- Email: [email protected]
MiR-650 regulates the proliferation, migration and invasion of human oral cancer by targeting growth factor independent 1 (Gfi1)
Accepted Manuscript MiR-650 regulates the proliferation, migration and invasion of human oral cancer by targeting Growth factor independent 1 (Gfi1) Sun Ningning, Sun Libo, Wu Chuanbin, Sun Haijiang, Zhou Qing PII:
S0300-9084(18)30272-4
DOI:
10.1016/j.biochi.2018.10.001
Reference:
BIOCHI 5516
To appear in:
Biochimie
Received Date: 22 June 2018 Accepted Date: 4 October 2018
Please cite this article as: S. Ningning, S. Libo, W. Chuanbin, S. Haijiang, Z. Qing, MiR-650 regulates the proliferation, migration and invasion of human oral cancer by targeting Growth factor independent 1 (Gfi1), Biochimie (2018), doi: https://doi.org/10.1016/j.biochi.2018.10.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Abstract Oral cancer being one of the lethal cancers is generally detected at advanced stages and causes significant mortality world over. The unavailability of the reliable biomarkers and therapeutic targets/agents forms a bottleneck in the treatment of oral cancer. MicroRNAs are considered of immense therapeutic potential for the treatment of cancer. Consistently, in this
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study the role and therapeutic potential of miR-650 was explored in oral cancer. The analysis of miR-650 expression by qRT-PCR revealed significant (p < 0.05) upregulation of miR-650 in oral cancer cell lines. Cell cycle analysis by flow cytometery revealed that suppression of miR-650 significantly (p < 0.05) inhibits the proliferation of the SCC-25 cells by prompting
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Sub-G1 cell cycle arrest. Further, miR-650 suppression also inhibited the migration and invasion of the SCC-25 oral cancer cells as revealed by transwell assays. TargetScan analysis showed that miR-650 targets Growth factor independent 1 (Gfi1). Moreover, the results of
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western blot analysis showed that miR-650 suppression inhibits the expression of Gfi1. Interestingly, suppression of Gfi1 exhibited similar effects on cell proliferation, migration and invasion of the oral cancer cells as that of miR-650 suppression. Nonetheless, miR-650 promoted the proliferation, migration and invasion of the SCC-25 cells by upregulating the expression of Gfi1. Moreover, overexpression of miR-650 could not rescue the effects of
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Gfi1 silencing on SCC-25 oral cancer cells. Conversely, overexpression of Gfi1 could rescue the effects of miR-650 inhibition on SCC-25 cell proliferation, migration and invasion. Additionally, miR-650 suppression could also inhibit the xenografted tumor growth in vivo by inhibiting the expression of Gfi1. Taken together, miR-650 may prove to be an important
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therapeutic target for the management of oral cancers.
ACCEPTED MANUSCRIPT 1
MiR-650 regulates the proliferation, migration and invasion of human oral cancer by
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targeting Growth factor independent 1 (Gfi1).
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Sun Ningning1, Sun Libo2, Wu Chuanbin1, Sun Haijiang1 and Zhou Qing1*
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University,117 Nanjing North Street, Shenyang110002, Liaoning, China
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Stomatological Hospital, Luzhou Sichuan, 646000, China
Department of Oral and Maxillofacial Surgery,
School
of
China
Medical
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Department of Oral and Maxillofacial Surgery, Southwest Medical University Affiliated
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*Corresponding author
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Zhou Qing, Department of Oral and Maxillofacial Surgery,
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School of Stomatology, China Medical University.
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117 Nanjing North Street, Shenyang110002, Liaoning, China
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Tel/Fax: 0086-024-31927721
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Email: [email protected]
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Stomatology,
Abstract
ACCEPTED MANUSCRIPT Oral cancer being one of the lethal cancers is generally detected at advanced stages and causes
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significant mortality world over. The unavailability of the reliable biomarkers and therapeutic
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targets/agents forms a bottleneck in the treatment of oral cancer. MicroRNAs are considered of
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immense therapeutic potential for the treatment of cancer. Consistently, in this study the role and
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therapeutic potential of miR-650 was explored in oral cancer. The analysis of miR-650
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expression by qRT-PCR revealed significant (p < 0.05) upregulation of miR-650 in oral cancer
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cell lines. Cell cycle analysis by flow cytometery revealed that suppression of miR-650
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significantly (p < 0.05) inhibits the proliferation of the SCC-25 cells by prompting Sub-G1 cell
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cycle arrest. Further, miR-650 suppression also inhibited the migration and invasion of the SCC-
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25 oral cancer cells as revealed by transwell assays. TargetScan analysis showed that miR-650
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targets Growth factor independent 1 (Gfi1). Moreover, the results of western blot analysis
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showed that miR-650 suppression inhibits the expression of Gfi1. Interestingly, suppression of
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Gfi1 exhibited similar effects on cell proliferation, migration and invasion of the oral cancer cells
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as that of miR-650 suppression. Nonetheless, miR-650 promoted the proliferation, migration and
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invasion of the SCC-25 cells by upregulating the expression of Gfi1. Moreover, overexpression
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of miR-650 could not rescue the effects of Gfi1 silencing on SCC-25 oral cancer cells.
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Conversely, overexpression of Gfi1 could rescue the effects of miR-650 inhibition on SCC-25
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cell proliferation, migration and invasion. Additionally, miR-650 suppression could also inhibit
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the xenografted tumor growth in vivo by inhibiting the expression of Gfi1. Taken together, miR-
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650 may prove to be an important therapeutic target for the management of oral cancers.
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Keywords: Oral cancer; MicroRNA; Growth factor independent 1; cell cycle arrest; cell
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1. Introduction
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Oral cancer is one of the frequently detected cancers of oral cavity and pharynx and is
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responsible for significant mortality and morbidity across the globe. Around 90% of oral cancers
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are oral squamous cell carcinoma (OSCC) which is common type of neck and head cancers [1].
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OSCC shows poor prognosis and frequently metastasises to the lymph nodes. It has been
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reported that major cause of OSCC related mortality is the invasion of the cancer to the distant
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body parts [2]. The chemotherapeutic agents used for the management of OSCC are generally
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inefficient and exhibit severe adverse effects on the overall health of the patients [3]. Moreover,
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the availability of reliable biomarker and therapeutic targets form a major bottleneck in the
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treatment of oral cancers [4]. Over the years, microRNAs (miRs) have gained tremendous
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ACCEPTED MANUSCRIPT attention as therapeutic targets for the management of several types of cancers [5]. The miRs are
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20 nucleotides non-coding RNA molecules which have been found to play vital functions in
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almost all biological pathways [6]. As such, miRs affect a wide array of cancer related processes
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which include, but are not limited to, proliferation, metastasis and cell cycle [7]. There are
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enormous concrete evidences related to the implications of miRs in the development of cancer
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[8]. It has been reported that miRs are often dysregulated in tumors due to a number of factors
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[9]. MiR-650 has been reported to regulate a number of cancer related processes. For instance, it
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controls the tumorigenesis and progression of gastric and hepatocelluar carcinoma [10-11].
