ORIGINAL ARTICLE

Role of miR-145 in human laryngeal squamous cell carcinoma Omer Faruk Karatas, MSc,1,2 Betul Yuceturk, BSc,1,3 Ilknur Suer, BSc,1 Mehmet Yilmaz, MD,4 Harun Cansiz, MD,4 Mustafa Solak, PhD,5 Michael Ittmann, MD, PhD,6 Mustafa Ozen, MD, PhD1,6,7* 1

Department of Medical Genetics, Cerrahpasa Medical School, Istanbul University, Istanbul, Turkey, 2Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum, Turkey, 3Advanced Genomics and Bioinformatics Research Center, The Scientific and Technological Research Council of Turkey (TUBITAK), Gebze, Kocaeli, Turkey, 4 Department of Otorhinolaryngology, Cerrahpasa Medical School, Istanbul University, Istanbul, Turkey, 5Department of Medical Genetics, Afyon Kocatepe University, Afyon, Turkey, 6Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, 7Department of Molecular Biology and Genetics, Biruni University, Topkapi, Istanbul, Turkey.

Accepted 14 October 2014 Published online 16 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23890

ABSTRACT: Background. Laryngeal squamous cell carcinoma (SCC), being an aggressive malignancy, is one of the most commonly diagnosed malignant types of head and neck SCC worldwide. Incidences of laryngeal SCC have been reported to increase recently. In this study, we aimed to explore the biological effects of miR-145 on laryngeal cancer cells. Methods. The relative miR-145 level in laryngeal SCC tumor tissues and normal samples was investigated. Then, Hep-2 cells were utilized for functional analysis of miR-145. The proliferation abilities of transfected cells were measured using MTS assay. Scratch assay and single colony migration assay were performed to observe the alterations in migration behavior of transfected cells. Caspase assay and cell cycle analysis were used to investigate the underlying reasons of proliferative inhibition in cells in which miR-145 is overexpressed. Moreover, expression of SOX2 was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis in Hep-2 cells upon miR-

INTRODUCTION Laryngeal squamous cell carcinoma (SCC) is an aggressive malignancy that constitutes about 2.4% of all cancer cases and 2.1% of all cancer deaths worldwide.1 It is reported as the second most common malignant neoplasm of head and neck SCC.2 Incidences of laryngeal SCC have been reported to increase recently with a considerably more frequency in middle-aged and elderly men.3 Treatment options for early and localized laryngeal SCC are considered as surgery, radiation, and chemotherapy, however, radiochemotherapy is the only option for the advanced cancer cases, which does not give positive clinical response for most of the cases. Besides, although there have been marked developments in terms of therapeutic strategies, no such enhancements could be

*Corresponding author: M. Ozen, Department of Medical Genetics/Molecular Biology and Genetics, Biruni University, Topkapi, Istanbul, Turkey. E-mail: [email protected] Contract grant sponsor: The studies presented in this article are supported by The Scientific and Technological Research Council of Turkey (TUBITAK; grant number: 210T009).

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145 transfection and its expression was evaluated in tumor and normal tissue sample of the larynx. Results. The miR-145 expression in laryngeal SCC tumor samples has been shown to be downregulated. The miR-145 overexpression caused inhibition of proliferation and migration in Hep-2 cells through induction of apoptosis and cell cycle arrest. The SOX2 level was demonstrated to be overexpressed in tumor samples and its expression was significantly decreased in miR-145 overexpressed Hep-2 cells. Conclusion. We have demonstrated the deregulation of miR-145 and SOX2 in laryngeal SCC. Based on these results, we propose that miR145, as an important regulator of SOX2, carries crucial roles in laryngeal C 2015 Wiley Periodicals, Inc. Head Neck 38: 260– SCC tumorigenesis. V 266, 2016

