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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.47 No.1 pp.13-23
DOI : https://doi.org/10.17779/KAOMP.2023.47.1.002

MicroRNA-126 Promotes the Proliferation and Migration of Oral Squamous Cell Carcinoma Cells Via HIF-1α Expression

Suyeon Park1), Ji-Hyeon Oh2), Sang Shin Lee1), Jongho Choi1)
1)Department of Pathology, College of Dentistry, Gangneung-Wonju National University
2)Department of Oral and Maxillofacial Surgery, College of Dentistry, Gangneung-Wonju National University
* Correspondence: Jongho Choi, Ph.D., Department of Oral Pathology, College of Dentistry Gangneung-Wonju National University, 7Jukheon-gil, Gangneung-si, Gangwon-do, Korea Tel: +82-33-640-2231 Email: jhchoi@gwnu.ac.kr
January 17, 2023 January 20, 2023 February 10, 2023

Abstract


Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer and is associated with high recurrence, poor treatment, and low survival rates. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates the response to hypoxia, a major factor in the tumor microenvironment that affects tumor development and progression in various cancer types. However, microRNA (miRNA) sequence analysis revealed that only a few miRNAs targeting HIF-1α had been discovered. In the present study, we investigated HIF-1α expression in OSCC and the effect of HIF-1α-targeting miRNAs on the progression and metastatic potential of OSCC. We analyzed public databases to explore which miRNAs target HIF-1α expression. In addition, the expression of proteins involved in the cell cycle, proliferation, and apoptosis in HSC-2 cells was analyzed after miRNA-126 mimic treatment. Furthermore, to investigate the effect of miRNA-126 on the proliferation and invasion ability of OSCC cells, 5-ethynyl-2′-deoxyuridine and Transwell assays were performed. The activities of MMP-2 and MMP-9 were evaluated via gelatin zymography. Our results showed that miRNA-126, which targets HIF-1α, enhances OSCC cell proliferation by regulating the cell cycle and reinforces the cell mobility of OSCC via HIF-1α expression. These findings suggest that miRNA-126 may be a novel marker for OSCC treatment and the development of new tools for patients with OSCC.


Oral squamous cell carcinoma , MicroRNAs , HIF-1α , Metastasis , Proliferation

MicroRNA-126은 HIF-1α의 발현을 통해 구강 편평상피세포암의 증식능과 이주능을 촉진한다

박 수연1), 오 지현2), 이 상신1), 최 종호1)
1)강릉원주대학교 치과대학 병리학교실
2)강릉원주대학교 치과대학 구강악안면외과학 교실

초록

    Ⅰ. INTRODUCTION

    Oral cancer largely includes tumors that occur in the lips, oral cavity, hard palate, tongue, buccal mucosa, and floor of the mouth.1,2) Data from the World Health Organization reported that oral cancer accounts for 377,713 (2.0%) new cancer cases involving the lip and oral cavity and 177,757 (1.8%) cases of cancer-related mortality worldwide.3) Oral squamous cell carcinoma (OSCC), generally referred to as oral cancer, has an estimated 7.9% crude incidence rate and 2.4% crude mortality rate per 1000,000 population in 2021 in Korea.4) The incidence of OSCC is lower than that of other cancers. In addition to advances in therapy, the lack of early diagnosis and advanced technology in OSCC contribute to its low incidence. However, the 5-year overall survival rate of patients with OSCC is as low as 50%.5) The dominant factor affecting this low 5-year survival rate is metastatic lymph nodes, which induces high recurrence and low survival rates.6,7,8)

    Hypoxia, a common characteristic of the malignant tumor microenvironment, is a key regulator of hypoxia-inducible factor-1α (HIF-1α) expression.9,10) Effect of HIF-1α, a transcription factor expressed in response to hypoxia, plays an important role in various biological processes. The oxygen- independent oncogenic characteristics of HIF-1α are associated with the regulation of metabolism, angiogenesis, cancer stemness, and the expression of several growth factors.11,12,13) In recent years, several studies have reported that miRNAs targeting HIF-1α regulate the cell cycle, cell proliferation, DNA damage repair, and mitochondrial function in human cancer cells.14,15) This evidence indicates that miRNAs play a significant role in the molecular mechanisms activated by hypoxia.16)