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Moreover, miR-650 exhibits oncogenic activity in case of prostate cancer via inhibition of
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cellular stress response 1 (CSR1) gene expression [12]. However, the role and therapeutic
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potential of miR-650 has not been explored in oral cancers. Against this backdrop, the present
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study was designed to investigate the role and therapeutic potential of miR-650 in oral cancer
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and here in miR-650 was found to regulation oral cancer proliferation by targeting Gfi1. Gfi1
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has been reported to be highly expressed in several types of cancers and has been shown to
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regulate the proliferation of many types of cancers [13, 14]. Moreover, it has been shown to play
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an important role in cell cycle [15]. Taken together, we propose that miR-650 may prove to an
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essential therapeutic target for the management of OSCC and as such warrants further
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evaluation.
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2. Materials and Methods
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2.1.Cell lines and culture conditions
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The normal (hTERT-OME) and oral cancer cell lines (SCC-15, SCC-4, SCC-9, SCC-25, CAL-
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27, FaDu, 019) were procured from American Type Culture Collection. All of these cell lines
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were maintained in Dulbeccoʼs modified Eagleʼs medium containing fetal 10% bovine serum,
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antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin), and 2 mM glutamine. The
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Cells were cultured in CO2 incubator (Thermo Scientific) at 37°C with 98% humidity and 5%
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CO2.
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2.2. Quantitative real-time PCR
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The total RNA was extracted from the oral cancer cell lines and the normal cell line with the
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assistance of RNeasy kits (Qiagen, Inc., Valencia, CA, USA). To reverse transcribe the cDNA,
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the Omniscript RT (Qiagen, Inc.) was employed using 1 µg of the extracted RNA. The cDNA
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was then used as a template for RT-qPCR analysis with the assistance of the Taq PCR Master
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Mix kit (Qiagen, Inc.) according to the manufacturer’s protocol. The reaction mixture consisted
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of 20 µl containing 1.5 mM MgCl2, 2.5 units Taq DNA Polymerase, 200 µM dNTP, 0.2 µM of
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each primer and 0.5 µg DNA. The cycling conditions were as follows: 95˚C for 20 sec, followed
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ACCEPTED MANUSCRIPT by 40 cycles of 95˚C for 15 sec, and 58˚C for 1 min. Actin was used as an internal control and
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the relative quantification (2-∆∆Cq) method was used to evaluate the quantitative variation
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between the samples
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2.3.Transfections
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As the oral cancer SCC-25 cells reached 80% confluence, they were transfected with inhibitor-
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NC (Negative control), miR-650 inhibitor and mimics (10 pmol, Shanghai GenePharma),
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siRNA-Gfi1 and pcDNA-Gfi1 (2 µg, Taijin Saier Biotechnology) with the help of Lipofectamine
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2000 (Invitrogen) as per manufacturer’s guidelines.
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2.4.Proliferation and colony formation assays
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The cell proliferation of the oral cancer cells was assessed by WST-1 colorimetric assay. Briefly,
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the SCC-25 oral cancer cells were seeded in ninety-six well plates at the density of 2 × 105
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cells/well. The cells were then incubated with WST-1 at 37○C for 4 h. The absorbance at 450 nm
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was then taken by a microplate reader to determine the viability of oral cancer cells. For
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assessment of the colony formation potential of the SCC-25 cells, the cells were collected at
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exponential phase of growth and the cells were then counted using a hemocytometer. The plating
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of the transfected cells was carried out at 200 cells /well. The plates were then kept at 37 ○C for 6
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days. After incubating the cells for about six days, they were subjected to washing with
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Phosphate buffered saline (PBS) and fixation with methanol. The SCC-25 cells were then
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stained with crystal violet followed by microscopy.
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2.5.Detection of apoptosis and cell cycle analysis
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To examine the apoptotic cell death, the nuclear morphology of the SCC-25 oral cancer cells was
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assessed by fluorescence microscopy after subjecting the cells to DAPI and annexin V/PI
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staining as described previously [16]. In brief, SCC-25 cells (0.6 × 106) were cultured in six well
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plates separately. Following transfection, the cells were incubated for 24 hours at 37 °C in CO2
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incubator with 5% CO2 for 24 h. The cells were the collected in flow tubes and centrifuged at
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1600 rpm for 5 min at 4 °C. Pellets were washed with PBS buffer and fixed in methanol for 30
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min at 4 °C and further suspended into 500 µl PBS. Cells were stained with 4’-6-diamidino-2-
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phenylindole (DAPI) 1µg/ml for 10 min and centrifuged. The cell pellet was resuspended in 50µl
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of mounting fluid (PBS: glycerol, 1:1) and 10 µl of this cell suspension was spread on clean glass
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slide and covered with coverslip. The slides were then observed for nuclear morphological
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alterations and apoptotic bodies under an inverted fluorescence microscope
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2.6.Cell cycle analysis
ACCEPTED MANUSCRIPT The cell cycle analysis was performed by flow cytometery as described in literature [17]. In
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brief, SCC-25 cells were cultured in 6-well cluster plates. Following transfection, the cells were
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cultured for 24 h, washed and stained with Annexin-V-FITC antibody and PI as per the
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instructions given by the manufacturer. The cells were scanned for fluorescence intensity in FL-1
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(FITC) and FL-2 (PI) channels. The fraction of cell population in different quadrants was
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analysed using quadrant statistics.
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2.7.Cell migration and invasion assay
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The cell migration and invasion of the oral cancer cells was evaluated by transwell assays as
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described previously [18]. In brief, after transfection for 24 h, SCC-25 cells were cultured in
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serum-free medium for 12 h. The cell concentration was adjusted to 4–5 × l05/ml. A transwell
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chamber with 8-µm pores (Corning, Corning, NY, USA) was used for the 24-well plates. RPMI-
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1640 medium (500 µl) containing 10% FBS was placed in the lower layer, and 200 µl of the cell
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suspension was placed in the upper chamber. Then, the cells were incubated for 10 h. The cells
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on the lower surface of the chamber were fixed with glacial acetic acid for 15–30 min and
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stained with crystal violet for 30 min, and 10 fields were selected randomly to count. The cells
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were covered with Matrigel (BD, Franklin, NJ, USA), and the same method was used to perform
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cell invasion assays.
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2.8.Target identification
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To identify the target, miR-145 was subjected to the online software TargetScan version 7.2
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(http://www.targetscan.org).
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2.9.In vivo study
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The mice xenografts male BALB/c nude mice (36 mice, 4-week-old) were maintained in the
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animal facility following the National Institutes of Health standards for the care and use of
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laboratory animals. The study was approved and supervised by The Ethics Committee of China
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Medical University. The mice were randomly divided into two groups (18 mice per group) and
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SCC-25 cells (approximately 1.0 × 107 cells/mouse) were stably transfected with either miR-650
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(Group 1) or NC (Group II), were subcutaneously injected into the back of mice. Tumor volume
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was monitored every week. At the end of the study, the mice were sacrificed, and the tumor
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weigh and volume was measured. Tumor tissues were subjected to protein isolation for further
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studies.
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2.10.
