Patient samples and Ethical statement
Laryngeal cancer tissues and matched adjacent non-cancer samples of 30 patients were collected from the Gongli Hospital (the Second Military Medical University), Ruijin Hospital (Shanghai Jiao Tong University School of Medicine) and Shanghai Ninth People’s Hospital (Shanghai Jiaotong University School of Medicine) between 2019 and 2021 year.
Immunohistochemical (IHC)
Laryngeal cancer tissues and matched adjacent non-cancer samples of 30 patients were first fixed in formalin and embedded in paraffin, then xylene-dewaxed and treated in citrate buffer (0.01 mol/L, pH 6.0). Rabbit anti-ROCK1 antibody (1:2000, Cell Signaling Technology) was used to stain those samples at 4°C all night. Then samples were incubated in secondary antibody for 30 min at 37°C, and in the and we visualized samples with DAB solution and counterstained them with haematoxylin. Two senior pathologists without knowing clinical information evaluated every sample, and a third pathologist was asked to Reevaluate the disputed samples. Five typical viewpoints were selected for IHC observation under a light microscope. The percentage of positive cells was divided into 4 grades (%): grade 0 (< 10%), grade 1 (10%-30%), grade 2 (30–50%), grade 3 (> 50%). The staining intensity score was also divided into 4 grades (intensity): grade 0 (no staining), grade 1 (weak staining), grade 2 (moderate staining) and grade 3 (strong staining). Total immunohistochemical score = positive cell score + staining intensity score. The total score was 0,1–2,3–4, and 5–6 were defined as negative (-), weak positive (±), moderate positive (+), and strong positive (++) respectively.
Cells line and cells culture
Cells were preserved by the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Dulbecco’s modified Eagle’s medium (DMEM, Gibco company, USA) with 10% Fetal Bovine Serum (FBS, Gibco) were used to culture the above cells with 37°C temperature and 5% CO2.
Immunofluorescent (IF)
5×104 TU686 and TU212 cells with different treatments were seeded into slides (Millipore, MA, USA) and 4% paraformaldehyde (PFA) was applied to fix cells for half an hour. PBS was used to rinse the slides for 3 times, and 5% BSA was applied to block the slides for 60 min at 37 degrees Celsius and primary antibodies were used to incubate the slides at 4°C for all night. The following day, slides were rinsed with PBS for 3 times and incubated with secondary antibodies in the dark at 37 degrees Celsius for 60 min. Anti-ROCK1 (Cell Signaling Technology) and Alexa Fluor® 488 goat (Abcam, Cambridge, MA, USA) and Alexa Fluor® 555 goat anti-rabbit IgG (Abcam, Cambridge, MA, USA) were used to IF staining. DAPI was used to visualized the nuclei of cells in the dark for 5 min. Analyzing slides by fluorescent microscopy (10x).
Transfections
3×105 TU686 and TU212 cells with different treatments were seeded into 6-well plates and incubated for all night. ROCK1 plasmid (Myc-ROCK1-Delta3 (1-727), Gene Pharma Company, Shanghai) by lipofectamine 2000 (Invitrogen) was used to transfect and transfected cells were selected via 1200 ug/ml G418. Western Blot and frozen were used to verify selected clones.
RNA Extraction and Quantitative Real-time PCR (qRT-PCR)
Total RNA of TU686 and TU212 cells with different treatments was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then was reverse transcribed to cDNA using Prime Script Reverse Transcriptase system (Promega, Madison, WI, USA). qRT-PCR was performed to quantify ROCK1 mRNA level with the SYBR Green PCR core Reagent kit (Applied Biosystems, Foster city, CA, USA). GAPDH was used as the endogenous reference. Data were analyzed by using the comparative Ct method. Specificity of resulting PCR products was confirmed by melting curves. The primers were designed using Primer Express v2.0 software (Applied Biosystems, Foster City, CA, USA). The primers used in this assay were: ROCK1 forward 5′-AGG AAG GCG GAC ATA TTA GTC CCT-3′ and reverse 5′-AGA CGA TAG TTG GGT CCC GGC-3′, β-actin forward 5′-TGA CGT GGA CAT CCG CAA AG-3′ and reverse 5′-CTG GAA GGT GGA CAG CGA GG-3′.