KEY WORDS: hsa-miR-145, SOX2, laryngeal squamous cell carcinoma, stem cell marker, cancer stem cell

achieved for therapeutic outcome, prognosis, and mortality rates of patients with laryngeal SCC.4,5 MicroRNAs (miRNAs) are 18 to 24 nucleotides long, endogenously synthesized, small noncoding RNAs, which are involved in the regulation of posttranscriptional mRNA expression. Up to now, 24,521 mature miRNAs have been identified and 2578 of them belong to homosapiens (miRBase, release 20.0, June 2013).6 Almost 60% of human genes are thought to be regulated by miRNAs7 and strong evidence suggest that their deregulation are strongly associated with several disorders, including cancer.8,9 Hsa-miR-145 (miR-145) is one of the most crucial tumor suppressor miRNAs, which has been shown to have reduced expression in various cancers, including colorectal, mammary, ovarian, prostate, and B-cell tumors.10 MiR-145 expression has also been demonstrated to be downregulated in laryngeal SCC (22605671). Bioinformatics and functional analysis have identified several potential targets for miR-145 and recently 30 untranslated region of SOX2, which plays a role in maintaining the self-renewal and pluripotency properties,11 has been demonstrated to be directly targeted by miR-145 in embryonic stem cells.12

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TABLE 1. Clinicopathological information of the patients.

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Kit (Roche, Switzerland) in accord with the manufacturer’s instructions.

Patients with laryngeal SCC

Age, y 60 >60 Sex Male Female Tumor location Supraglottic Subglottic Glottic Others T classification T1 and T2 T3 and T4 Tumor sizes 2 cm >2 cm

Proliferation assay 24 16 38 2 12 2 12 14 16 24 19 21

Abbreviation: SCC, squamous cell carcinoma.

In this study, we investigated the biological effects of miR-145 on laryngeal cancer cells. MiR-145 overexpression in Hep-2 cells resulted in inhibition of the proliferation and migration together with an increase in apoptosis and induction of G1 phase arrest. Further analysis showed that SOX2 expression was significantly decreased at both the mRNA and protein levels in Hep-2 cells in which miR-145 is ectopically expressed. Moreover, SOX2 expression in laryngeal SCC tumor samples has been demonstrated to be upregulated compared with those of corresponding adjacent normal larynx tissue samples.

The proliferation capacities of Hep-2 cells transfected with either miR-145 or nontargeting miRNA (nontargeting miR) were measured using MTS assay. Cells were seeded into 96-well plates in triplicate and 24 hours later they were transfected with miR-145 or nontargeting miR using X-treme gene transfection reagent. Proliferation rates were measured at the 24th and 48th hours subsequent to transfection. Cell proliferation reagent WST1 (Roche, Mannheim, Germany) was added to each well and incubated at 37 C for 3 hours in the dark. Cell viability was evaluated by measuring the absorbance at 490 nm with a Multiskan FC micro plate reader (Thermo, Rockford, IL).

Scratch assay Scratch assay was performed to evaluate the changes in migratory potential of transfected Hep-2 cells. The appropriate number of cells were seeded into 30 mm petri dishes in duplicate and, after the cells reached 60% to 70% confluency, they were transfected with miR-145 or nontargeting miR. Twenty-four hours after transfection, monolayer cells were scraped with a 200 ll pipette tip to create a scratch. Migration rates of controls and miR-145 transfected samples were analyzed with an inverted microscope (Leica, Wetzlar, Germany).

Single colony migration assay

This study has been approved by an institutional review board of Istanbul University, Cerrahpasa Medical School and patients were included in the study after each of them signed written informed consent forms. Forty normal and 40 tumor tissue samples were obtained from the Department of Otorhinolaryngology, Istanbul University, Cerrahpasa Medical School. None of the patients received radiotherapy, chemotherapy, or immunotherapy before the surgery. The clinicopathological characteristics of 40 patients are provided in Table 1. The percentage of tumor cells was at least 70% in all tumor tissue samples, which were collected after the surgery, immediately snap frozen, and kept in 280 C refrigerators until RNA isolation.

Single colony migration assay, as a novel technique to evaluate the migratory potential of cells, was used to investigate the alteration in the migration rate of Hep-2 cells transfected either with miR-145 or nontargeting miR. Hep-2 cells were seeded into 30 mm petri dishes in duplicate and, after the cells reached 60% to 70% confluency, they were transfected with miR-145 or nontargeting miR. Twenty-four hours after transfection, cells were harvested and reseeded in 96-well plates in 1 cell/well concentration. Then, the cells were incubated for 2 weeks to analyze the differences in migratory potential of the colonies formed from single cells. At the end of 2 weeks, colonies were fixed with absolute methanol for 10 minutes at 220 C and stained with crystal violet for 15 minutes. Migratory phenotypes were categorized as “–,” “1,” “11,” and “111” according to the degree of the migration phenotype. The total percentage of “11” and “111” were calculated and compared in cells transfected with either miR-145 or nontargeting miR.