    MicroRNAs (miRNAs) are small non-coding RNAs that are functional regulators of human gene expression. miRNAs consist of small, single-stranded RNAs 18–25 nucleotides long17,18) and interact with the 3′ untranslated regions (3′ UTRs) during the post-transcriptional regulation of gene expression 19,20). miRNAs can control the targeting of multiple genes, and several miRNAs can simultaneously regulate the expression of one gene.21,22) miRNAs play critical roles in various biological signaling pathways related to survival, cell cycle, apoptosis, migration, invasion, and metastasis23,24,25,26). However, aberrantly expressed miRNAs have either tumor- suppressive or oncogenic roles17,25,27) in human cancers. For example, miRNA-20528), miRNA-14029), miRNA-26a30), and miRNA-42931) act as tumor suppressors in breast cancer, human osteosarcoma and colon cancer, ovarian cancer, and renal carcinoma, respectively. On the other hand, miRNA-130b in bladder cancer32), and miRNA-196, miRNA-92, and miRNA-4286 in lung cancer 33) act as oncogenes. Recently, these findings have proposed that miRNA targets could be useful biomarkers for the diagnosis and prognosis of various cancers33).

    MicroRNA-126 is located on chromosome 9q34.3 of the epidermal growth factor-like domain 7 (EGFL-7) gene34). It was reported as an endothelium-specific miRNA35) and has been studied in endothelial cells and keratinocytes36). Moreover, miRNA-126 is a component of developmental angiogenesis 35,37), cell proliferation, migration, and vascular formation.38) Although many studies have been conducted on miRNA-126, its hypoxia-inducible function at the cellular level in OSCC remains to be elucidated.

    Therefore, this study aimed to investigate whether miRNA-126 targeting HIF-1α is involved in OSCC development. We found that miRNA-126 functions as a regulator of cell growth and mobility in OSCC cell lines and controls the expression of tumor suppressor genes and oncogenes as oncogenic drivers. Thus, miRNA-126 could provide novel insights into biomarkers, pathological diagnosis, and treatment of OSCC.

    Ⅱ. MATERIALS AND METHODS

    1. OSCC cell lines and cell culture

    The human OSCC cell line HSC-2 (JCRB, Ibaraki, Osaka, Japan) was cultured in DMEM/F-12 (Gibco) medium with high glucose (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (P/S) in a humidified atmosphere of 5% CO2 at 37 ℃. To mimic hypoxic conditions, cells were cultured in 5% CO2 and 1.5% O2 balanced with N2 at 37 ℃. To overexpress miRNA-126, cells were cultured with 25 nM of miRNA-126 mimic (Bioneer, Daejeon, Korea) or miRNA negative control (Bioneer) using Lipofectamine 2000 (Invitrogen).

    2. RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

    Total RNA from OSCC cells treated with miRNA-126 was isolated using the TRIzol reagent (Invitrogen). After the isolation of total RNA, an Accupower® RocketScriptTM Cycle RT PreMix kit (Bioneer) was used for cDNA synthesis according to the manufacturer’s instructions. The thermocycling conditions for cDNA amplification were as follows: 50 °C for 60 min, 95 °C for 5 min, and hold at 4 °C. Next, qRT-PCR was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) to evaluate the expression of miRNA and mRNA. The PCR amplification conditions were: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s, with a final step at 95 °C for 15 s, 65 °C for 5 s, and 95 °C for 30 s. The miRNA amplification conditions were: 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s, with a final step at 95 °C for 1 min, 65 °C for 5 s, and 95 °C for 30 s. All gene expression levels were normalized to the expression of GAPDH or U6 and were calculated using the 2-ΔΔCT method, which were then reported as fold-changes. The following primer sequences were used: HIF-1α forward, 5′-AGC CGA GGA AGA ACT ATG A-3′; HIF-1α reverse, 5′-CAC ACT GAG GTT GGT TAC TG-3′; GAPDH forward, 5′-CAA AGT TGT CAT GGA TGA CC-3′; GAPDH reverse, 5′-CCA TGG AGA AGG CTG GGG-3′; has-miRNA-126: 5′-CAU UAU UAC UUU UGG UAC GCG-3′; and U6 reverse, 5′- AAA ATA TGG AAC GCT TCA CGA -3′. All experiments were performed at least three times.

