MiR-194 functions as a tumor suppressor in laryngeal squamous cell carcinoma by targeting Wee1

The emerging roles of microRNAs (miRs) have been deeply investigated in cancer. However, the role of miR-194 in human laryngeal squamous cell carcinoma (LSCC) is still unclear. Here, we have demonstrated that miR-194 is significantly downregulated in LSCC tissues and cells, and overexpression of miR-194 inhibits the proliferation, migration, invasion, and drug resistance in LSCC cells. Moreover, Wee1 is identified as a novel direct target of miR-194. Ectopic expression of Wee1 at least in part overcomes the suppressive impacts of miR-194 on the malignant phenotypes of LSCC. Overall, our study provides new sights into the role of miR-194/Wee1 axis in LSCC and suggests a novel miR-194/Wee1-based clinical application for LSCC patients. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0402-6) contains supplementary material, which is available to authorized users.

polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA and incubated with the indicated primary antibodies. Corresponding horseradish peroxidase-conjugated secondary antibodies were used against each primary antibody.
GAPDH or α-Tubulin was used as a loading control. Signals were detected using the ChemiDoc XRS chemiluminescent gel imaging system (Bio-RAD).

Wound healing assay
Briefly, cells (5 × 10 5 /well) were seeded into six-well dishes. Till the cells reached 80-90% confluence, the cell monolayer was wounded using a sterilized 10 μl pipette tip and washed with PBS two times. Cells were allowed to migrate for 24 hours and 48 hours in serum-free medium, and the wounds were observed and captured. The gap lengths were measured from the photomicrographs.

Transwell assay
Cells were plated in the upper compartment of a modified Boyden chamber (Corning) containing matrigel-coated polycarbonate membrane filter (6.5 mm diameter, 8 μm pore size), and the lower chamber contained medium with 10% FBS, and allowed to migrate for 24 hours at 37°C in 5% CO 2 . Cells on the upper surface of the membrane were wiped off, and cells invaded to the bottom surface were photographed and counted.

Sphere formation assay
Cells were trypsinized, suspended in medium containing 0.3% agar and 10% FBS and seeded at a density of 5× 10 2 cells/well in a 12-well plate. The agar-cell mixture was plated onto a bottom layer with 0.5% agar. Then treated cells were incubated in a humidified incubator and fresh medium was added every 3 days. Two weeks later, colonies were analyzed microscopically.

Cell cycle assay
Cells were digested, harvested and then washed twice with cold phosphate-buffered saline (PBS), then permeabilized with 70% cold ethanol for 2 hours at 4°C. After washing twice in PBS, cells were resuspended with 0.5 ml PBS containing PI (50 μg/ml), 0.1% Triton X-100, 0.1% sodium citrate, and DNase-free RNase (100 μg/ml), and assessed by FCM after incubation at room temperature in the dark for 15 minutes.
Fluorescence was measured at an excitation wavelength of 480 nm through a FL-2filter (585 nm). Data were analyzed using ModFit LT 3.0 software.

Plasmid construction and lentivirus production
The synthesized precursor hsa-miR-194 (Supplementary Table 1) was cloned into lentiviral vector pLKO.1-GFP to generate the hsa-miR-194 lentivirus construct. The Wee1 cDNA was cloned into lentiviral vector LV5. Lentivirus was packaged in HEK293T cells and collected from the medium supernatant. Stable cell lines were established by infecting lentivirus into Hep-2 and KB-3-1 cells, followed by puromycin selection.

Nude mice tumorigenesis assay
Balb/c nude mice were obtained from the Shanghai SLAC Laboratory Animal Co and maintained with sterilized food and water. Five female nude mice with 5 weeks old and 16-18 g weight were used for each group. Hep-2 cells, as well as KB-3-1 cells, infected with control or has-miR-194 lentivirus, respectively, were suspended 4 × 10 6 in 100 μl of DMEM for each mouse and were injected subcutaneously under the shoulder of the female nude mice, five mice per group. The body weights of the animals and the two perpendicular diameters (a and b) of tumor were recorded every five days. The tumor volume (V) was calculated as: The mice were anaesthetized after experiment,, and tumors were removed, weighed, and sectioned, followed by qRT-PCR analysis and IHC analysis. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University.

Luciferase reporter assay
The pmirGLO, pmirGLO-Wee1 3'-UTR-wt or pmirGLO-Wee1 3'-UTR-mut (Promega) vectors were cotransfected into Hep-2 and KB-3-1 cells respectively with the hsa-miR-194 vector or empty vector. Cell lysates were collected at 24 hours posttransfection. Luciferase and renilla signals were measured using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer.

Phalloidin staining assay
Cells were seeded on glass cover slips for 24 hours and then fixed in 4% paraformaldehyde for 20 minutes and permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. The coverslips were incubated in the dark with 100 nM rhodamine-phalloidin at room temperature for 30 minutes. Nuclei were counterstained with 100 nM DAPI. The coverslips were rinsed in PBS and inverted on a drop of anti-fade mounting media on a glass slide. Then, these slides were sealed with neutral balsam and viewed under the confocal microscope.

Statistical analysis
All statistical analyses were performed using the SPSS 20.0 statistical software package.

Supplementary Table 1. Relationship between miR-194 expression level and clinicopathologic parameters in LSCC.
or Kruskal-Wallis test (for > 2 groups).