Cell culture and transfection of cells with miRNAs

Cell cycle analysis

The Hep-2 cell line was utilized for functional analysis of miR-145. Cells were grown within Roswell Park Memorial Institute medium (Gibco-BRL, Bethesda, MA) containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin streptomycin amphotericin at 37 C in a humidified and 5% CO2 incubator. Mature miR-145 and nontargeting miRNA was purchased from SwitchGear Genomics (Carlsbad, CA). Transfection experiments were optimized and performed with X-treme Gene Transfection

Cell cycle analysis was performed to evaluate the changes in cell cycle distribution of transfected Hep-2 cells. Twenty-four hours after miR-145 overexpression, Hep-2 cells transfected with nontargeting miR or miR145 were harvested and fixed in 80% ethanol at 220 C. After overnight fixation, cells were washed with phosphate-buffered saline and stained with propidium iodide for 5 minutes. Then, cells were analyzed by FACSCalibur (BD Biosciences, San Jose, CA) flow

MATERIALS AND METHODS Collection of tumor and normal tissue samples

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FIGURE 1. (A) Relative expression levels of miR-145 in each tumor and normal sample, and (B) mean relative expression levels miR-145 in tumor samples with respect to normal samples.

cytometer, and BD CellQuest Pro Software (BD Biosciences) was used to analyze the results.

analysis was done by d-delta-Ct method, as described previously.14

Caspase assay

Western blot analysis

Caspase assay was used to evaluate the apoptotic activity of Hep-2 cells after miR-145 transfection. Cells were seeded into 96-well plates in duplicate. Colorimetric caspase 3 activity assay was performed using the CaspACE Assay System, Colorimetric (Promega) at 24 hours after transfection, in accord with the manufacturer’s instructions. For each measurement, caspase assay mix was added to each well and incubated at 37 C for 4 hours in the dark. Color development was evaluated by measuring the absorbance at 405 nm with a Multiskan FC microplate reader (Thermo).

Hep-2 cells were seeded in duplicate into 6-well plates. Forty-eight hours later, the transfection of cells with miR145 cells were scraped from the plates and washed in phosphate-buffered saline twice. Cells were lysed using Radioimmunoprecipitation lysis buffer containing 10 mM phenylmethylsulphonyl fluoride, and 13 protease inhibitors (Pierce protease inhibitor tablets; Thermo). Equal amounts of protein extracts (20 lg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane and probed with the primary antibodies diluted in 13 TRIS-buffered salineTween-20 (10 mM Tris–HCl; pH 7.5; 150 mM NaCl, and 0.1% Tween-20), 3% skim milk: m-a-SOX2 (1:2000, Abcam), and rb-a-b-tubulin (1:3000; Thermo). Data were normalized to b-tubulin. Signals were detected with Pierce ECL Western blotting substrate (Thermo) using corresponding horseradish peroxidase-conjugated secondary antibodies (1:3000; Pierce, Thermo). Quantification of bands was performed using the Image J program.

RNA isolation, cDNA synthesis, and quantitative real-time polymerase chain reaction Total RNA from miRNA-transfected samples, tumor, and normal tissue specimens was isolated using TRIzol (Invitrogen, San Diego, CA) reagent. For miRNA quantitative real-time polymerase chain reaction (qRT-PCR) experiments, equal amounts of total RNA were used for first strand DNA (cDNA) synthesis using miRNA specific primers purchased from Applied Biosystems (Foster City, CA) and the TaqMan microRNA reverse transcription kit, in accord with the manufacturer’s protocol (Applied Biosystems). For gene expression analysis, cDNAs were synthesized using the Transcriptor high fidelity cDNA synthesis kit (Roche, Switzerland) according to the manufacturer’s instructions. The qRT-PCR was carried out in a Roche LightCycler480-II real-time thermal cycler (Roche, Switzerland). For miRNA expression analysis, TaqMan Universal Master Mix (Applied Biosystems) was used and miRNA-specific probes were purchased from Applied Biosystems (Denmark). MiRNA expression data were normalized to RNU43. For gene expression analysis, SYBR Green Master Mix of Roche (Switzerland) was used. Each experiment was performed in duplicate and expression data were normalized to b-actin. Primer sequences used for qRT-PCR were as follows: SOX2: forward 50 -CTCCGGGACATGATCAGC-30 , reverse 50 GGTAGTGCTGGGACATGTGAA-30 13; b-actin: forward 50 -GCCTCGCCTTTGCCGATC-30 , and reverse 50 -CCCA CGATGGAGGGGAAG-30 . The relative quantification 262