    3. Western blotting

    Total protein from HSC-2 cells was harvested using a lysis buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Equal amounts of protein were loaded onto 8-15% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, transferred to nitrocellulose membranes (Bio-Rad Nobel Drive Hercules, CA, USA), and incubated overnight at room temperature. The next day, the membranes were blocked using a solution containing 5% bovine serum albumin (Sigma) diluted with 0.01% Tween-20 (PBS-T) for 30 min, then washed with 0.01% PBS-T twice at room temperature for 3 min each time. Next, the membranes were incubated with primary antibodies diluted in 0.01% PBS-T for 2 h and 30 min, washed with 0.01% PBS-T twice for 3 min each time, and then incubated with the corresponding secondary antibodies diluted with 0.01% PBS-T for 1 h at room temperature. The primary antibodies used were anti- HIF-1α (GeneTex; 1:1,000 dilution), anti-Cyclin D1 (Santa Cruz; 1:1,000 dilution), anti-Cyclin E (Santa Cruz; 1:1,000 dilution), anti-Cyclin A (Santa Cruz; 1:1,000 dilution), anti-Cyclin B1 (Santa Cruz; 1:1,000 dilution), anti-Caspase-3 (Santa Cruz; 1:1,000 dilution), anti-Caspase-7 (Santa Cruz; 1:1,000 dilution), anti-PCNA (Cell Signaling; 1:1,000 dilution) and anti-GAPDH (ABFrontier, Seoul, Republic of Korea; 1:3,000 dilution). The secondary antibodies used were anti-mouse IgG (Cell Signaling; 1:5,000 dilution) and anti-rabbit-IgG (Cell Signaling, 1:5,000 dilution). After washing the membranes with 0.01% PBS-T twice for 5 min each time, the proteins were visualized using a detectable solution (Millipore Corporation, St. Louis, MO, USA) and an imaging system (Vilber, Eberhardzell, Germany). The intensity of the protein bands was analyzed using ImageJ software. All experiments were performed at least thrice.

    4. 5-Ethynyl-2′-deoxyuridine (EdU) assay

    Cell proliferation was assessed using an EdU Staining Proliferation Kit (Fluor 488) (Abcam, Waltham, MA, USA) according to the manufacturer’s instructions. After the cells were plated on coverslips, miRNA-126 mimic or miRNA negative control was used to treat the cells. After 48 h of culture, half of the spent medium was removed, fresh culture medium containing 10 μM EdU labeling solution was added, and the cells were incubated for another 2 h at 37 ℃. The cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, the fixative solution was removed, and treated with 0.5% Triton® X-100 (Sigma) for 20 min at room temperature. Next, the cells were stained with 500 μL Click-iT reaction buffer for 1 h at room temperature. EdU-stained cells were mounted with 0.7 μL of 4’,6-diamidino-2-phenylindole (DAPI) and observed via fluorescence microscopy (Olympus IX73, Tokyo, Japan) with a 40× objective. All experiments were performed at least thrice.

    5. Migration assay using Transwell inserts

    The migration ability of OSCC cells was determined using a 24-well cell culture (Corning Inc., Corning, NY, USA). Next, 3 × 104 cells suspended in 0.3 mL Opti-MEM medium were cultured in the upper chamber of each insert, and 0.7 mL culture medium containing 5% FBS was added to the lower chamber. The cells were then treated with or without miRNAs using Lipofectamine RNAiMAX (Invitrogen) to induce miRNA-126 expression and incubated for 6 h. Afterward, the culture medium was changed, and the cells were incubated for another 24 h at 37 ℃ and 5% CO2. The next day, the spent medium was removed from the upper chamber, and 0.7 mL methanol was added and fixed for 15 min to fix the cells. The cells were then washed twice with DPBS and stained with Mayer’s hematoxylin staining reagent (Dako, Santa Clara, CA, USA) for 10 min at room temperature. After washing twice with DPBS, the staining reagent was removed using cotton swabs. The number of cells was counted in at least eight random images using an inverted light microscope (Olympus IX73, Tokyo, Japan) with a 20× objective. All experiments were performed at least thrice.

    6. Gelatin zymography

    The activities of MMP-2 and MMP-9 were detected in HSC-2 cells treated with or without miRNA-126 mimics via gelatin zymography. The supernatants of the Transwell migration assay were harvested and incubated with a zymogram sample buffer (0.125 M Tris-HCl [pH 6.8] containing 4% SDS, 0.01% bromophenol, and 20% glycerol) for 15 min at room temperature. The mixtures were then loaded onto a 12% SDS-PAGE gel containing 1% gelatin (Sigma-Aldrich). The gels were then washed twice for 15 min with 1× renaturation buffer (Bio-Rad) at room temperature and incubated 1× developing buffer (Bio-Rad) overnight at 37 ℃. The next day, gels were stained with a gel fixing solution containing 40% methanol, 10% acetic acid, 50% distilled water, and 0.5% Coomassie brilliant blue G-250 (Sigma) for 3 h at room temperature. To detect the activity of MMP-2 and MMP-9, the gels were washed with a fixative solution for 1 h at room temperature. The density of the gel bands was measured using ImageJ software. All experiments were performed at least thrice.