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The oral cancer SCC-25 were lysed with the help of the ice-cold hypotonic buffer. After
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estimating the protein concentrations in each of the cell extracts, the samples containing the
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proteins were loaded and separated on SDS–PAGE. This was followed by transference to a
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Western blot analysis
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nitrocellulose membrane and incubation with the primary antibody (1:1000) for 24 h at 4 ○C.
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Thereafter the membrane was incubated with HRP-conjugated secondary antibody (1:1000) for
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at 24○C for about 1 h. The visualization of the proteins was carried out by enhanced chemi-
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luminescence reagent.
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2.11.
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Statistical analysis was performed using student’s t test (for comparisons between two groups)
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and one way analysis of variance followed by Tukeys’s post-hoc test (For comparison between
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more than two groups) using SPSS software package v9.05 (SPSS, Inc., Chicago, IL, USA).
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Data are presented as the mean ± standard deviation,and P < 0.05 was considered to indicate a
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statistically significant difference.
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3. Results
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3.1.MiR-650 is significantly upregulated in OSCC
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The expression of miR-650 was examined in seven different OSCC cell lines and one normal
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cell line by qRT-PCR. The results showed that the expression of miR-650 is significantly
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upregulated in all the OSCC cell lines in comparison to the non-cancerous cells. The expression
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of miR-650 was found to up to 6.5 folds relative to the expression in the normal cells (Figure
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1A). The highest expression of miR-650 was observed in case of SCC-25 cell line and as such
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this cell line was selected for further experimentation.
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3.2.Suppression of miR-650 inhibits the proliferation SCC-25 cells by Sub-G1 arrest
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To decipher the role of miR-650 in oral cancer cells, its expression was suppressed in SCC-25
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cells (Figure 1B). The results revealed that the suppression of miR-650 in SCC-25 cells caused
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significant and time dependent decrease in the proliferation rate as well colony formation
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potential of the SCC-25 oral cancer cells (Figure 1C and D). Next, to investigate the mechanism
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underlying the inhibition of cell proliferation, we performed the DAPI and annexin V/PI staining
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(Figure 1E). However, the results revealed that miR-650 suppression did not exerted anti-
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proliferative via induction of apoptotic cell death. Hence, we carried out the cell cycle analysis of
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miR-650 inhibitor and inhibitor-NC transfected SCC-25 cells and it was observed that miR-650
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cells inhibited the proliferation of SCC-25 cells by prompting Sub-G1 cell cycle arrest cells
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(Figure 1F). As compared to the 0.95% sub-G1 cell populations in inhibitor-NC transfected cells,
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the percentage of the sub-G1 cells was found to be 41.16 % in miR-650 inhibitor transfected
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cells. To sum up, these results indicate that miR-650 suppression inhibits the proliferation of the
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SCC-25 cells by triggering Sub-G1 cell cycle arrest.
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3.3.Suppression of miR-650 inhibits the migration and invasion of SCC-25 cells.
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Statistical analysis
ACCEPTED MANUSCRIPT The effect of miR-650 suppression was also examined on the migration and invasion of the SCC-
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25 cells by determining the cell migration and invasion by transwell assays. The results revealed
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that miR-650 suppression lead to the inhibition of the migration and invasion of the SCC-25 cells
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(Figure 2A). Further, we examined the effects of miR-650 suppression on the cell migration and
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invasion of the oral non-cancerous cells and it was observed that miR-650 suppression had no
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significant effects on the migration and invasion of the non-cancerous cells (Figure 2B). Taken
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together, we conclude that miR-650 specifically regulates the migration an invasion of the cancer
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cells.
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3.4.MiR-650 exerts its effects by targeting Gfi1
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To identify the potential target, miR-650 was screened by TargetScan online software and Gfi1
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was identified as the potential target of miR-650 (Figure 3A). This was further confirmed by
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immunoblotting wherein it was found that miR-650 suppression caused significant
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downregulation in the expression of Gfi1 (Figure 3B). Moreover, Gfi1 suppression had similar
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effects on the proliferation, migration and invasion of the SCC-25 cells as that of miR-650
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suppression (Figure 3C-E). These results clearly indicate the miR-650 may target Gfi1 in SCC-
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25 cells.
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3.5.Overexpression of miR-650 promotes proliferation, migration and invasion of SCC-25 cells
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Next, the effects of miR-650 overexpression were also investigated on the Gfi1 expression and
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proliferation of the SCC-25 cells. For this miR-650 mimics were transfected into the SCC-25
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cells and overexpression was confirmed qRT-PCR (Figure 4A). It was found that miR-650
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overexpression also caused significant upregulation in the expression of Gfi1 (Figure 4B).
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Additionally, miR-650 also promoted the cell proliferation, migration and invasion of the SCC-
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25 cells (Figure 4C-E).
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3.6.Overexpression of Gfi1 rescues the effects of miR-650 suppression on proliferation,
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migration and invasion of SCC-25 cells.
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Next, to further confirm that the miR-650 exerted effects on cell proliferation, migration and
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invasion is via modulation of Gfi1 expression, miR-650 mimics and Si-Gfi1 were co-
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transformed into the SCC-25 cells. The results revealed that overexpression of miR-650 in Si-
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Gfi1 transfected SCC-25 cells did not rescued the effects of Gfi1 silencing on the proliferation,
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migration and invasion (Figure 5A-C). On the other hand, overexpression of Gfi1 in SCC-25
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cells transfected with miR-650 inhibitor, could rescue the effects of miR-650 suppression on cell
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proliferation, migration an invasion (Figure 6A-C). Taken together these results clearly indicate
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the miR-650 regulates the proliferation, migration and invasion of oral cancer SCC-25 cells at
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least in part by modulating the expression of Gfi1.
ACCEPTED MANUSCRIPT 3.7.Suppression of MiR-650 inhibits the tumor growth in vivo
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Next, the effect of miR-650 suppression was also examined on the oral tumor growth in vivo.
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The NC or miR-650 inhibitor transfected SSC-25 cells were subcutaneously injected into male
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BALB/c-A nude mice. The results revealed that miR-650 suppression significantly inhibited the
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tumor weight and volume in vivo (Figure 7A-C). Further, miR-650 suppression in oral cancer
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caused significant inhibition of Gfi1 expression (Figure 7D).
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4. Discussion
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The cancers of the oral cavity and pharynx are currently ranked as the sixth most prevalent
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types of cancers. Despite the recent advancements in cancer research, the five-year survival rate
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for oral cancer is still 62% which is very poor in comparison to other lethal cancer such as
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breast cancer (89%) and prostate cancer (99%).
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identification of efficient therapeutic targets/agents and post-therapeutic monitoring is urgently
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required [19-21]. Studies have reported that the expression of around 30% of the genes in human
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are controlled by miRs and as such they are involved in a wide array of cellular, developmental
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and physiological processes [22]. Moreover, the expression of miRs has been reported to be
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frequently dysregulated in cancers which include but are not limited to OSCC and miRNA
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signatures are considered imperative from diagnosis to treatment [22]. Among others, miR-650
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has been implicated in the development and progression of several types of cancers which
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include but are not limited to gastric, hepatocelluar and prostate cancers [10-12]. However, the
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role of miR-650 has not been investigated in case of oral cancer. In this study, it was observed
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that the expression of miR-650 is significantly upregulated in oral cancer cell lines. Suppression
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of miR-650 in SCC-25 oral cancer cells caused the inhibition of SCC-25 cell proliferation,
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migration an invasion. These findings are also supported by previous investigations [23-25]. For
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instance, the expression of miR-650 is upregulated in colorectal cancer cells and regulates their
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proliferation [23]. Further, miR-650 has also been reported to regulate the proliferation and
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invasion of rheumatoid arthritis synovial fibroblasts [24]. In yet another study, miR-650 has been
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found to promote the proliferation, migration and invasion of non-small lung cancer cells [25].
