Ethics statement and patients
The Review Board of the Affiliated Hospital of Jiangnan University approved the clinical samples for research purposes (NO.LS2018020). This study was confirmed to the principles contained in the World Medical Association Declaration of Helsinki. Informed consent was requested as anonymous specimens and was given by all human participants in this study. The human colorectal cancer samples (n = 106, without chemical treatments, paraffin embedding tissues) were randomly collected from Affiliated Hospital of Jiangnan University. Patients were recruited between 2010 and 2012.
Cell culture
The human colon cancer cell lines SW620 was purchased from the American Type Culture Collection (ATCC, VA, USA). HCT-116 and HT-29 were purchased from the Chinese Academy of Sciences cell bank (CBCCCAS, Shanghai, China). Cell lines were maintained in McCoy’s 5A or Leibovitz’s L-15 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific, MA, USA). Cells were cultured at 37 °C with 5% CO2 in a humidified incubator.
RT-qPCR analysis
Total RNA was isolated by Trizol (Invitrogen, CA, USA). Reverse transcription polymerase chain reaction (RT-PCR) was performed with the PrimeScript™ RT reagent kit (Takara, China) following the manufacturer’s instructions. qPCR was performed using QuantiNova SYBR Green PCR Master Mix (QIAGEN, Hilden, Germany) in the LightCycler 480 Real Time PCR system (Roche, Basel, Sweden). Primer sequences are listed in the supplemental Table S1.
siRNA transfection
Cells in 12-well plate were transiently transfected with 25 nM siRNA using DharmaFECT 1 Transfection Reagent (GE Dharmacon, CO, USA) according to the manufacturer’s instructions. Briefly, adding 5 μL of 5 μM siRNA to 95 μL of serum free medium in tube 1. Adding 5 μL of DharmaFECT 1 Transfection Reagent to 95 μL of serum free medium in tube 2. Gently mix and incubate for 5 min. Then add the contents of tube1 to tube 2, and add 800 μL of antibiotic-free complete medium for a total volume of 1000 μL transfection medium. TRPV4 silencing was performed using siRNAs targeting the following sequences: siRNA#1: 5′-AUCUUGGUAACAAACUUGG-3′, and siRNA#2: ON-TARGET plus SMART pool against human TRPV4 siRNA (GE Dharmacon, CO, USA). ZEB1 silencing was performed using siRNA targeting the following sequences: 5′-CAGUGUUCCAUGCUUAAGA-3′. AKT silencing was performed using siRNA targeting the following sequences: 5′-GCACCUUC AUUGGCUACAATT-3′.
Plasmid transfection
The plasmid carrying human TRPV4 (GenBank NM_021625.4) was obtained from GeneCopoeia, Inc. Cells were transfected with the TRPV4 plasmid (TRPV4) or Vector using Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CO, USA) according to the manufacturer’s instructions.
Western blot analysis
Western blots were performed as previously described [17]. Primary antibodies against ACTB (Santa, TX, USA), TRPV4 (alomone labs, Israel), ZEB1, E-cadherin, N-cadherin and Vimentin (Cell Signaling Technology, MA, USA) were incubated at 4 °C overnight with constant shaking. HRP labeled secondary antibodies (Cell Signaling Technology, MA, USA) were incubated at room temperature for 1 h.
Wound healing assay
The wound healing assay was used a monolayer denudation assay as described previously [18]. Briefly, cells cultured in 12-well plates as confluent monolayers were mechanically scratched using a 200 μL pipette tip to create the wound. Cells were washed with PBS to remove the detached cells and then cultured to allow migration. Photographs were taken and the percent of wound closure was calculated.
Migration and invasion assays
Cell migration assay was determined using the transwell chamber (Corning) as described previously [18]. Briefly, cells were harvested, washed, resuspended and then seeded into the upper chamber in serum-free medium. The medium containing 10% FBS was placed in the lower chamber and the cells were further incubated for 24 h, cells in the upper chamber were removed with a cotton swab, and the rest of the membrane had invaded the cells. Cells migrated through the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. Cell invasion was determined with Matrigel matrix (BD Biosciences) coated on the upper surface of the transwell chamber. Cells were seeded, fixed and stained as described in migration assay.
Immunohistochemistry staining
Immunohistochemistry staining was performed as previously described [19]. Primary antibodies against TRPV4 (ACC-034, alomone labs, Israel) and ZEB1(#70512, Cell Signaling Technology, MA, USA) were used. All staining was assessed by pathologists blinded to the origin of the samples and patient outcomes. The widely-accepted German semi-quantitative scoring system was used to assess the staining intensity and proportion of stained cells. Each specimen was assigned a score according to the intensity of staining (0, none; 1, weak; 2, moderate; 3, strong) and the proportion of stained cells (0, 0%; 1, 1–24%; 2, 25–49%; 3, 50–74%; 4, 75–100%). The final score for immunoreactivity was determined by multiplying the intensity by the proportion, ranging from 0 to 12.
Statistical analyses
Statistical analysis was performed using two-tailed Student’s t-test or one-way ANOVA. All analyses were carried out with the GraphPad Prism software version 5.0 (GraphPad Software, CA, USA). Data were expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. The difference was considered significant if p value< 0.05.