    7. Statistical analysis

    Statistical analysis was performed using SPSS 20.0 statistical software (SPSS, Inc., Chicago, IL, USA). All data are presented as the mean ± standard error of the mean. Statistical differences were analyzed using a two-tailed unpaired Student’s t-test. Statistical significance was set at p < 0.05. All experiments were performed at least thrice.

    Ⅲ. RESULTS

    1. miRNA-126 targets HIF-1α and upregulates the expression of HIF-1α in OSCC cells

    To investigate the target genes of miRNA-126, we analyzed three databases, TargetScan, miRDB, and PicTar. We then identified which genes in the representative database TargetScan had 3′ UTRs containing potential miR-126 binding sites with the target gene HIF-1α (Fig. 1a-b). Next, we confirmed the expression of miRNA-126 in HSC-2 cells under hypoxic conditions. The expression of miRNA-126 was significantly higher in HSC-2 cells under hypoxic conditions than under normoxic conditions (p < 0.05) (Fig. 1c). To investigate the effect of miRNA-126 on HIF-1α expression in OSCC cell lines, we confirmed the protein expression of HIF-1α. Our results showed that the expression of HIF-1α was significantly increased in HSC-2 cells treated with miRNA-126 mimics compared to those treated with the miRNA negative control (p < 0.05) (Fig. 1d).

    2. miRNA-126 regulates the cell cycle in OSCC cells

    To investigate the effect of miRNA-126 on the cell cycle, we confirmed the expression of cell cycle markers. Western blot analysis showed that the expressions of Cyclin D1, A, and B were significantly increased in OSCC cell lines treated with miRNA-126 compared to those treated with the miRNA negative control (p < 0.05). However, the expression of Cyclin E was significantly decreased in OSCC cell lines treated with miRNA-126 mimics compared to that in the miRNA negative control (p < 0.05) (Fig. 2a-b). Collectively, these results indicated that miRNA-126 affects the cell cycle of OSCC cell lines.

    3. miRNA-126 regulates the apoptosis and cell proliferation rates of OSCC cells

    To determine the effect of miRNA-126 on cell proliferation and apoptosis in OSCC cells, we confirmed the expression of markers related to apoptosis and cell proliferation. The results of western blotting showed that the expression of Caspase-3 and -7 was significantly decreased in OSCC cell lines treated with miRNA-126 mimics compared to those treated with the miRNA negative control (p < 0.05) (Fig. 3a-b). In contrast, the expression of proliferating cell nuclear antigen (PCNA) was significantly increased in OSCC cell lines treated with miRNA-126 mimics compared to that in the miRNA negative control (p < 0.05) (Fig. 3a-b). Quantifying protein expression indicated increased Caspase-3 and Caspase-7 and decreased PCNA expression in OSCC cells after treatment of miRNA-126 mimics (Fig. 3b). These results suggest that miRNA-126 suppresses apoptosis and promotes the proliferation of OSCC cell lines.

    4. miRNA-126 regulates cell proliferation OSCC cells

    To further evaluate the influence of miRNA-126 on the proliferation in OSCC cells, we performed an EdU incorporation assay, which does not require DNA denaturation to detect the incorporated nucleoside39). Our results showed that the number of EdU-positive cells was significantly increased in OSCC cell lines treated with miRNA-126 mimics compared to those treated with the miRNA negative control (p < 0.05) (Fig. 4a-b). Taken together, these data indicate that miRNA-126 treatment enhances the proliferation of OSCC cell lines.

    5. miRNA-126 regulates the migration ability of OSCC cells

    We performed a migration assay to explore the functional effects of miRNA-126 on the migration ability of OSCC cell lines. Our results showed that the number of OSCC cells that migrated from the upper chamber was significantly increased after treatment with miRNA-126 mimics than the miRNA negative control (p < 0.05) (Fig. 5a-b). Furthermore, gelatin zymography analysis was conducted to detect the activities of MMP-2 and -9, which were identified as white bands against a deep blue background40). The activities of MMP-2 and MMP-9 did not change regardless of miRNA-126 treatment (Fig. 5c-d). These results suggest that miRNA-126 promotes the migration of OSCC cells.

