with Sp1 to regulate OSA cell aggressiveness.

Figure 3.

Figure 3

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miR-29b inhibits Sp1 expression. A. Wild-type AKT (WT) and mutant (MUT) forms of putative miR-29 target sequences of the Sp1 3’-UTR. B. Analysis of the luciferase activity of psicheck-2-Sp1 3’-UTR WT and MUT vectors in OSA cells; **P < 0.01. C. Sp1 mRNA levels in the indicated cells were analyzed by qRT-PCR; *P < 0.05. D. Sp1 protein levels in the indicated cells were examined by western blotting. E. miR-29b levels were analyzed by qRT-PCR in OSA cells transfected with Sp1; *P < 0.05. F. Sp1 protein levels were detected by western blotting analysis after transfection of KHOS and U2OS cells with si-Sp1 and Sp1. G, H. Trypan blue assay and MTT assay were performed using KHOS and U2OS cells transfected with si-Sp1 and Sp1; *P < 0.05.

miR-29b activates the PTEN/AKT pathway by suppressing the Sp1 loop in OSA cells

miR-29b inhibits proliferation, migration, and invasion of tongue tumor cells via phosphatase and tensin homolog (PTEN)-AKT-SP1 pathway [30]. To examine whether miR-29b acts as a potential tumor suppressor in OSA cells, we performed pathway analysis of PTEN-AKT-SP1 and found that miR-29b significantly increased PTEN protein levels and downregulated phosphorylated AKT expression without affecting the total AKT levels (Figure 4A). We next transfected KHOS and U2OS cells with miR-29b expression plasmid and a luciferase reporter containing the wild-type or mutant PTEN promoter. A marked increase in the luciferase/Renilla ratio was found in cells transfected with the wild-type PTEN promoter reporter but not in those with the mutant reporter (Figure 4B). To ascertain whether miR-29b upregulates PTEN expression by targeting Sp1, we determined whether knockdown of endogenous Sp1 activity mimics the effects of exogenous miR-29b overexpression. There was a marked increase in the luciferase/renilla ratio in cells transfected with the wild-type PTEN promoter reporter, but not with the mutant reporter (Figure 4C). As expected, siRNA-mediated Sp1 knockdown in OSA cells increased PTEN expression and consequently decreased AKT phosphorylation (Figure 4D). In addition, PI3K inhibition by LY294002 increased miR-29b and decreased Sp1 and p-AKT (S473) protein levels, so miR-29b was negatively regulated by PI3K/AKT (Figure 4E). Overexpression of AKT suppressed the miR-29b-dependent decrease in Sp1 mRNA levels (Figure 4F). Transfection of the synthetic miR-29b mimic also resulted in reduced phosphorylation of the AKT substrate glycogen synthase kinase-3b (p-GSK-3b (S9)) (Figure 4G). Moreover, we provided preliminary evidence that transfection of cells with miR-29b mimics inhibits mTOR-mediated signals, as evidenced by the reduced p-4EBP1 (S65) (Figure 4G). These findings demonstrate that miR-29b acts as a negative regulator of the PI3K/AKT pathway.

Figure 4.

Figure 4

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miR-29b activates the PTEN/AKT pathway via Sp1 suppression. A. PTEN, p-AKT, and AKT protein levels were analyzed by western blotting of two OSA cell lines transfected with miR-29b. PTEN gene expression in Scr- and miR-29b-transfected cells by qRT-PCR; *P < 0.05. B. The Sp1 binding site (middle) on the PTEN promoter (lower).Mutation of Sp1 binding site (upper). C. Relative luciferase activity in control or miR-29b-expressing OSA cell lines co-transfected with a wild-type or mutant PTEN promoter; **P < 0.01. D. PTEN, p-AKT, and AKT protein levels were analyzed by western blotting of two OSA cell lines transfected with si-Sp1 RNA. PTEN gene expression in Scr- and si-Sp1-transfected cells by qRT-PCR; *P < 0.05; **P < 0.01. E. qRT-PCR of miR-29b in OSA cell lines treated with 20 mM LY294002 or vehicle (DMSO) for 48 h. Immunoblotting shows the protein levels of Sp1 and phosphorylated AKT at S473 in LY294002-treated cells; *P < 0.05, **P < 0.01. F. Cells were transfected with pcDNA.3.1-HA-myr-AKT dominant active construct or the empty vector pcDNA3.1 and analyzed after 48 h for Sp1 expression levels by qRT-PCR; **P < 0.01. G. The indicated antibodies were used for western blotting of OSA cells transfected with miR-29b.