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The miRs exerts their effects by targeting other genes and in this study Growth factor
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independent 1 (Gfi1) was found to be the potential target of miR-650 in oral cancer cells. It was
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found that miR-650 inhibition causes suppression of the Gfi1 expression. Previously, miR-650
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has also been reported to modulate the expression of Gfi1 in myeloid leukemia cells further
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authenticating our findings [26]. Gfi1 is a zinc transcription factor (located on the chromosome
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lp22) which has been showed to be play a diversity of biological functions. Gfi1 has also been
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reported to play a role in the development and metastasis of several types of cancers. For
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Therefore, markers for early detection,
ACCEPTED MANUSCRIPT example, upregulation of Gfi1 has been found to be associated with the development of
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medulloblastoma [27]. In this study we observed that suppression of Gfi1 could inhibit the
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proliferation, migration and invasion of oral cancer cells. These effects on proliferation,
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migration an invasion were similar to those of miR-650 suppression in SCC-25 cells. The fact
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that miR-650 suppression inhibits proliferation of the oral cancer cells by triggering sub-G1 cell
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cycle arrest could be explained by the fact that Gfi1 has been reported to play vital role in cell
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cycle progression [28]. Overexpression of miR-650 could promote the proliferation, migration
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and invasion of the SCC-25 cells by enhancing the expression of Gfi1. Further, it was also
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observed that although miR-650 could not rescue the effects of Gfi1 silencing in SCC-25 cells.
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However, overexpression of Gfi1 could rescue the effects of miR-650 suppression on SCC-25
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cell migration and invasion, suggesting that Gfi1 is essential for the regulation of proliferation,
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migration and invasion of oral cancer cells both in vitro and in vivo.
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It is well known that miRs exert their effects by suppressing the expression of the target genes.
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However in the present study we observed that miR-650 inhibition suppressed the expression of
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Gfi1 and overexpression of miR-650 upregulated the expression of Gfi1, which is opposite to the
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expected. This may be because miR-650 may also target some repressor of the Gfi1, or induces a
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protection from the degradation. Moreover, there are reports wherein miRs have been reported to
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induce the expression of the target genes. For example, miR-373 has been reported to induce the
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expression of the target gene E-cadherin [29]. More studies directed at miR-650 will enable
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better understanding of miR-650 and its target Gfi1 in oral cancer. Nonetheless, miR-650 may
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prove to be beneficial for the management of oral cancers.
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Conflict of interest
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The authors declare that there are no conflicts of interests.
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20. Brinkman BM, Wong DT. Disease mechanism and biomarkers of oral squamous cell carcinoma. Current opinion in oncology. 2006 May 1;18(3):228-33.
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Figure 1: Expression analysis and effect of miR-650 inhibition on proliferation of oral
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cancer cells. (A) Expression of miR-650 in oral cancer and normal cell line as determined by
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qRT-PCR, (B) Expression of miR-650 in SCC-25 oral cancer cells transfected with NC or miR-
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650 mimics (C) Cell viability of SCC-25 cells transfected with NC or miR-650 mimics, (D)
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Colony formation of SCC-25 cells transfected with NC and miR-650 mimics, (E) Detection of
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apoptosis in NC and miR-650 mimics transfected SCC-25 cells by DAPI and annexin V/PI
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staining and (F) Cell cycle analysis of NC and miR-650 mimics transfected SCC-25 cells . The
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values are mean of three biological replicates and expressed as mean ± SD. (figure 1A, *p < 0.05
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for normal cell line (hTRET-OME) Vs oral cancer cell lines; figure 1B to 1F *p < 0.05 for NC
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Vs miR-650 inhibitor).
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Figure 2: miR-650 inhibition suppresses the migration and invasion of SCC-25 cells (A)
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Effect of miR-650 overexpression on (A) SCC-25 oral cancer cell migration and invasion (B)
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non-cancerous hTERT-OME cell migration and invasion. The values are mean of three
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biological replicates and expressed as mean ± SD (*p < 0.05 for NC Vs miR-650 inhibitor).
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Figure 3: miR-650 exerts its effects by targeting Gfi1 in SCC-25 cells. (A) Identification of
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Gfi1 as potential target of miR-650 (B) Expression of Gfi1 in NC and miR-650 mimics
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transfected SCC-25 cells, (C) Effect of Gfi1 suppression on cell proliferation and (D) Colony
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formation (E) Effect of Gfi1 suppression on migration and invasion of SCC-25 cells. The values
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are mean of three biological replicates and expressed as mean ± SD. (*p < 0.05 for NC Vs Si-
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Gfi1).
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Figure 4: Overexpression of miR-650 promotes the proliferation, migration and invasion of
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SCC-25 cells. (A) Expression of miR-650 in NC and miR-650 transfected cells (B) Expression
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of Gfi1 in NC and miR-650 mimics transfected cells (C) Effect of miR-650 overexpression on
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ACCEPTED MANUSCRIPT (C) proliferation (D) cell viability (E) migration and invasion of the SCC-25 cells. The values are
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mean of three biological replicates and expressed as mean ± SD. (*p < 0.05 for NC Vs miR-650
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mimics).
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Figure 5: miR-650 overexpression does not rescue the effects of Gfi1 silencing on
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proliferation, migration and invasion of SCC-25 cells. Effect of miR-650 mimics on (A)
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proliferation (B) colon formation and (C) migration and invasion of Si-NC or Si-Gfi1 transfected
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SCC-25 cells. The values are mean of three biological replicates and expressed as mean ± SD.
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(*p < 0.05 for NC Vs Si-Gfi1 or Si-Gfi1 + miR-650-mimics).
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Figure 6: Gfi1 overexpression rescues the effects of miR-650 inhibition on the proliferation
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migration and invasion of SCC-25 cells. Effect of Gfi1overexpression (pcDNA-Gfi1) on (A)
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proliferation (B) colony formation (C) migration and invasion of NC or miR-650 inhibitor
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transfected SCC-25 cells. The values are mean of three biological replicates and expressed as
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mean ± SD. (*p < 0.05 for miR-650 inhibitor Vs miR-650-inhibitor + pcDNA-Gfi1).
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Figure 7: miR-650 inhibition suppresses the xenografted tumor growth. In vivo effects of
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miR-650 suppression on tumor growth (A) images of the NC and miR-650 inhibitor tumors (B)
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Effect of NC and miR-650 inhibitor transfection on xenografted tumor volume (C) Effect of NC
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and miR-650 inhibitor transfection on xenografted tumor weight (D) expression of Gfi1 in NC
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and miR-650 inhibitor tumors. The values are mean of three biological replicates and expressed
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as mean ± SD. (*p < 0.05 for NC Vs miR-650 inhibitor).
