2.1 Patient samples

Clinical LMS tissues and their corresponding normal tissues were collected from 7 patients. The gathered specimens were rapidly frozen in liquid nitrogen and stored at a temperature of −80 °C.

An expert pathologist examined formalin-fixed and paraffin-embedded (FFPE) tissue blocks obtained from specimens including 32 patients with LMS and 7 patients with leiomyoma who underwent surgical treatment at our hospital. To construct tissue microarrays (TMA), 1 to 3 cores were acquired from viable tissue areas of each tissue block. TRPV4-specific antibodies were used to perform immunohistochemistry (IHC) on 4 μm tissue sections using the Leica Bond-Max autostainer platform. Sufficient cores were deemed suitable for analysis if there was a minimum of 25% of tumoral tissue area per spot available for scoring. The average optical density (AOD) of positive staining was measured by ImageJ software [24, 25]. All procedures of this study were approved by the Ethics Committee of Zhongshan Hospital, Fudan University (Approval Number: B2020-338).

2.2 IHC

IHC identified the presence of LMS tissues. To summarize, the slices underwent a process of wax removal and dehydration initially. The slices were immersed in citrate buffer and subjected to high temperature for a duration of 15 min, after which they were allowed to cool down to the ambient temperature. The slices were immersed in 3% H2O2 in the dark for a duration of 15 min. After being cleaned with PBS, the slices were incubated with 3% BSA at room temperature for 30 min. Then, incubation was performed with a primary antibody at 4 °C overnight. Afterwards, the secondary antibody that corresponds to it was combined and left to incubate for a duration of 1 h. The DAB (Servicebio, Wuhan, China) working solution was discarded and left to incubate at room temperature for 3–5 min. Following the removal of moisture using alcohol, the tablets were subsequently immersed in ethanol and xylene, hermetically sealed with neutral gum, and examined under a microscope.

2.3 The Cancer Genome Atlas (TCGA) dataset

TCGA is a comprehensive collection of genomic data related to cancer. Data on mRNA expression and survival were acquired from TCGA dataset (https://portal.gdc.com). The ggstatsplot package in R software was employed to visualize the relationship between TRPV4 expression and LMS patients’ survival.

2.4 Cell culture and transfection

Two human LMS cell lines, MES-SA cells and SK-UT-1 cells, were purchased from KeyGen Biotech (Nanjing, China) and Fenghui Biotechnology Co., Ltd (Hunan, China), respectively. FuHeng Biology provided the H8 human cervical epithelial cells. MES-SA cells were cultured in McCoy’s 5 A (Modified) Medium (KeyGen). SK-UT-1 cells were maintained in Minimum Essential Media (MEM; KeyGen). H8 cells were using Dulbecco modified Eagle medium (DMEM; KeyGen). All of the above-mentioned cells were grown in a humidified incubator at 37 °C, supplemented with 10% fetal bovine serum (Gibco, USA) and kept in an atmosphere of 5% CO2. Cells were used in the logarithmic growth phase.

MES-SA and SK-UT-1 cells were infected with the lentivirus vector by adding Hitrans G (GeneChem, Shanghai, China). The efficiency of inhibition or overexpression was evaluated by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB). The sequence targeting shTRPV4 (#1: 5′-GCTGGAGTCCACCCTATATGA-3′; #2: 5′-GCGAGGTCATTACGCTCTTCA-3′; #3: 5′-GGATGAATGCCCTTTACTTCA-3′) and a non-targeting control shRNA (TTCTCCGAACGTGTCACGT) were synthesized by Genechem. Transfection was performed using Lipofectamine 3000 (Thermo Scientific, Waltham, MA, Cat#L3000015) following the manufacturer’s guidelines.