Carbon ion beam irradiation in combination with miR-29b mimic decreases OSA cell viability and increases apoptosis induction

To evaluate the effects of miR-29b on the radiosensitivity of OSA cells, cell viability assays were performed as mentioned in Methods. OSA cells were first treated with carbon ion beam alone, γ-ray alone, or in combination with miR-29b pretreatment for 24 h. miR-29b pretreatment significantly reduced the cell growth of OSA cells following 72 h of irradiation (Figure 5A), indicating that miR-29b markedly enhances the radiosensitivity of OSA cells. In addition, carbon-ion beam + miR-29b mimic reduced the survival fraction of OSA cells significantly more than carbon ion beam irradiation alone (Figure 5B). We also found that apoptotic events induced by combination treatment of carbon ion beam irradiation and miR-29b mimic were associated with increased caspase-3 activation and activated PARP (Figure 5C, 5D). Consistent with our assumption, antagomiR-29b-expressing cells showed proliferative characteristics and were less susceptible to carbon ion beam-induced growth inhibition (Figure 5E).

Figure 5.

Figure 5

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Carbon ion beam irradiation combined with miR-29b mimic decreased OSA cell survival and enhanced apoptosis induction. A. Trypan blue assay of OSA cells after transfection with synthetic miR-29b or carbon ion or X-ray irradiation + miR-29b mimic combination treatment; *P < 0.05, **P < 0.01. B. Colony formation assay of OSA cells after transfection with synthetic miR-29b or carbon ion or X-ray irradiation + miR-29b mimic combination treatment. C. Analysis of caspase activity in both OSA cell lines 48 h after carbon ion beam treatment alone or in combination with miR-29b. Data were collected using Multiskan EX at 405 nm (upper); *P < 0.05, **P < 0.01. D. Cleaved-PARP protein level was analyzed by western blotting of two OSA cell lines with the indicated treatment (lower). E. Two OSA cells were transduced with the empty vector or miR-29b mimic or antagomiR-29b and then treated with irradiation for 48 h. Viable cells were determined using trypan blue assay; **P < 0.01.

Carbon ion beam irradiation combined with the miR-29b mimic enhances radiosensitivity via Sp1 suppression in OSA cells

To investigate the molecular mechanism underlying the enhanced carbon ion radiosensitivity of OSA cells after treatment with the miR-29b mimic, we examined the changes in Sp1 mRNA and protein levels in OSA cells. Compared with γ-ray irradiation, carbon ion beam irradiation dramatically downregulated Sp1 expression in KHOS cells and U20S cells (Figure 6A). In addition, carbon ion beam irradiation significantly upregulated miR-29b expression compared with γ-ray irradiation in both KHOS and U2OS cells (Figure 6B). As shown in Figure 6C, miR-29b overexpression along with siSP1 treatment caused a significant inhibition in cell growth and invasive ability of both OSA cell lines starting at 48 h after transfection (Figure 6D). Western blotting results further confirmed that transcription factors associated with epithelial-mesenchymal transition proteins were dramatically decreased in carbon ion beam + miR-29b-treated cells (Figure 6E). Next, we examined the effect of mithramycin A, a Sp1-specific inhibitor, on the carbon ion radiosensitivity of OSA cells (Figure 6F). Mithramycin A significantly reduced the growth of OSA cells after a carbon ion beam. Furthermore, carbon ion irradiation showed a significant inhibition of cell viability than gamma-ray irradiation. These findings indicate that Sp1 is essential for the radioresistance of OSA cells and that decreased Sp1 expression may be involved in miR-29b-induced carbon ion radiosensitivity.

Figure 6.

Figure 6

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Carbon ion beam combined with miR-29b mimic enhanced radiosensitivity in OSA cells via Sp1 suppression. A. Immunoblotting using the indicated antibodies for both OSA cell lines treated for 24 h with each radiation. B. miR-29b levels after γ-ray or carbon ion beam irradiation were analyzed by qRT-PCR; *P < 0.05, **P < 0.01. C. Trypan blue assay of both OSA cell lines 48 h after treatment with the indicated treatment ; *P < 0.05. D. Transwell migration and invasion assays were performed using the two OSA cell lines transfected with miR-29b or siSP1; *P < 0.05, **P < 0.01. E. Immunoblotting of the indicated antibodies 48 h after synthetic miR-29b transfection of KHOS/NP cells combined with carbon ion beam treatment. F. Trypan blue assay of miR-29b or carbon ion or Gamma-ray or irradiation + miR-29b mimic combination treatment in both OSA cells treated with mithramycin-A (100 nmol/L) or vehicle (DMSO) for 15 h; **P < 0.01 Mith decreased the cell number of OSA cells treated with Carbon ion; *P < 0.05, **P < 0.01.