    Ⅳ. DISCUSSION

    OSCC is the most common malignancy worldwide, with invasive metastasis resulting in high mortality rates41). Thus, there is a need to identify biomarkers for the early prediction and diagnosis of OSCC. Although several studies have demonstrated the role of miRNAs in tumor growth and metastasis, functional studies in OSCC are still few. miR-126 is an endothelial-specific miRNA that regulates endothelial proliferation and migration, resulting in angiogenesis38,42). Furthermore, as a tumor suppressor, miR-126 expression has been reported in diverse cancer cells, including bladder cancer, prostate cancer, gastric cancer, and leukemia43,44,45). Recent findings have demonstrated that miR-126 produced by endothelial cells is delivered to vascular smooth muscle cells46), suggesting that physiological changes induced by miR-126 are not restricted to endothelial cells. Sasahira et al. showed that miR-126 expression is a negative regulator of vascular endothelial growth factor (VEGF)-A, which is a functional marker of endothelial cell activation and is associated with tumor angiogenesis, progression, and poor prognosis in OSCC47. However, the specific role of miR-126 in the proliferation and invasion of OSCC cells has not been extensively studied.

    HIF-1α is an upstream target of VEGF, a transcription factor that controls the endothelial cell response under hypoxia 48). HIF-1α and hypoxia are critical for angiogenesis and metastasis, which determines tumor development49). HSC-2 cells were derived from patients with OSCC with metastatic sites. These cells are appropriate for studying OSCC proliferation and invasion because of their rapid growth and invasion into the surrounding tissues50). Previous reports have demonstrated that these cells are characterized by their higher expression of HIF-1α and more invasive properties than other OSCC cell lines51). Therefore, in the present study, we aimed to elucidate the functional role of miR-126 and its target genes in the proliferation and invasion abilities of HSC-2 cells in vitro.

    Recently, many studies have highlighted the possibility of a link between HIF-1a and miR-126; however, the relationship between HIF-1α and miR-126 remains controversial. Alique et al. showed that the inhibition of miR-126 downregulates HIF-1α expression, suppressing the migration, proliferation, and tube formation of endothelial cells; in contrast, the inhibition of HIF-1α did not affect the expression of miR-12652). Song et al. showed that HIF-1α expression triggers the expression of miR-126 in endothelial cells under normoxic and hypoxic conditions via the PI3K/AKT/eNOS and MAPK signaling pathways53). In the present study, we found that miR-126 expression was activated in HSC-2 cells under hypoxic conditions compared with normoxic conditions, while miR-126 expression was unchanged after the inhibition of HIF-1α using small interfering RNA (data not shown). These findings suggest that miR-126 regulates hypoxia- induced HIF-1α expression.

    Cyclins bind to specific catalytic partners, such as cyclin- dependent kinase (CDK), to control the balance between cell cycle arrest, apoptosis, and proliferation, which are unregulated in cancers54). In particular, HIF-1α is involved in tumor cell proliferation and cell cycle progression mediated by p21 via the modulation of CDK expression54). Mizokami et al. showed that the negative correlation between HIF-1α and p21 is involved in the apoptotic index and progression of gastric cancer55). Together, these results suggest that cyclin and p21 may modulate cyclins and the cell cycle to regulate OSCC proliferation. We found that miR-126-induced HIF-1α expression regulated the proliferation of HSC-2 cells by promoting p21, Cyclin A, B, and D and inhibiting Cyclin E expression. Mazumder et al. suggested that Cyclin E is often deregulated in cancer cells, which is irregularly over- or underexpressed in different types of cancers56).

    In conclusion, the results of the present study provide evidence for the hypothesis that miR-126 regulates the proliferation and invasion ability of OSCC cells and that the interaction between miR-126 and HIF-1α regulates the cell cycle, leading to OSCC progression and metastasis.

    ACKNOWLEDGMENTS

    This research was supported by the Co-operative Research Program (CR1902) of the Gangneung-Wonju University Dental Hospital.

    Figure

    KAOMP-47-1-13_F1.gif

    Identification of the involvement of miRNA-126 with HIF-1α in OSCC. (a) Venn diagram displaying the potential targets of HIF-1α and miRNAs as predicted by the Target Scan, miRDB, and PicTar databases. (b) Schematic drawing showing which part of the miRNA-126 sequence interacts with HIF-1α, showing a binding sequence (red) and a binding site (yellow). (c) The expression of miRNA-126 in HSC-2 cells under hypoxic conditions compared to normoxic conditions. (d) Protein expression of HIF-1α, PTTG1, and p21 in HSC-2 cells treated with miR-126 as detected using western blotting. GAPDH was used as the loading control. *indicates a significant difference in expression between HSC-2 cells treated with the NC and miR-126 (p < 0.05). All experiments were performed at least in triplicate. NC: negative control; miRNA-126: microRNA-126; HIF-1α: hypoxia-inducible factor-1 alpha; PTTG1: pituitary tumor-transforming gene 1; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