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MiR-650 is significantly upregulated in oral cancer cells Inhibition of miR-650 suppresses the proliferation, migration and invasion of the oral cancer cells by modulating the expression of Gfi1 Gfi1 overexpression rescues the inhibitory effects of miR-650 on the proliferation, migration and invasion of the oral cancer cells Overexpression of miR-650 promotes the proliferation, migration and invasion of the oral cancer cells Inhibition of miR-650 suppresses the tumor growth in xenografted mice
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DOI:
10.1016/j.biochi.2018.10.001
Reference:
BIOCHI 5516
To appear in:
Biochimie
Received Date: 22 June 2018 Accepted Date: 4 October 2018
Please cite this article as: S. Ningning, S. Libo, W. Chuanbin, S. Haijiang, Z. Qing, MiR-650 regulates the proliferation, migration and invasion of human oral cancer by targeting Growth factor independent 1 (Gfi1), Biochimie (2018), doi: https://doi.org/10.1016/j.biochi.2018.10.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Abstract Oral cancer being one of the lethal cancers is generally detected at advanced stages and causes significant mortality world over. The unavailability of the reliable biomarkers and therapeutic targets/agents forms a bottleneck in the treatment of oral cancer. MicroRNAs are considered of immense therapeutic potential for the treatment of cancer. Consistently, in this
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study the role and therapeutic potential of miR-650 was explored in oral cancer. The analysis of miR-650 expression by qRT-PCR revealed significant (p < 0.05) upregulation of miR-650 in oral cancer cell lines. Cell cycle analysis by flow cytometery revealed that suppression of miR-650 significantly (p < 0.05) inhibits the proliferation of the SCC-25 cells by prompting
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Sub-G1 cell cycle arrest. Further, miR-650 suppression also inhibited the migration and invasion of the SCC-25 oral cancer cells as revealed by transwell assays. TargetScan analysis showed that miR-650 targets Growth factor independent 1 (Gfi1). Moreover, the results of
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western blot analysis showed that miR-650 suppression inhibits the expression of Gfi1. Interestingly, suppression of Gfi1 exhibited similar effects on cell proliferation, migration and invasion of the oral cancer cells as that of miR-650 suppression. Nonetheless, miR-650 promoted the proliferation, migration and invasion of the SCC-25 cells by upregulating the expression of Gfi1. Moreover, overexpression of miR-650 could not rescue the effects of
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Gfi1 silencing on SCC-25 oral cancer cells. Conversely, overexpression of Gfi1 could rescue the effects of miR-650 inhibition on SCC-25 cell proliferation, migration and invasion. Additionally, miR-650 suppression could also inhibit the xenografted tumor growth in vivo by inhibiting the expression of Gfi1. Taken together, miR-650 may prove to be an important
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therapeutic target for the management of oral cancers.
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MiR-650 regulates the proliferation, migration and invasion of human oral cancer by
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targeting Growth factor independent 1 (Gfi1).
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Sun Ningning1, Sun Libo2, Wu Chuanbin1, Sun Haijiang1 and Zhou Qing1*
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1
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University,117 Nanjing North Street, Shenyang110002, Liaoning, China
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2
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Stomatological Hospital, Luzhou Sichuan, 646000, China
Department of Oral and Maxillofacial Surgery,
School
of
China
Medical
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Department of Oral and Maxillofacial Surgery, Southwest Medical University Affiliated
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*Corresponding author
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Zhou Qing, Department of Oral and Maxillofacial Surgery,
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School of Stomatology, China Medical University.
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117 Nanjing North Street, Shenyang110002, Liaoning, China
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Tel/Fax: 0086-024-31927721
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Email: [email protected]
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Stomatology,
Abstract
ACCEPTED MANUSCRIPT Oral cancer being one of the lethal cancers is generally detected at advanced stages and causes
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significant mortality world over. The unavailability of the reliable biomarkers and therapeutic
37
targets/agents forms a bottleneck in the treatment of oral cancer. MicroRNAs are considered of
38
immense therapeutic potential for the treatment of cancer. Consistently, in this study the role and
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therapeutic potential of miR-650 was explored in oral cancer. The analysis of miR-650
40
expression by qRT-PCR revealed significant (p < 0.05) upregulation of miR-650 in oral cancer
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cell lines. Cell cycle analysis by flow cytometery revealed that suppression of miR-650
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significantly (p < 0.05) inhibits the proliferation of the SCC-25 cells by prompting Sub-G1 cell
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cycle arrest. Further, miR-650 suppression also inhibited the migration and invasion of the SCC-
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25 oral cancer cells as revealed by transwell assays. TargetScan analysis showed that miR-650
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targets Growth factor independent 1 (Gfi1). Moreover, the results of western blot analysis
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showed that miR-650 suppression inhibits the expression of Gfi1. Interestingly, suppression of
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Gfi1 exhibited similar effects on cell proliferation, migration and invasion of the oral cancer cells
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as that of miR-650 suppression. Nonetheless, miR-650 promoted the proliferation, migration and
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invasion of the SCC-25 cells by upregulating the expression of Gfi1. Moreover, overexpression
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of miR-650 could not rescue the effects of Gfi1 silencing on SCC-25 oral cancer cells.
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Conversely, overexpression of Gfi1 could rescue the effects of miR-650 inhibition on SCC-25
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cell proliferation, migration and invasion. Additionally, miR-650 suppression could also inhibit
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the xenografted tumor growth in vivo by inhibiting the expression of Gfi1. Taken together, miR-
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650 may prove to be an important therapeutic target for the management of oral cancers.
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Keywords: Oral cancer; MicroRNA; Growth factor independent 1; cell cycle arrest; cell
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1. Introduction
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Oral cancer is one of the frequently detected cancers of oral cavity and pharynx and is
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responsible for significant mortality and morbidity across the globe. Around 90% of oral cancers
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are oral squamous cell carcinoma (OSCC) which is common type of neck and head cancers [1].
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OSCC shows poor prognosis and frequently metastasises to the lymph nodes. It has been
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reported that major cause of OSCC related mortality is the invasion of the cancer to the distant
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body parts [2]. The chemotherapeutic agents used for the management of OSCC are generally
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inefficient and exhibit severe adverse effects on the overall health of the patients [3]. Moreover,
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the availability of reliable biomarker and therapeutic targets form a major bottleneck in the
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treatment of oral cancers [4]. Over the years, microRNAs (miRs) have gained tremendous
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ACCEPTED MANUSCRIPT attention as therapeutic targets for the management of several types of cancers [5]. The miRs are
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20 nucleotides non-coding RNA molecules which have been found to play vital functions in
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almost all biological pathways [6]. As such, miRs affect a wide array of cancer related processes
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which include, but are not limited to, proliferation, metastasis and cell cycle [7]. There are
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enormous concrete evidences related to the implications of miRs in the development of cancer
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[8]. It has been reported that miRs are often dysregulated in tumors due to a number of factors
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[9]. MiR-650 has been reported to regulate a number of cancer related processes. For instance, it
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controls the tumorigenesis and progression of gastric and hepatocelluar carcinoma [10-11].
