A total of 51 pairs of LSCC tissues and adjacent normal epithelial tissues were obtained from patients who received primary surgical resection of LSCC between September 2012 and July 2015 in the Department of Otolaryngology, Head and Neck Surgery, Tianjin Union Medical Center (Tianjin, China). None of the LSCC patients were treated with radiotherapy or chemotherapy prior to surgery. Tissue samples were snap-frozen in liquid nitrogen immediately following resection and stored at −80°C until use. The present study was approved by the Ethics Committee of Tianjin Union Medical Center (Tianjin, China), and all patients gave their informed written consent.

Three human LSCC cell lines (Hep-2, AMC-HN-8 and Tu-177), a normal human keratinocyte cell line (HaCaT) and 293T cell line were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 or Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and were grown in a humidified atmosphere at 37°C with 5% CO₂.

miR-195 mimics and miRNA mimics negative control (miR-NC) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Small interfering (si)RNA targeting Rho-associated kinase (ROCK)1 (si-ROCK1) and its control siRNA (si-NC) were chemical synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). For cell transfection, cells (8×10⁵ cells/well) were seeded in 6-well plates. Following overnight incubationat 37°C with 5% CO₂, cells were transfected with miRNA mimics or siRNA by using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following to the manufacturer' sinstructions.

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer' sprotocol. The concentration and purity of total RNA was measured using a NanoDrop^® ND-1000 spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). For miR-195 expression, the One Step PrimeScript miRNA cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan) was used to perform reverse transcription, followed by qPCR with SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.). The temperature protocol for reverse transcription was as follows: 37°C for 15 min and 85°C for 5 sec. qPCR was performed with the following thermo cycling conditions: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec.

For ROCK1 mRNA expression, cDNA was synthesized from RNA by using cDNA Synthesis kit (Takara Bio, Inc.). qPCR was carried out using SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.). The temperature protocol for reverse transcription was as follows: 37°C for 60 min and 85°C for 5 sec. The thermocycling conditions for qPCR was as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 and GAPDH were used as control for miR-195 and ROCK1 mRNA expression, respectively. The primers were designed as follows: miR-195, 5′-ACACTCCAGCTGGGTAGCAGCACAGAAAT-3′ (forward) and 5′-TGGTGTCGTGGAGTCG-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); ROCK1, 5′-AGGAAGGCGGACATATTGATCCCT-3′ (forward) and 5′-AGACGATAGTTGGGTCCCGGC-3′ (reverse); and GAPDH, 5′-CCCCTTCATTGACCTCAACT-3′ (forward) and 5′-ATGAGTCCTTCCACGATACC-3′ (reverse). The data were calculated using the 2^−ΔΔCq method (22).

At 24 h post-transfection, cells were collected and seeded into 96-well plates at a density of 2,000 cells/well. Cells were then cultured for 24, 48, 72 and 96 h. At each time point, MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) assay was carried out. A total volume of 20 µl MTT solution was added to each well and incubated at 37°C with 5% CO₂ for another 4 h. The culture medium was then removed and 150 µl DMSO was added to each well. Following incubation at 37°C for 10 min with a constant shaking, the absorbance at 490 nm was determined by using a microtiter plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell migration and invasion assays were performed using Trans well chambers (Corning Life Sciences, Corning, NY, USA) and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) coated Trans well chambers, respectively. At 48 h post-transfection, cells were collected and suspended in FBS-free culture medium. A total of 1×10⁵ cells were seeded into the upper chamber, whereas 500 µl culture medium containing 20% FBS was added in the lower chamber. After incubation at 37°C with 5% CO₂ for 48 h, those cells left on the upperchamber were removed with cotton swabs. The migrated or invaded cells were fixed with 95% ethanol for 20 min and stained with 0.1% crystal violet for 10 min. After washing with PBS (Gibco; Thermo Fisher Scientific, Inc.), the migrated and invaded cells were photographed and quantified using an inverted microscope.

The putative target genes of miR-195 were predicted using the TargetScan (http://www.targetscan.org) and miRanda (http://www.microrna.org/microrna/). Based onbioinformatics analysis, ROCK1 was identified as a potential target of miR-195. Thewild type (Wt) or mutant (Mut) 3′-untranslated region (UTR) of ROCK1 harboring the miR-195 binding site was cloned into pGL3 control vector. For luciferase reporter assay, pGL3-ROCK1-3′UTR Wt or pGL3-ROCK1-3′UTR Mut together with miR-195 mimics or miR-NC were injected into 293T cells by using Lipofectamine 2000, according to the manufacturer's instructions. Luciferaseactivities were determined 48 h post-transfection using the Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). The results were expressed as relative luciferase activities (firefly luciferase/Renilla luciferase).