    KAOMP-47-1-13_F2.gif

    The expression of miRNA-126 is related to the cell cycle in HSC-2 cells. (a) Western blotting results of the relative expression of proteins related to the cell cycle, including Cyclin D1, Cyclin E, Cyclin A, and Cyclin B1 in HSC-2 cells treated with miR-126. GAPDH was used as an internal control. (b) Graphs showing relative intensity were calculated for each pixel image using ImageJ software. All experiments were performed at least three times. *indicates a significant difference in expression between HSC-2 cells treated with the NC and miR-126 (p < 0.05). All experiments were performed at least in triplicate. NC: negative control; miRNA-126: microRNA-126; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

    KAOMP-47-1-13_F3.gif

    The expression of miRNA-126 affects apoptosis and proliferation in HSC-2 cells. (a) Western blotting results of the relative expression of proteins related to apoptosis and proliferation, including Caspase-3, Caspase-7, and PCNA in HSC-2 cell lines treated by miRNA-126. GAPDH was used as an internal control. (b) Graphs showing relative intensity were calculated for each pixel image using ImageJ software. All experiments were performed at least three times. *indicates a significant difference in expression between HSC-2 cells treated with the NC and miR-126 (p < 0.05). All experiments were performed at least in triplicate. NC: negative control; miRNA-126: microRNA-126; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; PCNA: Proliferating cell nuclear antigen.

    KAOMP-47-1-13_F4.gif

    Viability of HSC-2 cells after treatment with miRNA-126. (a) Representative images comparing the cell viability of HSC-2 cells treated with NC and miR-126 using the EdU assay (Scale bar: 200 μm; original magnification: 10×). (b) Graph indicating the number of proliferating cells. Six regions were randomly selected to count cells (the mean ± SD was calculated). *indicates a significant difference between HSC-2 cells treated with the NC and miR-126 (p < 0.05). All experiments were performed at least in triplicate. NC: negative control; miRNA-126: microRNA-126.

    KAOMP-47-1-13_F5.gif

    The effect of treatment with miRNA-126 on mobility in HSC-2 cells. (a) Migration ability of HSC-2 cells after treatment with the NC and miR-126 (Scale bar: 200 μm; original magnification: 10×). (b) Graph representing the number of migrating cells after treatment with miR-126. Five regions were randomly selected to count cells (the mean ± SD was calculated). (c) The activities of MMP-2 and MMP-9 in HSC-2 cells treated with miR-126 as detected via zymography. (d) The fold intensity of MMP-2 and MMP-9 activity was calculated for pixel images using ImageJ software. *indicates a significant difference between HSC-2 cells treated with the NC and miR-126 (p < 0.05). All experiments were performed at least in triplicate. NC: negative control; miRNA-126: microRNA-126; MMP: matrix metalloproteinase.