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Moreover, miR-650 exhibits oncogenic activity in case of prostate cancer via inhibition of
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cellular stress response 1 (CSR1) gene expression [12]. However, the role and therapeutic
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potential of miR-650 has not been explored in oral cancers. Against this backdrop, the present
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study was designed to investigate the role and therapeutic potential of miR-650 in oral cancer
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and here in miR-650 was found to regulation oral cancer proliferation by targeting Gfi1. Gfi1
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has been reported to be highly expressed in several types of cancers and has been shown to
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regulate the proliferation of many types of cancers [13, 14]. Moreover, it has been shown to play
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an important role in cell cycle [15]. Taken together, we propose that miR-650 may prove to an
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essential therapeutic target for the management of OSCC and as such warrants further
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evaluation.
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2. Materials and Methods
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2.1.Cell lines and culture conditions
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The normal (hTERT-OME) and oral cancer cell lines (SCC-15, SCC-4, SCC-9, SCC-25, CAL-
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27, FaDu, 019) were procured from American Type Culture Collection. All of these cell lines
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were maintained in Dulbeccoʼs modified Eagleʼs medium containing fetal 10% bovine serum,
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antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin), and 2 mM glutamine. The
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Cells were cultured in CO2 incubator (Thermo Scientific) at 37°C with 98% humidity and 5%
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CO2.
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2.2. Quantitative real-time PCR
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The total RNA was extracted from the oral cancer cell lines and the normal cell line with the
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assistance of RNeasy kits (Qiagen, Inc., Valencia, CA, USA). To reverse transcribe the cDNA,
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the Omniscript RT (Qiagen, Inc.) was employed using 1 µg of the extracted RNA. The cDNA
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was then used as a template for RT-qPCR analysis with the assistance of the Taq PCR Master
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Mix kit (Qiagen, Inc.) according to the manufacturer’s protocol. The reaction mixture consisted
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of 20 µl containing 1.5 mM MgCl2, 2.5 units Taq DNA Polymerase, 200 µM dNTP, 0.2 µM of
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each primer and 0.5 µg DNA. The cycling conditions were as follows: 95˚C for 20 sec, followed
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the relative quantification (2-∆∆Cq) method was used to evaluate the quantitative variation
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between the samples
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2.3.Transfections
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As the oral cancer SCC-25 cells reached 80% confluence, they were transfected with inhibitor-
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NC (Negative control), miR-650 inhibitor and mimics (10 pmol, Shanghai GenePharma),
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siRNA-Gfi1 and pcDNA-Gfi1 (2 µg, Taijin Saier Biotechnology) with the help of Lipofectamine
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2000 (Invitrogen) as per manufacturer’s guidelines.
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2.4.Proliferation and colony formation assays
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The cell proliferation of the oral cancer cells was assessed by WST-1 colorimetric assay. Briefly,
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the SCC-25 oral cancer cells were seeded in ninety-six well plates at the density of 2 × 105
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cells/well. The cells were then incubated with WST-1 at 37○C for 4 h. The absorbance at 450 nm
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was then taken by a microplate reader to determine the viability of oral cancer cells. For
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assessment of the colony formation potential of the SCC-25 cells, the cells were collected at
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exponential phase of growth and the cells were then counted using a hemocytometer. The plating
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of the transfected cells was carried out at 200 cells /well. The plates were then kept at 37 ○C for 6
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days. After incubating the cells for about six days, they were subjected to washing with
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Phosphate buffered saline (PBS) and fixation with methanol. The SCC-25 cells were then
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stained with crystal violet followed by microscopy.
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2.5.Detection of apoptosis and cell cycle analysis
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To examine the apoptotic cell death, the nuclear morphology of the SCC-25 oral cancer cells was
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assessed by fluorescence microscopy after subjecting the cells to DAPI and annexin V/PI
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staining as described previously [16]. In brief, SCC-25 cells (0.6 × 106) were cultured in six well
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plates separately. Following transfection, the cells were incubated for 24 hours at 37 °C in CO2
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incubator with 5% CO2 for 24 h. The cells were the collected in flow tubes and centrifuged at
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1600 rpm for 5 min at 4 °C. Pellets were washed with PBS buffer and fixed in methanol for 30
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min at 4 °C and further suspended into 500 µl PBS. Cells were stained with 4’-6-diamidino-2-
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phenylindole (DAPI) 1µg/ml for 10 min and centrifuged. The cell pellet was resuspended in 50µl
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of mounting fluid (PBS: glycerol, 1:1) and 10 µl of this cell suspension was spread on clean glass
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slide and covered with coverslip. The slides were then observed for nuclear morphological
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alterations and apoptotic bodies under an inverted fluorescence microscope
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2.6.Cell cycle analysis
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brief, SCC-25 cells were cultured in 6-well cluster plates. Following transfection, the cells were
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cultured for 24 h, washed and stained with Annexin-V-FITC antibody and PI as per the
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instructions given by the manufacturer. The cells were scanned for fluorescence intensity in FL-1
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(FITC) and FL-2 (PI) channels. The fraction of cell population in different quadrants was
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analysed using quadrant statistics.
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2.7.Cell migration and invasion assay
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The cell migration and invasion of the oral cancer cells was evaluated by transwell assays as
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described previously [18]. In brief, after transfection for 24 h, SCC-25 cells were cultured in
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serum-free medium for 12 h. The cell concentration was adjusted to 4–5 × l05/ml. A transwell
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chamber with 8-µm pores (Corning, Corning, NY, USA) was used for the 24-well plates. RPMI-
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1640 medium (500 µl) containing 10% FBS was placed in the lower layer, and 200 µl of the cell
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suspension was placed in the upper chamber. Then, the cells were incubated for 10 h. The cells
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on the lower surface of the chamber were fixed with glacial acetic acid for 15–30 min and
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stained with crystal violet for 30 min, and 10 fields were selected randomly to count. The cells
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were covered with Matrigel (BD, Franklin, NJ, USA), and the same method was used to perform
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cell invasion assays.
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2.8.Target identification
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To identify the target, miR-145 was subjected to the online software TargetScan version 7.2
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(http://www.targetscan.org).
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2.9.In vivo study
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The mice xenografts male BALB/c nude mice (36 mice, 4-week-old) were maintained in the
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animal facility following the National Institutes of Health standards for the care and use of
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laboratory animals. The study was approved and supervised by The Ethics Committee of China
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Medical University. The mice were randomly divided into two groups (18 mice per group) and
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SCC-25 cells (approximately 1.0 × 107 cells/mouse) were stably transfected with either miR-650
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(Group 1) or NC (Group II), were subcutaneously injected into the back of mice. Tumor volume
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was monitored every week. At the end of the study, the mice were sacrificed, and the tumor
164
weigh and volume was measured. Tumor tissues were subjected to protein isolation for further
165
studies.
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2.10.