Discussion

In this study, we aimed to elucidate the molecular mechanism underlying miR-29b-induced carbon ion beam radiosensitization of OSA cells. We found that carbon ion beam irradiation combined with miR-29b mimic significantly increased caspase-3 activation compared with that of carbon ion beam irradiation alone, suggesting that the enhanced radiosensitization of miR-29b may be partially due to an increased induction of apoptosis. Regarding the molecular mechanisms underlying the radiosensitizing effects of miR-29b, the miR-29b-Sp1 loop has been shown to play a major role in this process.

First, we analyzed miR-29b expression in primary OSA and non-tumors from 14 patients and found that miR-29b expression was lower in tumor tissues than in the corresponding non-tumor tissues. Our recent study involving miRNA microarray profiling from four paired samples of OSA versus matched nonmalignant tissues also showed the same results. This finding is also consistent with previous reports demonstrating that miR-29b expression is decreased in various tumors, such as multiple myeloma, lung tumors, and gastric cancer, and acts as a tumor suppressor gene [28,35,38-46]. Then, we confirmed that treatment with miR-29b mimic alone significantly inhibited cell proliferation and survival and suppressed cell migration and invasion, accompanied by enhanced p21 expression and decreased CDK6 and MCL1 expression in KHOS and U2OS cells. This finding is consistent with previous reports demonstrating that miR-29b acts as a tumor suppressor in various cancers, such as prostate, breast, glioblastoma, and lung cancers [40-43]. Moreover, the miR-29b mimic significantly reduced Sp1 expression by binding to its target site at the 3’-UTR of Sp1. In contrast, Sp1 overexpression reduced miR-29b expression, whereas silencing of Sp1 increased miR-29b levels, consequently suppressing the proliferation, migration, and invasion of OSA cells. These results demonstrate that miR-29b inhibits the growth and invasion of OSA cells, at least in part, by downregulating Sp1. Therefore, we speculate that a miR-29b-Sp1 regulatory feedback loop occurs in OSA cells and radiation-treated OSA cells, and it could be a potential therapeutic target. This finding is in agreement with previous findings where miR-29b was found to be negatively correlated with Sp1 expression [44-46].

In the present study, miR-29b mimic significantly increased PTEN expression and reduced phosphorylated AKT levels without affecting total AKT levels. Sp1 knockdown increased PTEN expression and subsequently decreased AKT phosphorylation in OSA cells. Moreover, inhibition of PI3K by LY294002 decreased Sp1 protein expression and increased miR-29b levels as further proof for the negative regulation exerted by PI3K/AKT on miR-29b expression. These findings demonstrate that miR-29b acts as a negative regulator of the PI3K/AKT pathway. miR-29b inhibits AKT phosphorylation by suppressing PI3K and increasing PTEN expression [42,43]. Carbon ion beam irradiation effectively inhibited Sp1 expression. Carbon ion beam irradiation in combination with miR-29b mimic showed stronger cell killing effects and suppression of OSA cell migration and invasion than γ-ray irradiation. These results present novel evidence on the major molecular mechanisms underlying the enhancement of carbon ion beam radiosensitivity of OSA cells by miR-29b.

In summary, carbon ion beam irradiation in combination with miR-29b mimic efficiently suppressed the invasion, migration, and proliferation of OSA cells and promoted apoptosis by inhibiting AKT phosphorylation via Sp1/PTEN-AKT pathway (Figure 7). The miR-29b mimic enhanced radiosensitivity of OSA cells to carbon ion beam irradiation via PTEN-AKT-Sp1 pathway, suggesting a strategy for the development of novel combination CIRT.

Figure 7.

Figure 7

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Molecular mechanisms underlying the increased carbon ion radiosensitivity of OSA cells via modulation of the miR-29b-PTEN/AKT-SP1 pathway.

Acknowledgements

This study was part of a research project with Heavy-ion at NIRS-HIMAC. This work was supported by the National Research Foundation of Korea (NRF; grant no. NRF-2019M2A2B4095150) and by the Rare Isotope Science Project of Institute for Basic Science funded by Ministry of Science and ICT and NRF of Korea (2013M7A1A1075764).

Disclosure of conflict of interest

None.

Abbreviations

CIRT

carbon-ion radiotherapy

CSC

cancer stem cell

LET

linear transfer energy

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

GSK-3b

glycogen synthase kinase-3b

HIMAC

heavy ion medical accelerator in Chiba

KIRAMS

Korea Institute of Radiological and Medical Sciences

miRNAs

microRNAs

NIRS

National Institute of Radiological Sciences

OSA

Osteosarcoma

QST

National Institutes for Quantum and Radiological Science and Technology

UTR

untranslated region

PTEN

phosphatase and tensin homolog

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