    Table

    Reference

    1. Soluk-Tekkesin M, Wright JM: The World Health Organization Classification of Odontogenic Lesions: A Summary of the Changes of the 2022 (5th) Edition. Turk Patoloji Derg 2022;38:168-184.
    2. Bewley AF, Farwell DG: Oral leukoplakia and oral cavity squamous cell carcinoma. Clin Dermatol 2017; 35:461-467.
    3. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A: Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209-249.
    4. Jung KW, Won YJ, Hong S, Kong HJ, Im JS, Seo HG: Prediction of Cancer Incidence and Mortality in Korea, 2021. Cancer Res Treat 2021;53:316-322.
    5. Geum DH, Roh YC, Yoon SY, Kim HG, Lee JH, Song JM: The impact factors on 5-year survival rate in patients operated with oral cancer. J Korean Assoc Oral Maxillofac Surg 2013;39:207-216.
    6. Tajmirriahi N, Razavi SM, Shirani S, Homayooni S, Gasemzadeh G: Evaluation of metastasis and 5-year survival in oral squamous cell carcinoma patients in Isfahan (2001-2015). Dent Res J (Isfahan) 2019;16: 117-121.
    7. Wang B, Zhang S, Yue K, Wang XD: The recurrence and survival of oral squamous cell carcinoma: a report of 275 cases. Chin J Cancer 2013;32:614-618.
    8. Sim YC, Hwang JH, Ahn KM: Overall and disease-specific survival outcomes following primary surgery for oral squamous cell carcinoma: analysis of consecutive 67 patients. J Korean Assoc Oral Maxillofac Surg 2019;45:83-90.
    9. Mazure NM, Chen EY, Laderoute KR, Giaccia AJ: Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element. Blood 1997;90:3322-3331.
    10. Petrova V: Annicchiarico-Petruzzelli M, Melino G, Amelio I: The hypoxic tumour microenvironment. Oncogenesis 2018;7:10.
    11. Masoud GN, Li W: HIF-1α pathway: role, regulation and intervention for cancer therapy. Acta Pharm Sin B 2015;5:378-389.
    12. Ziello JE, Jovin IS, Huang Y: Hypoxia-Inducible Factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia. Yale J Biol Med 2007;80:51-60.
    13. Jun JC, Rathore A, Younas H, Gilkes D, Polotsky VY: Hypoxia-Inducible Factors and Cancer. Curr Sleep Med Rep 2017;3:1-10.
    14. Dang K, Myers KA: The role of hypoxia-induced miR-210 in cancer progression. Int J Mol Sci 2015; 16:6353-6372.
    15. Jing X, Yang F, Shao C, Wei K, Xie M, Shen H: Role of hypoxia in cancer therapy by regulating the tumor microenvironment. Mol Cancer 2019;18:157.
    16. He C, Wang L, Zhang J, Xu H: Hypoxia-inducible microRNA-224 promotes the cell growth, migration and invasion by directly targeting RASSF8 in gastric cancer. Mol Cancer 2017;16:35.
    17. Peng Y, Croce CM: The role of MicroRNAs in human cancer. Signal Transduct Target Ther 2016;1:15004.
    18. Ying SY, Chang DC, Lin SL: The microRNA (miRNA): overview of the RNA genes that modulate gene function. Mol Biotechnol 2008;38:257-268.
    19. Matoulkova E, Michalova E, Vojtesek B, Hrstka R: The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells. RNA Biol 2012;9:563-576.
    20. Lai EC, Tomancak P, Williams RW, Rubin GM: Computational identification of Drosophila microRNA genes. Genome Biol 2003;4:R42.
    21. Shivdasani RA. MicroRNAs: regulators of gene expression and cell differentiation. Blood 2006;108: 3646-3653.
    22. Hua Z, Lv Q, Ye W, Wong CK, Cai G, Gu D: MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia. PLoS One 2006;1: e116.
    23. Jones KB, Salah Z, Del Mare S, Galasso M, Gaudio E, Nuovo GJ: miRNA signatures associate with pathogenesis and progression of osteosarcoma. Cancer Res 2012;72: 1865-1877.
    24. Ma L, Teruya-Feldstein J, Weinberg RA: Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 2007;449:682-688.
    25. Zhang B, Pan X, Cobb GP, Anderson TA: microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302:1-12.
    26. Hwang HW, Mendell JT: MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 2006;94:776-780.
    27. Shenouda SK, Alahari SK: MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer Metastasis Rev 2009;28:369-378.
    28. Wu H, Mo YY: Targeting miR-205 in breast cancer. Expert Opin Ther Targets 2009;13:1439-1448.
    29. Song B, Wang Y, Xi Y, Kudo K, Bruheim S, Botchkina GI: Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells. Oncogene 2009;28:4065-4074.
    30. Gao S, Bian T, Su M, Liu Y, Zhang Y: miR‑26a inhibits ovarian cancer cell proliferation, migration and invasion by targeting TCF12. Oncol Rep 2020;43: 368-374.
    31. Su Z, Jiang G, Chen J, Liu X, Zhao H, Fang Z: MicroRNA-429 inhibits cancer cell proliferation and migration by targeting AKT1 in renal cell carcinoma. Mol Clin Oncol 2020;12:75-80.