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Statistical analysis Data were plotted as mean 6 SE and the statistical significances were tested by t test. A p value of .05 or below was accepted as statistically significant.

RESULTS MiR-145 is downregulated in laryngeal tumor samples Forty laryngeal SCC tumor samples and 40 corresponding adjacent normal laryngeal tissues were included in this study to investigate the expression status of miR-145 in 40 patients with laryngeal SCC whose diagnosis had been confirmed histopathologically in Istanbul University, Cerrahpasa Medical School. The clinicopathological information of the patients is provided in Table 1. The expression status of miR-145 in laryngeal tumor samples and the adjacent normal tissues obtained from the same patients was analyzed by qRT-PCR. Results showed that, of all 40 sample-pairs tested, the expression level of miR-145 was significantly downregulated in tumor samples in comparison to the corresponding normal tissues (Figure 1A). Figure 1B shows the mean relative expression data for miR-145 in tumor and normal tissue

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ANALYSIS OF MIR–145 IN LARYNGEAL CANCER

FIGURE 2. (A) Relative expression of miR145 in Hep-2 cells transfected with miR-145 at 24, 72, 96, and 120 hours after transfection. MiRNA levels were normalized to RNU43. (B) Proliferation assay in miR-145 transfected Hep-2 cells. (C–G) Scratch assay showing migration capabilities in miR-145 transfected Hep-2 cells. (H–L) Single cell migration assay showing migration capabilities in miR-145 transfected Hep-2 cells. *p < .05.

samples. Normalized to RNU43, the miR-145 level in laryngeal SCC (n 5 40) and normal tissues (n 5 40) was 0.21 6 0.14 and 1, respectively (Figure 1B; p < .0001).

MiR-145 overexpression reduces proliferation in Hep-2 cells To investigate the role of miR-145 in laryngeal SCC tumorigenesis, we analyzed the functional effects of miR145 upon its ectopic expression. To explore its effects on the proliferation capacity of Hep-2 cells, they were transfected with miR-145. The effectiveness of miR-145 transfection and its durability were controlled with microRNA qRT-PCR, which demonstrated that mature miR-145 molecules are expressed efficiently when Hep-2 cells were

transfected with miR-145 but not with nontargeting control miRNA. Besides, even after 5 days, miR-145 levels were kept quite high in miR-145 transfected Hep-2 cells, which gave the opportunity to analyze the effects of miR-145 for longer periods (Figure 2A). The proliferation assay showed that ectopic overexpression of miR-145 in the Hep-2 cell line brought about a significant decrease in the proliferation rate. The viability of Hep-2 cells diminished significantly 24 and 48 hours after miR-145 overexpression (Figure 2B).

MiR-145 overexpression inhibits migratory potential of Hep-2 cells To investigate the impact of miR-145 on the migratory potential of Hep-2 cells, we initially performed a scratch HEAD & NECK—DOI 10.1002/HED

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FIGURE 3. (A–C) Cell cycle distribution in miR-145 transfected Hep-2 cells. (D) Relative caspase-3 levels in miR-145 transfected PC3 cells and corresponding controls at 24 hours after transfection. *p < .05.