At 72 h after transfection, total protein was extracted from transfected cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). The protein concentration was determined by using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated by electrophoresis on a 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membrane (EMD Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk in 0.1% TBS and 0.05% Tween-20 (TBST; Beyotime Institute of Biotechnology) for 2 h and incubated with primary antibodies at 4°C overnight, including mouse anti-human monoclonal ROCK1 antibody (1:1,000 dilution; sc-365628; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse anti-human monoclonal GADPH antibody (1:1,000 dilution, sc-365062; Santa Cruz Biotechnology, Inc.). After washing three times with TBST, the membrane was incubated with corresponding horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The protein bands were visualized with enhanced chemiluminescence detection system (Amersham; GE Healthcare Life Sciences, Chalfont, UK). ROCK1 protein expression was normalized to total GAPDH.

The SPSS software (version, 17.0; SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All data were expressed aspresented as mean ± standard deviation, and were compared by a Student's t test to determine the statistical significance. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-195 in LSCC tissues and adjacent normal epithelial tissues were determined using RT-qPCR. The results indicated that miR-195 was lower in LSCC tissues than adjacent normal epithelial tissues (Fig. 1A, P<0.05). Furthermore, the authors analyzed miR-195 expression in LSCC cell lines. The data indicated that miR-195 was significantly downregulated in LSCC cell lines compared with normal human keratinocyte cell line (Fig. 1B, P<0.05).

To further investigate whether there was an association between miR-195 expression and LSCC prognosis, statistical analysis was performed to analyze the correlation between miR-195 expression and the clinicopathological factors of LSCC. As presented in Table I, there were significantly association of miR-195 expression with lymph node metastasis (P = 0.011) and TNM stage (P = 0.015). However, there were no statistically correlation between miR-195 expression and other clinicopathological features, including sex distribution, age, location, alcohol history, pathological differentiation and T classification (all P>0.05).

To investigate the biological roles of miR-195 on LSCC cancer cells, Hep-2 and AMC-HN-8 cells were transfected with miR-195 mimics or miR-NC. RT-qPCR showed that miR-195 was markedly upregulated in Hep-2 and AMC-HN-8 cells transfected with miR-195 mimics (Fig. 2A, P<0.05). The effect of miR-195 on proliferation of LSCC cells was assessed using MTT assay. As demonstrated in Fig. 2B, upregulation of miR-195 suppressed Hep-2 and AMC-HN-8 cell proliferation (P<0.05). Then, the author sex amined the effects of miR-195 on the migration and invasion capacities of LSCC cells by using cell migration and invasion assays. The results revealed that restoration of miR-195 obviously decreased the migration and invasion abilities of Hep-2 and AMC-HN-8 cells compared with miR-NC groups (Fig. 2C, P<0.05). These data suggested that overexpression of miR-195 suppressed growth and metastasis of LSCC cells.

The potential molecular mechanism on how miR-195 suppressed cell growth and metastasis of LSCC was analyzed by exploring its direct target genes. Based onbioinformatics analysis with public databases, ROCK1 was identified as a potential target of miR-195 (Fig. 3A).

To verify whether ROCK1 was a direct target gene of miR-195, aluciferase reporter assay was performed. 293T cells were transfected with miR-195 mimics or miR-NC as well as pGL3-ROCK1-3′UTR Wt or pGL3-ROCK1-3′UTR Mut. The results indicated that miR-195 overexpression reduced luciferase activities of vector containing wild type ROCK1 3′UTR (Fig. 3B, P<0.05), while miR-195 had no regulation effect on mutant type of ROCK1 3′UTR, suggesting that this binding site in ROCK1 3′UTR was essential for the regulation by miR-195.

Moreover, the authors assessed the effects of miR-195 overexpression on the expression of ROCK1. RT-qPCR and western blotting indicated that ectopic of miR-195 expression suppressed ROCK1 mRNA (Fig. 3C, P<0.05) and protein (Fig. 3D, P<0.05) expression level in Hep-2 and AMC-HN-8 cells. Taken together, miR-195 can directly decrease ROCK1 expression through targeting the binding site in the 3′UTR of ROCK1.

To study the effects of ROCK1 on LSCC, Hep-2 and AMC-HN-8 cells were transfected with si-ROCK1 or si-NC. After transfection, RT-qPCR and western blotting were used to evaluate its transfection efficiency. The results demonstrated that si-ROCK1 significantly decreased ROCK1 expression in Hep-2 and AMC-HN-8 cells at both mRNA (Fig. 4A, P<0.05) and protein (Fig. 4B, P<0.05) levels. Moreover, MTT assay, cell migration and invasion assays were used to investigate the effects of ROCK1 underexpression on LSCC cell proliferation, migration and invasion, respectively. The data revealed that inhibition of ROCK1 has similar effects to that of miR-195 overexpression, since it obviously suppressed growth (Fig. 4C, P<0.05) and metastasis (Fig. 4D, P<0.05) of Hep-2 and AMC-HN-8 cells. The results suggested that miR-195 overexpression suppressed proliferation, migration and invasion of LSCC cells through downregulation of ROCK1.