MicroRNA-625 inhibits cell invasion and epithelial–mesenchymal transition by targeting SOX4 in laryngeal squamous cell carcinoma

Introduction: Laryngeal squamous cell carcinoma (LSCC) is a highly aggressive malignant cancer, but the molecular mechanisms underlying its development and progression remain largely elusive. The purpose of the present study is to investigate the expression profile and functional role of microRNA-625 (miR-625) in LSCC. Materials and methods: LSCC tissues and adjacent normal tissues were collected from 86 LSCC patients. The expression levels of miR-625 and SOX4 mRNA in tissues and cells were detected by RT-qPCR analysis. The expression levels of SOX4 and EMT-related proteins were detected by western blot analysis. In vitro cell proliferation, migration, and invasion were detected by MTT assay, colony formation assay, wound healing assay, and transwell invasion assay, respectively. Dual-luciferase reporter assay was performed to verify the binding relationship between miR-625 and the 3′-UTR of SOX4. Results: The results demonstrated that miR-625 is significantly down-regulated in clinical LSCC tissues, and its low expression may be closely associated with unfavorable clinicopathological characteristics of LSCC patients. Overexpression of miR-625 significantly suppressed the proliferation, migration, invasion, and EMT of LSCC cells. Furthermore, SOX4 was validated as a direct target of miR-625 in LSCC cells, and rescue experiments suggested that restoration of SOX4 blocked the tumor suppressive role of miR-625 in LSCC cells. Conclusions: Taken together, these findings highlighted a critical role of miR-625 in the pathogenesis of LSCC, and restoration of miR-625 could be considered as a potential therapeutic strategy against this fatal disease.


Introduction
Laryngeal cancer is one of the most common head and neck cancers, and laryngeal squamous cell carcinoma (LSCC) accounts for approximately 85-90% of all laryngeal cancer cases [1]. In spite of recent advances in diagnostic and therapeutic methods, the long-term prognosis of LSCC patients remains largely unsatisfactory [2]. Accordingly, a complete understanding of the molecular mechanisms that are involved in LSCC development and progression is of critical importance.
MicroRNAs (miRNAs), initially identified in 1993, are a class of endogenous small (∼22 nucleotide) noncoding RNAs that play critical regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression [3,4]. A growing body of evidence indicates that miRNAs can function as potential oncogenes or tumor suppressive genes in cancer biology [5]. For example, the tumor suppressive role of miR-625 has been widely explored. Decreased expression level of miR-625 is found in breast cancer [6], melanoma [7], and glioma [8]. But the functional role of this miRNA in LSCC remains largely elusive. In the present study, we aimed to examine the expression profile of miR-625 in LSCC tissues and cell lines, evaluate the biological functions of miR-625 in LSCC cells, and identify the downstream target gene of miR-625 in LSCC. We believed that our findings might open new avenues for the treatment of this malignant disease.

Materials and methods
Patients and tissue samples 86 pairs of LSCC tissues and adjacent normal tissues (at least 1 cm from the neoplastic edge) were collected from LSCC patients who underwent partial or total laryngectomy at Affiliated Hospital of Hangzhou Normal University (Hangzhou, China) between March 2017 and May 2018. All recruited patients did not receive chemotherapy or radiotherapy preoperatively. The collected tissue samples were confirmed histologically by two experienced pathologists, snap-frozen in liquid nitrogen, and stored at −80 • C. The research protocol was approved by the Ethics Committee of Affiliated Hospital of Hangzhou Normal University, and written informed consent was obtained from all patients or their relatives. The clinicopathological characteristics of LSCC patients are recorded in Table 1. Synthetic miR-625 mimics (5 -AGGGGGAAAGUUCUAUAGUCC-3 ) and mimics negative control (5 -CAGUACUUUUGUGUAGUACAA-3 ) were obtained from Guangzhou Ribobio Co., Ltd (Guangzhou, China). The SOX4 overexpression plasmid was established by inserting the full-length CDS sequence of SOX4 into the pcDNA3.1 vector (Invitrogen). An empty vector was considered as a negative control. Cells were seeded in six-well plates at 80% confluence, and transfected with the plasmids and oligonucleotides using Lipofectamine 2000 (Invitrogen). After 48 h, the transfection efficiency was validated by RT-qPCR analysis.

Colony formation assay
Cells were seeded on six-well plates at a density of 500 cells/well. The culture medium was replaced every 3 days. Ten days later, the colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted.

Wound healing assay
Cells were seeded into 12-well plate and grown until 90% confluent. The cellular monolayer was then wounded using a sterilized 200 μl pipette tip, and then the cells were cultured in serum-free medium. Images were captured at 0 and 24 h following the initial scratch under an optical microscope.

Transwell invasion assay
Cells were suspended in 200 ml of serum-free medium and placed in the upper chamber of the transwell filters (8 μm pore size; BD Biosciences, San Jose, CA, U.S.A.) that were coated with Matrigel (BD Biosciences). The lower chamber was filled with 600 μl medium containing 10% FBS as a chemoattractant. Following incubation for 24 h, the cells remaining on the upper side of the membrane were wiped off with cotton swabs, and the cells that moved to the bottom surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of invaded cells was counted randomly in five fields of each membrane.

Dual-luciferase reporter assay
The fragment from SOX4 3 -UTR containing the predicted miR-625 binding site was amplified by PCR and cloned into the psiCHECK2 vector (Promega, Madison, WI, U.S.A.). HEK293T cells were cultured in 24-well plates and co-transfected with 400 ng of luciferase reporter vectors comprising SOX4-WT or SOX4-MUT, 50 nmol/l of miR-625 mimics or mimics control, and 50 ng of pRL-TK plasmid (Promega) using Lipofectamine 2000. The pRL-TK Renilla luciferase plasmid was used as internal control. Forty eight hours after transfection, the luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega), and the firefly luciferase activity was normalized to the Renilla luciferase activity.