assay. Scratch assay revealed that the migration rate of miR145 overexpressing Hep-2 cells decreased compared to that of the control cells (Figure 2C–2F). To demonstrate the inhibition of migration quantitatively, we measured the percentage of coverage of the scratch at the end of 24 hours after miR-145 overexpression. Results showed that, although control cells covered more than 30% of the scratch, miR-145 transfected Hep-2 cells could only cover 15% (Figure 2G; p < .001). To further validate the migration inhibitory potential of miR-145 in Hep-2 cells, we performed single colony migration assay. Single cells, transfected either with miR145 or nontargeting miR, were seeded in 96-well plates and monitored for migration from the colonies at the end of 2 weeks. Colonies with no migration or very few migrations were counted as “–” and “1,” respectively (Figure 2H and 2I). Colonies with higher migration potential were designated as “11” or “111,” according to the number of cells migrated from the colonies (Figure 2J and 2K). As a result, single colony migration assay demonstrated that the percentage of the colonies with a “11” and “111” migration phenotype in miR-145 transfected Hep-2 cells was significantly reduced compared to that of control cells (Figure 2L; p 5 .02). Overall results clearly show that miR-145 overexpression in Hep-2 cells inhibits their migratory potentials.

MiR-145 induces G1-phase arrest and inhibits proliferation through apoptosis To reveal which mechanisms are involved in the inhibition of proliferation, miR-145 transfected Hep-2 cells have been 264

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evaluated for cell cycle distribution and apoptosis. Cell cycle analysis showed that the number of cells residing in the G0/ G1 phase significantly increases in miR-145 transfected cells (p 5 .02). Moreover, a marked reduction in the number of cells residing in the G2/M phase has been reported (p 5 .02; Figure 3A–3C). Furthermore, to evaluate apoptosis in miR145 transfected Hep-2 cells, caspase-3 activity, which rises as a result of the increasing number of early apoptotic cells, has been measured through a colorimetric method 24 hours after treatment with miR-145. The caspase-3 activity assay revealed that the elevated expression of miR-145 in Hep-2 cells caused an approximately 1.5-fold increase in the caspase-3 activity in comparison to that of the control cells (Figure 3D). Overall results concerning cell cycle and apoptosis analysis suggest that both cell cycle arrest and induction of apoptosis are involved in the inhibition of proliferation in Hep-2 cells upon miR-145 overexpression.

SOX2 expression is downregulated in both mRNA and protein level upon miR-145 overexpression SOX2 has been previously shown to be directly targeted by miR-145 and its downregulation has been reported during embryonic stem cell differentiation in response to upregulation of miR-145.12 The expression status of SOX2 was analyzed by qRT-PCR 24 hours after miR-145 overexpression in Hep-2 cells. SOX2 level has been shown to be significantly downregulated in miR-145 transfected cells (p < .001). Figure 4A shows the relative expression data for SOX2. To further validate the downregulation of

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ANALYSIS OF MIR–145 IN LARYNGEAL CANCER

FIGURE 4. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of SOX2 levels in miR-145 transfected Hep-2 cells compared with the control cells. The mRNA levels were normalized to b-actin. (B) Western blot analysis in SOX2 level in miR-145 transfected Hep-2 cells compared to the control cells. Protein levels were normalized to b-tubulin. (C) Relative expression levels of SOX2 in each tumor and normal samples and (D) mean relative expression levels of SOX2 in tumor samples with respect to normal samples. *p < .05.

SOX2, miR-145 transfected Hep-2 cells were lysed 48 hours after transfection and lysates were analyzed by Western blot. In parallel to the qRT-PCR results, the SOX2 protein level was similarly reduced as a result of miR-145 overexpression in Hep-2 cells (Figure 4B; p < .05). These results clearly suggest that reduced levels of SOX2 mRNA and protein levels accompany the ectopic expression of miR-145 in Hep-2 cells.

Reduced miR-145 expression in laryngeal squamous cell carcinoma tumor tissues accompany elevated SOX2 mRNA levels Because the expression level of miR-145 was significantly reduced in laryngeal SCC tumor tissue samples in all 40 sample-pairs tested, we investigated the expression status of SOX2 in a 20 sample-pair set by qRT-PCR. Results demonstrated that the expression of SOX2 was upregulated in most of the tumor tissue samples compared with the adjacent normal tissues (Figure 4A). Normalized to b-actin, the SOX2 mRNA level in laryngeal SCC and normal tissues were 2.12 6 0.30 and 1, respectively (Figure 4B; p < .001). The expression level of SOX2 mRNA was significantly elevated in laryngeal SCC tissues compared to the corresponding tumor-adjacent normal tissues. We further analyzed the correlation of miR-145 and SOX2 expressions and found that there is a negative correlation (R 5 20.204).