Western Blot analysis
Total protein TU686 and TU212 cells with different treatments were lysed with RIPA buffer (Pierce, Rockford, USA). And BCA Protein Assay Kit was performed to measure the concentration of protein. Proteins with an equal amount (100 µg/sample) were electrophoresed by 10% SDS-PAGE for 2 h and transferred onto 0.22um PVFD membranes (Millipore, MA, USA). Incubating membranes with primary antibodies which included anti-ROCK1 (1:2000, Cell Signaling Technology), anti-p-FAK (1:2000, Cell Signaling Technology), anti-FAK (1:2000, Cell Signaling Technology) and GAPDH (1:5000, Abcam). After membranes incubated for all night at 4 degrees Celsius. Then incubating membranes with secondary antibody (1:5000, Cell Signaling Technology) at 4 degrees Celsius for 2 h, the proteins were visualized using enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ, USA).
Plate colony formation assay
Seeding TU686 and TU212 cells with different treatments into 6-well plates at 1×103 and 2×103 cells/well. Cells were cultured in DMEM with 10% FBS for 3 weeks, washed twice with PBS and stained with crystal violet for 30 min. Cell colonies were counted in every well.
Apoptosis levels using Annexin V/PI staining
TU686 and TU212 cells with different treatments were treated with celastrol (1, 2 and 4 µM) for 48 h, then cells were harvested and washed with PBS for 3 times. Then TU686 and TU212 cells with different treatments were resuspended with 1X binding buffer prior to staining with Annexin V for half an hour at 37 degrees Celsius in the dark. TU686 and TU212 cells with different treatments were then double-stained with PI for 30 min at 37 degrees Celsius in the dark. The apoptotic TU686 and TU212 cells with different treatments were quantitatively counted by a flow cytometer.
In Vitro cell migration and invasion assays
2×105 TU686 and TU212 cells with different treatments after for all night starvation were plated in the coated filters in 100 µl of serum-free medium. And 600µl of medium containing 10% FBS was added to the lower chamber. The insert chambers’ membrane was coated by Diluted Matrigel (BD Biosciences) for measuring the cells invasion. TU686 and TU212 cells with different treatments were counted under a high-power objective (10x) in random fields. For migration assays, the upper chamber membranes were plated on top of uncoated (Matrigel-free) filters.
Animal experiments
Animals were obtained from the Institute of Zoology, Chinese Academy of Sciences. Animal experiments were carried out in accordance with the Guidelines for The Care and Utilization of Experimental Animals issued by the Institute of Experimental Animal Research. The following protocols had been approved by the medical center IRB. Animal care and treatment were conducted in accordance with NIH guidelines for the Care and Utilization of Laboratory Animals. At the end of the experiment, an overdose of sodium pentobarbital (4%, 200 mg/kg; Sigma, Shanghai, China) was performed to kill the animals by intraperitoneal injection. And lung specimens were collected from each group for further analysis.
In vivo metastasis
A 4-week-old male immunodeficient mouse reared at the Animal Resource Facility of Shanghai Jiao Tong University School of Medicine. The animal care and experiment were carried out in accordance with the Guidelines for “The Care and Utilization of Experimental Animals” and “The Principles for the Care and Utilization of Vertebrates”, and approved by the Experimental Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. Experimental animals were grouped according to the randomization formula. In the allocation process, researchers did not understand the group allocation at different stages of the experiment, the experimental progress, the results evaluation, and the data analysis. The mice were randomly divided into groups (5 in each group): TU686 and TU212 cells with different treatments were inoculated via tail vein into the mice. After 6 weeks, H&E staining was performed to count pulmonary metastatic nodules. For IH staining, the target recovery solution was used to thermally induce epitope recovery in microwave for 6 min. At high magnification (×400), the number of tumors was calculated in the field.
Statistical analysis
Data were analyzed by Graph-Pad Prism 6 software and displayed by means ± SD. Student’s t test and one-way analysis of variance (ANOVA) were used to analyze the data and the significance level was set at P < 0.05.