167
The oral cancer SCC-25 were lysed with the help of the ice-cold hypotonic buffer. After
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estimating the protein concentrations in each of the cell extracts, the samples containing the
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proteins were loaded and separated on SDS–PAGE. This was followed by transference to a
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Western blot analysis
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nitrocellulose membrane and incubation with the primary antibody (1:1000) for 24 h at 4 ○C.
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Thereafter the membrane was incubated with HRP-conjugated secondary antibody (1:1000) for
172
at 24○C for about 1 h. The visualization of the proteins was carried out by enhanced chemi-
173
luminescence reagent.
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2.11.
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Statistical analysis was performed using student’s t test (for comparisons between two groups)
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and one way analysis of variance followed by Tukeys’s post-hoc test (For comparison between
177
more than two groups) using SPSS software package v9.05 (SPSS, Inc., Chicago, IL, USA).
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Data are presented as the mean ± standard deviation,and P < 0.05 was considered to indicate a
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statistically significant difference.
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3. Results
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3.1.MiR-650 is significantly upregulated in OSCC
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The expression of miR-650 was examined in seven different OSCC cell lines and one normal
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cell line by qRT-PCR. The results showed that the expression of miR-650 is significantly
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upregulated in all the OSCC cell lines in comparison to the non-cancerous cells. The expression
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of miR-650 was found to up to 6.5 folds relative to the expression in the normal cells (Figure
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1A). The highest expression of miR-650 was observed in case of SCC-25 cell line and as such
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this cell line was selected for further experimentation.
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3.2.Suppression of miR-650 inhibits the proliferation SCC-25 cells by Sub-G1 arrest
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To decipher the role of miR-650 in oral cancer cells, its expression was suppressed in SCC-25
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cells (Figure 1B). The results revealed that the suppression of miR-650 in SCC-25 cells caused
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significant and time dependent decrease in the proliferation rate as well colony formation
192
potential of the SCC-25 oral cancer cells (Figure 1C and D). Next, to investigate the mechanism
193
underlying the inhibition of cell proliferation, we performed the DAPI and annexin V/PI staining
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(Figure 1E). However, the results revealed that miR-650 suppression did not exerted anti-
195
proliferative via induction of apoptotic cell death. Hence, we carried out the cell cycle analysis of
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miR-650 inhibitor and inhibitor-NC transfected SCC-25 cells and it was observed that miR-650
197
cells inhibited the proliferation of SCC-25 cells by prompting Sub-G1 cell cycle arrest cells
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(Figure 1F). As compared to the 0.95% sub-G1 cell populations in inhibitor-NC transfected cells,
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the percentage of the sub-G1 cells was found to be 41.16 % in miR-650 inhibitor transfected
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cells. To sum up, these results indicate that miR-650 suppression inhibits the proliferation of the
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SCC-25 cells by triggering Sub-G1 cell cycle arrest.
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3.3.Suppression of miR-650 inhibits the migration and invasion of SCC-25 cells.
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Statistical analysis
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25 cells by determining the cell migration and invasion by transwell assays. The results revealed
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that miR-650 suppression lead to the inhibition of the migration and invasion of the SCC-25 cells
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(Figure 2A). Further, we examined the effects of miR-650 suppression on the cell migration and
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invasion of the oral non-cancerous cells and it was observed that miR-650 suppression had no
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significant effects on the migration and invasion of the non-cancerous cells (Figure 2B). Taken
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together, we conclude that miR-650 specifically regulates the migration an invasion of the cancer
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cells.
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3.4.MiR-650 exerts its effects by targeting Gfi1
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To identify the potential target, miR-650 was screened by TargetScan online software and Gfi1
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was identified as the potential target of miR-650 (Figure 3A). This was further confirmed by
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immunoblotting wherein it was found that miR-650 suppression caused significant
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downregulation in the expression of Gfi1 (Figure 3B). Moreover, Gfi1 suppression had similar
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effects on the proliferation, migration and invasion of the SCC-25 cells as that of miR-650
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suppression (Figure 3C-E). These results clearly indicate the miR-650 may target Gfi1 in SCC-
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25 cells.
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3.5.Overexpression of miR-650 promotes proliferation, migration and invasion of SCC-25 cells
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Next, the effects of miR-650 overexpression were also investigated on the Gfi1 expression and
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proliferation of the SCC-25 cells. For this miR-650 mimics were transfected into the SCC-25
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cells and overexpression was confirmed qRT-PCR (Figure 4A). It was found that miR-650
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overexpression also caused significant upregulation in the expression of Gfi1 (Figure 4B).
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Additionally, miR-650 also promoted the cell proliferation, migration and invasion of the SCC-
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25 cells (Figure 4C-E).
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3.6.Overexpression of Gfi1 rescues the effects of miR-650 suppression on proliferation,
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Next, to further confirm that the miR-650 exerted effects on cell proliferation, migration and
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invasion is via modulation of Gfi1 expression, miR-650 mimics and Si-Gfi1 were co-
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transformed into the SCC-25 cells. The results revealed that overexpression of miR-650 in Si-
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Gfi1 transfected SCC-25 cells did not rescued the effects of Gfi1 silencing on the proliferation,
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migration and invasion (Figure 5A-C). On the other hand, overexpression of Gfi1 in SCC-25
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cells transfected with miR-650 inhibitor, could rescue the effects of miR-650 suppression on cell
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proliferation, migration an invasion (Figure 6A-C). Taken together these results clearly indicate
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the miR-650 regulates the proliferation, migration and invasion of oral cancer SCC-25 cells at
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least in part by modulating the expression of Gfi1.
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Next, the effect of miR-650 suppression was also examined on the oral tumor growth in vivo.
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The NC or miR-650 inhibitor transfected SSC-25 cells were subcutaneously injected into male
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BALB/c-A nude mice. The results revealed that miR-650 suppression significantly inhibited the
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tumor weight and volume in vivo (Figure 7A-C). Further, miR-650 suppression in oral cancer
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caused significant inhibition of Gfi1 expression (Figure 7D).
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4. Discussion
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The cancers of the oral cavity and pharynx are currently ranked as the sixth most prevalent
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types of cancers. Despite the recent advancements in cancer research, the five-year survival rate
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for oral cancer is still 62% which is very poor in comparison to other lethal cancer such as
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breast cancer (89%) and prostate cancer (99%).
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identification of efficient therapeutic targets/agents and post-therapeutic monitoring is urgently
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required [19-21]. Studies have reported that the expression of around 30% of the genes in human
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are controlled by miRs and as such they are involved in a wide array of cellular, developmental
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and physiological processes [22]. Moreover, the expression of miRs has been reported to be
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frequently dysregulated in cancers which include but are not limited to OSCC and miRNA
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signatures are considered imperative from diagnosis to treatment [22]. Among others, miR-650
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has been implicated in the development and progression of several types of cancers which
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include but are not limited to gastric, hepatocelluar and prostate cancers [10-12]. However, the
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role of miR-650 has not been investigated in case of oral cancer. In this study, it was observed
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that the expression of miR-650 is significantly upregulated in oral cancer cell lines. Suppression
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of miR-650 in SCC-25 oral cancer cells caused the inhibition of SCC-25 cell proliferation,
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migration an invasion. These findings are also supported by previous investigations [23-25]. For
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instance, the expression of miR-650 is upregulated in colorectal cancer cells and regulates their
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proliferation [23]. Further, miR-650 has also been reported to regulate the proliferation and
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invasion of rheumatoid arthritis synovial fibroblasts [24]. In yet another study, miR-650 has been
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found to promote the proliferation, migration and invasion of non-small lung cancer cells [25].