Statistical analysis
All statistical analyses were conducted using the GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, U.S.A.) and SPSS 17.0 (SPSS, Inc., Chicago, IL, U.S.A.). Differences between experimental groups were assessed by Student's t-test or one-way analysis of variance (ANOVA). The association between miR-625 expression and clinicopathological characteristics of LSCC patients was evaluated using the chi-square test. All P-values were two-sided, and P<0.05 was considered statistically significant.

miR-625 is down-regulated in LSCC tissues and cell lines
First, remarkably lower miR-625 expression was observed in LSCC tissues than that in the adjacent normal tissues ( Figure 1A). In addition, the expression levels of miR-625 in the LSCC cell lines (AMC-HN-8 and Tu-177) were also significantly decreased compared with normal HaCaT cells ( Figure 1B).
According to the average value of miR-625 expression, the LSCC patients were divided into high expression group (n=44) and low expression group (n=42). As listed in Table 1, low miR-625 expression was distinctly associated with advanced clinical stage (P=0.010) and lymph node metastasis (P=0.032) of LSCC patients.

miR-625 inhibits LSCC cell proliferation
To further investigate the regulatory role of miR-625 in LSCC, we then transfected the LSCC cell lines (AMC-HN-8 and Tu-177 cells) with miR-625 mimics, and the ectopic expression of miR-625 was validated by RT-qPCR analysis (Figure 2A). The growth curves determined by MTT assay demonstrated that the proliferation abilities of AMC-HN-8 and Tu-177 cells were significantly impaired when miR-625 was overexpressed ( Figure 2B). Besides, as demonstrated in Figure 2C, miR-625 overexpression also remarkably reduced the number of colonies formed by AMC-HN-8 and Tu-177 cells.

miR-625 inhibits LSCC cell migration and invasion
Through wound healing assay, we found that the migratory abilities of AMC-HN-8 and Tu-177 cells were significantly inhibited by miR-625 overexpression ( Figure 3A). We also performed transwell assay to examine cell invasive ability, and as shown in Figure 3B, miR-625 overexpression remarkably reduced the number of invaded AMC-HN-8 and Tu-177 cells.
EMT is a key event in cancer metastasis. As shown in Figure 3C, E-cadherin expression was significantly increased while the expression levels of N-cadherin and Vimentin were remarkably decreased in AMC-HN-8 and Tu-177 cells when miR-625 was overexpressed.

miR-625 directly targets SOX4 in LSCC cells
To gain further insights into the functional role of miR-625 in LSCC, we then searched for its target genes. Through TargetScan website (http://www.targetscan.org) [10], we found that SOX4 3 -UTR contains a complimentary binding site of miR-625 ( Figure 4A). In order to further confirm the prediction, we then performed dual-luciferase reporter assay. The results showed that miR-625 mimics significantly reduced the luciferase activity driven by SOX4-WT in HEK293T cells, but had no obvious effect on the activity of SOX4-MUT ( Figure 4B). We further found that overexpression of miR-625 significantly decreased the protein expression levels of SOX4 in AMC-HN-8 and Tu-177 cells ( Figure 4C). Furthermore, in LSCC tissues, remarkably higher SOX4 mRNA expression was observed ( Figure 4D), and Pearson correlation analysis indicated that the expression levels of miR-625 and SOX4 mRNA were negatively correlated in LSCC tissues (r = −0.24, P=0.026; Figure 4E).

SOX4 blocks the tumor suppressive role of miR-625 in LSCC cells
Then we analyzed whether miR-625 inhibited the migratory and invasive capacities of LSCC cells partly by negative regulation of SOX4. As shown in Figure 5A, SOX4 overexpression rescued the impaired EMT in miR-625 mimics-transfected AMC-HN-8 and Tu-177 cells. Moreover, we also uncovered that SOX4 overexpression remarkably abrogated the inhibitory role of miR-625 on the migration and invasion of AMC-HN-8 and Tu-177 cells ( Figure  5B,C).

Discussion
Initiation and progression of cancer are often caused by an imbalance between oncogenes and tumor suppressive genes. From The Cancer Genome Atlas (TCGA) database, a number of differentially expressed miRNAs in LSCC have been identified [11]. Several miRNAs, including miR-26a and miR-153, emerged as powerful regulators in LSCC, as indicated in recent studies [12,13]. One major finding of our study is that miR-625 was markedly decreased in LSCC tissue specimens and cell lines. Besides, decreased expression of miR-625 was associated with adverse clinicopathologic features of LSCC patients. In line with our clinical results, the in vitro experimental data also revealed that the ectopic expression of miR-625 led to the inhibition of LSCC cell proliferation, migration, and invasion. These results clearly implied that miR-625 may function as a tumor suppressor in LSCC.
Local invasion and distant metastasis are the main causes of death in LSCC patients [14]. EMT is a complex trans-differentiation process by which cancer cells gain migratory and invasion abilities [15]. We observed that miR-625 overexpression inhibited EMT in LSCC cells by increasing the expression levels of E-cadherin and decreasing the expression levels of N-cadherin and Vimentin. A miRNA can modulate a large number of target genes [16]. We then predicted the target gene of miR-625 using bioinformatics analysis and considered SOX4 as a potential target gene in LSCC. Sex-determining region Y-box 4 (SOX4), a master regulator of EMT, serves a fundamental role in tumorigenesis and metastasis [17,18]. Further rescue experiments confirmed that the tumor suppressive role of