    32. Yu X, Wang ZL, Han CL, Wang MW, Jin Y, Jin XB: LncRNA CASC15 functions as an oncogene by sponging miR-130b-3p in bladder cancer. Eur Rev Med Pharmacol Sci 2019;23: 9814-9820.
    33. Lin Y, Zhang L, Ding X, Chen C, Meng M, Ke Y: Relationship between the microRNAs and PI3K/AKT/mTOR axis: Focus on non-small cell lung cancer. Pathol Res Pract 2022;239:154093.
    34. Hansen TF, Nielsen BS, Jakobsen A: Sørensen FB. Intra-tumoural vessel area estimated by expression of epidermal growth factor-like domain 7 and microRNA-126 in primary tumours and metastases of patients with colorectal cancer: a descriptive study. J Transl Med 2015;13:10.
    35. Wang S, Aurora AB, Johnson BA, Qi X, McAnally J, Hill JA: The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Dev Cell 2008;15:261-271.
    36. Chang L, Liang J, Xia X, Chen X: miRNA-126 enhances viability, colony formation, and migration of keratinocytes HaCaT cells by regulating PI3 K/AKT signaling pathway. Cell Biol Int 2019;43:182-191.
    37. Alhasan L: MiR-126 Modulates Angiogenesis in Breast Cancer by Targeting VEGF-A -mRNA. Asian Pac J Cancer Prev 2019;20:193-197.
    38. Schober A, Nazari-Jahantigh M, Wei Y, Bidzhekov K, Gremse F, Grommes J: MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat Med 2014;20:368-376.
    39. Guo J, Li D, Zhang Y: Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: using 5-ethynyl-2' -deoxyuridine. Cardiovasc Diabetol 2012;11: 150.
    40. Snoek-van Beurden PA, Von den Hoff JW: Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 2005;38:73-83.
    41. Kalogirou EM, Tosios KI, Christopoulos PF.:The Role of Macrophages in Oral Squamous Cell Carcinoma. Front Oncol 2021;11:611115.
    42. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281-297.
    43. Feng R, Chen X, Yu Y, Su L, Yu B, Li J: miR-126 functions as a tumour suppressor in human gastric cancer. Cancer Lett 2010;298:50-63.
    44. Li Z, Lu J, Sun M, Mi S, Zhang H, Luo RT: Distinct microRNA expression profiles in acute myeloid leukemia with common translocations. Proc Natl Acad Sci U S A 2008;105:15535-15540.
    45. Musiyenko A, Bitko V, Barik S: Ectopic expression of miR-126*, an intronic product of the vascular endothelial EGF-like 7 gene, regulates prostein translation and invasiveness of prostate cancer LNCaP cells. J Mol Med(Berl) 2008;86:313-322.
    46. Zhou J, Li YS, Nguyen P, Wang KC, Weiss A, Kuo YC: Regulation of vascular smooth muscle cell turnover by endothelial cell-secreted microRNA-126: role of shear stress. Circ Res 2013;113:40-51.
    47. Sasahira T, Kurihara M, Bhawal UK, Ueda N, Shimomoto T, Yamamoto K: Downregulation of miR-126 induces angiogenesis and lymphangiogenesis by activation of VEGF-A in oral cancer. Br J Cancer 2012;107:700-706.
    48. Lv X, Li J, Zhang C, Hu T, Li S, He S: The role of hypoxia-inducible factors in tumor angiogenesis and cell metabolism. Genes Dis 2017;4:19-24.
    49. Chaudary N, Hill RP: Hypoxia and metastasis in breast cancer. Breast Dis 2006;26:55-64.
    50. Momose F, Araida T, Shioda S, Sasaki S: Variant sublines with different metastatic potentials selected in nude mice from human oral squamous cell carcinomas. J Oral Pathol Med 1989; 18:391-395.
    51. Kim S, Park S, Oh JH, Lee SS, Lee Y, Choi J: MicroRNA-18a regulates the metastatic properties of oral squamous cell carcinoma cells via HIF-1alpha expression. BMC Oral Health 2022;22:378.
    52. Alique M, Bodega G, Giannarelli C, Carracedo J, Ramirez R: MicroRNA-126 regulates Hypoxia- Inducible Factor-1alpha which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence. Sci Rep 2019;9:7381.
    53. Song W, Liang Q, Cai M, Tian Z.:HIF-1alpha-induced up-regulation of microRNA-126 contributes to the effectiveness of exercise training on myocardial angiogenesis in myocardial infarction rats. J Cell Mol Med 2020;24:12970-12979.
    54. Ivanovska I, Ball AS, Diaz RL, Magnus JF, Kibukawa M, Schelter JM: MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression. Mol Cell Biol 2008;28:2167-2174.
    55. Mizokami K, Kakeji Y, Maehara Y: Relationship of hypoxia- inducible factor 1alpha and p21WAF1/ CIP1 expression to cell apoptosis and clinical outcome in patients with gastric cancer. World J Surg Oncol 2006;4:94.
    56. Mazumder S, DuPree EL, Almasan A: A dual role of cyclin E in cell proliferation and apoptosis may provide a target for cancer therapy. Curr Cancer Drug Targets 2004;4:65-75.
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