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The miRs exerts their effects by targeting other genes and in this study Growth factor
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independent 1 (Gfi1) was found to be the potential target of miR-650 in oral cancer cells. It was
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found that miR-650 inhibition causes suppression of the Gfi1 expression. Previously, miR-650
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has also been reported to modulate the expression of Gfi1 in myeloid leukemia cells further
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authenticating our findings [26]. Gfi1 is a zinc transcription factor (located on the chromosome
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lp22) which has been showed to be play a diversity of biological functions. Gfi1 has also been
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reported to play a role in the development and metastasis of several types of cancers. For
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ACCEPTED MANUSCRIPT example, upregulation of Gfi1 has been found to be associated with the development of
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medulloblastoma [27]. In this study we observed that suppression of Gfi1 could inhibit the
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proliferation, migration and invasion of oral cancer cells. These effects on proliferation,
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migration an invasion were similar to those of miR-650 suppression in SCC-25 cells. The fact
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that miR-650 suppression inhibits proliferation of the oral cancer cells by triggering sub-G1 cell
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cycle arrest could be explained by the fact that Gfi1 has been reported to play vital role in cell
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cycle progression [28]. Overexpression of miR-650 could promote the proliferation, migration
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and invasion of the SCC-25 cells by enhancing the expression of Gfi1. Further, it was also
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observed that although miR-650 could not rescue the effects of Gfi1 silencing in SCC-25 cells.
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However, overexpression of Gfi1 could rescue the effects of miR-650 suppression on SCC-25
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cell migration and invasion, suggesting that Gfi1 is essential for the regulation of proliferation,
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migration and invasion of oral cancer cells both in vitro and in vivo.
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It is well known that miRs exert their effects by suppressing the expression of the target genes.
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However in the present study we observed that miR-650 inhibition suppressed the expression of
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Gfi1 and overexpression of miR-650 upregulated the expression of Gfi1, which is opposite to the
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expected. This may be because miR-650 may also target some repressor of the Gfi1, or induces a
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protection from the degradation. Moreover, there are reports wherein miRs have been reported to
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induce the expression of the target genes. For example, miR-373 has been reported to induce the
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expression of the target gene E-cadherin [29]. More studies directed at miR-650 will enable
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better understanding of miR-650 and its target Gfi1 in oral cancer. Nonetheless, miR-650 may
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prove to be beneficial for the management of oral cancers.
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Conflict of interest
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The authors declare that there are no conflicts of interests.
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Figure 1: Expression analysis and effect of miR-650 inhibition on proliferation of oral
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cancer cells. (A) Expression of miR-650 in oral cancer and normal cell line as determined by
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qRT-PCR, (B) Expression of miR-650 in SCC-25 oral cancer cells transfected with NC or miR-
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650 mimics (C) Cell viability of SCC-25 cells transfected with NC or miR-650 mimics, (D)
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Colony formation of SCC-25 cells transfected with NC and miR-650 mimics, (E) Detection of
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apoptosis in NC and miR-650 mimics transfected SCC-25 cells by DAPI and annexin V/PI
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staining and (F) Cell cycle analysis of NC and miR-650 mimics transfected SCC-25 cells . The
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values are mean of three biological replicates and expressed as mean ± SD. (figure 1A, *p < 0.05
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for normal cell line (hTRET-OME) Vs oral cancer cell lines; figure 1B to 1F *p < 0.05 for NC
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Vs miR-650 inhibitor).
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Figure 2: miR-650 inhibition suppresses the migration and invasion of SCC-25 cells (A)
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Effect of miR-650 overexpression on (A) SCC-25 oral cancer cell migration and invasion (B)
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non-cancerous hTERT-OME cell migration and invasion. The values are mean of three
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biological replicates and expressed as mean ± SD (*p < 0.05 for NC Vs miR-650 inhibitor).
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Figure 3: miR-650 exerts its effects by targeting Gfi1 in SCC-25 cells. (A) Identification of
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Gfi1 as potential target of miR-650 (B) Expression of Gfi1 in NC and miR-650 mimics
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transfected SCC-25 cells, (C) Effect of Gfi1 suppression on cell proliferation and (D) Colony
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formation (E) Effect of Gfi1 suppression on migration and invasion of SCC-25 cells. The values
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are mean of three biological replicates and expressed as mean ± SD. (*p < 0.05 for NC Vs Si-
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Gfi1).
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Figure 4: Overexpression of miR-650 promotes the proliferation, migration and invasion of
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SCC-25 cells. (A) Expression of miR-650 in NC and miR-650 transfected cells (B) Expression
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of Gfi1 in NC and miR-650 mimics transfected cells (C) Effect of miR-650 overexpression on
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mean of three biological replicates and expressed as mean ± SD. (*p < 0.05 for NC Vs miR-650
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mimics).
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Figure 5: miR-650 overexpression does not rescue the effects of Gfi1 silencing on
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proliferation, migration and invasion of SCC-25 cells. Effect of miR-650 mimics on (A)
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proliferation (B) colon formation and (C) migration and invasion of Si-NC or Si-Gfi1 transfected
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SCC-25 cells. The values are mean of three biological replicates and expressed as mean ± SD.
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(*p < 0.05 for NC Vs Si-Gfi1 or Si-Gfi1 + miR-650-mimics).
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Figure 6: Gfi1 overexpression rescues the effects of miR-650 inhibition on the proliferation
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migration and invasion of SCC-25 cells. Effect of Gfi1overexpression (pcDNA-Gfi1) on (A)
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proliferation (B) colony formation (C) migration and invasion of NC or miR-650 inhibitor
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transfected SCC-25 cells. The values are mean of three biological replicates and expressed as
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mean ± SD. (*p < 0.05 for miR-650 inhibitor Vs miR-650-inhibitor + pcDNA-Gfi1).
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Figure 7: miR-650 inhibition suppresses the xenografted tumor growth. In vivo effects of
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miR-650 suppression on tumor growth (A) images of the NC and miR-650 inhibitor tumors (B)
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Effect of NC and miR-650 inhibitor transfection on xenografted tumor volume (C) Effect of NC
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and miR-650 inhibitor transfection on xenografted tumor weight (D) expression of Gfi1 in NC
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and miR-650 inhibitor tumors. The values are mean of three biological replicates and expressed
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as mean ± SD. (*p < 0.05 for NC Vs miR-650 inhibitor).
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MiR-650 is significantly upregulated in oral cancer cells Inhibition of miR-650 suppresses the proliferation, migration and invasion of the oral cancer cells by modulating the expression of Gfi1 Gfi1 overexpression rescues the inhibitory effects of miR-650 on the proliferation, migration and invasion of the oral cancer cells Overexpression of miR-650 promotes the proliferation, migration and invasion of the oral cancer cells Inhibition of miR-650 suppresses the tumor growth in xenografted mice
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