DISCUSSION Laryngeal SCC is an aggressive malignancy that constitutes a big portion of the malignant type of head and

neck SCC. It has been reported to demonstrate an increasing incidence rate and poor prognosis. Recently, there have been enormous developments made in laryngeal SCC therapy applications targeting early and nonmetastasized laryngeal SCC cases; however, the need for therapeutic approaches against advanced laryngeal SCC cases still remains. Therefore, there is an urgent need for understanding the underlying mechanisms of laryngeal SCC pathogenesis and development of therapeutic approaches against advanced laryngeal SCC cases. There are now several reports that show evidence for the participation of miRNAs in laryngeal carcinogenesis. Aberrant expression of miRNAs has been detected in laryngeal SCC cell lines and clinical samples.15,16 These alterations are suggested to have significant roles in the laryngeal SCC pathogenesis. Now more than 2500 miRNAs have been identified that are expressed in homosapiens and they are thought to target and modulate the expression of more than 60% of human genes.6,7 Because miRNAs are among the most promising targets to develop therapeutic approaches, investigation of certain miRNAs’ involvement in laryngeal SCC remains very crucial for understanding the miRNA mechanisms associated with laryngeal carcinogenesis. MiR-145 is one of the significant tumor suppressor miRNAs whose expression is downregulated in various tumor types, including colorectal, mammary, ovarian, B-cell tumors, and prostate cancer.10,17,18 In a recent study, expression of miR-145 has been also shown to be reduced in laryngeal SCC tumor specimens.19 Functional characterization of miR-145, however, has not been studied in HEAD & NECK—DOI 10.1002/HED

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laryngeal SCC until now. We, therefore, validated the downregulation of miR-145 in laryngeal SCC tumor samples in comparison to the tumor-adjacent normal tissues and further analyze its functions in Hep-2 cells. We showed that ectopic expression of miR-145 brought about significant reduction in the proliferation rate in Hep-2 cells. Moreover, the migratory potential of Hep-2 cells was also significantly inhibited upon overexpression of miR-145. Cell cycle analysis showed that miR-145 overexpression in Hep-2 cells induces G1-phase arrest. In addition, caspase assay demonstrated that apoptosis was significantly increased in Hep-2 cells upon overexpression of miR-145. All these functional studies suggest that downregulation of miR-145 in laryngeal SCC tumor tissues may cause an increase in aggression, early recurrence, and fast progression of laryngeal SCC. In embryonic stem cells, miR-145 has shown to directly target the 30 untranslated region of SOX2 and elevated levels of miR-145 have been associated with inhibition of human embryonic stem cell self-renewal.12 Therefore, we wanted to investigate the expression level of SOX2 both in Hep-2 cells upon miR-145 overexpression and in laryngeal tumors and normal tissue specimens for understanding its role in laryngeal SCC oncogenesis. The qRT-PCR and Western blot analysis demonstrated that SOX2 expression in both mRNA and the protein level is significantly reduced upon ectopic miR-145 overexpression in Hep-2 cells. Knowing the fact that involvement of SOX2 in the tumorigenesis of various cancer types has been recently reported,20–22 we further compared the expression level of SOX2 in laryngeal SCC tumor tissues and their corresponding tumor adjacent normal tissues and showed that SOX2 levels in tumor samples were significantly upregulated in tumor specimens. This finding supports a recent report showing that the number of SOX2 expressing cells in prostate cancer tissues has been shown to outnumber those in benign prostate hyperplasia or normal prostate tissues.23 Moreover, being one of the crucial stemness factors,24,25 SOX2 overexpression along with miR-145 downregulation in laryngeal SCC samples might be postulated as causing enrichment of cancer stem cells in the laryngeal SCC tumors. As a result, we propose that downregulation of miR145 in laryngeal SCC accompany overexpression of SOX2, which is a validated target of miR-145, and these expressional alterations would be a key step in the laryngeal SCC pathogenesis. Further studies, however, are needed to elucidate the roles of miRNAs, especially the miR-145, in more detail in laryngeal SCC pathogenesis.

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Role of miR-145 in human laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (SCC), being an aggressive malignancy, is one of the most commonly diagnosed malignant types of head and neck SCC wo...
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