2.5 Immunofluorescence (IF)

Cells at an appropriate density were grown in gelatin-coated coverslips in 24-well plates. Following the treatment, the cells were rinsed three times with PBS that was chilled on ice, then fixed by 4% paraformaldehyde (Servicebio) for 15 min. After blocking with 1% BSA, the cells were incubated overnight at 4 °C with a primary antibody. Next day cells were washed with PBS. After being incubated with a secondary antibody labeled with Alexa Fluor 594 (Yeasen, Shanghai, China) for 1 h, the cells were washed with PBS. The cells were then incubated with diluted DAPI (Beyotime, Shanghai, China) at room temperature in the dark for 10 min. Subsequently, the cells were washed with PBS. Finally, the coverslip was mounted on a slide using antifade mounting medium, and fluorescence microscope (Olympus, Tokyo, Japan) was used to capture images.

2.6 WB

Cells or tissues were washed using PBS and collected into RIPA buffer (Beyotime) containing protease (Beyotime) and phosphatase inhibitors (Beyotime). The protein was obtained by centrifuging at 12,000 rpm and 4 °C for 30 min, and its concentration was measured using a BCA Protein Assay Kit from Beyotime (Cat#P0012). The cell lysates (containing 20 µg of protein) were separated using 10% SDS-PAGE. Following standard procedures, proteins were transferred onto a PVDF membrane and subsequently western blotting was performed. The diluted primary antibodies were as follows: GAPDH (1: 100000, Proteintech, Hubei, China), TRPV4 (1:1000, Abcam, Cambridge, UK), ECM1 (1: 1000, Proteintech), AKT (1:1000, Cell Signal Technology, Boston, MA, USA), pAKT (1:1000, Cell Signal Technology), FAK (1:1000, Cell Signal Technology), pFAK (1:1000, Cell Signal Technology), PI3K (1:1000, Proteintech), pPI3K (1:1000, Abmart, Shanghai, China), GSK3β (1:1000, Cell Signal Technology), pGSK3β (1:1000, Cell Signal Technology), β-catenin (1:1000, Abcam). The antibodies were used overnight at a temperature of 4 °C. Following incubation with the secondary antibody, the protein bands were identified by utilizing the ECL substrate from Yeasen.

2.7 qRT-PCR

The Trizol reagent (Merck, Darmstadt, Germany) was used according to the manufacturer’s protocols to isolate Total RNA from LMS cells. The NanoDrop 2000 was used to assess the amount and integrity of RNA. The RNA was converted into cDNA using PrimeScript™RT reagent Kit (TaKaRa, Osaka, Japan) according to the instructions provided by the manufacturer. The qRT-PCR machine was used to detect the presence of TB Green® Premix Ex Taq™ (TaKaRa). All primers were purchased from Sangon Biotech (Shanghai, China), and the sequences are shown as follows: (1) TRPV4, forward: 5′-CAATGAACTGCTGCGGGACAAG-3′, reverse: 5′-GTAGTAGGCGGTGAGAGTGAAGATG-3′; (2) ACTB, forward: 5′-GGCACCACACCTTCTACAATGAGC-3′, reverse: 5′-GATAGCACAGCCTGGATAGCAAGG-3′.

2.8 Intracellular Ca2+ measurement

The treatment group was exposed to HC067047 [26, 27] (HC, a TRPV4 inhibitor, 10 µM, Selleck, Houston, Texas, USA) for 30 min. Subsequently, the Fluo-4 AM dye, functioning as Ca2+ probe (2 µM, Beyotime) was added to cells and incubated at 37 °C for 20 min. The cells were washed once with PBS and cultured in PBS for 20 min. The fluorescence intensity of intracellular Ca2+ in LMS cells was observed and recorded by a fluorescence microscope (Olympus, Tokyo, Japan) with cellSens software (Olympus).

2.9 Cell proliferation, cell cycle and migration assays

The growth of MES-SA and SK-UT-1 cells was assessed using a CCK-8 Kit (Beyotime) following the manufacturer’s instructions, with data collected at 450 nm (Thermo Fisher, Waltham, MA, USA). Colony formation was evaluated after 2 weeks of incubation in 6- or 12-well plates, followed by fixation with 4% paraformaldehyde, crystal violet staining, and colony counting.

Cells were fixed by 70% ethanol at 4 °C for over 18 h and spun down at 1000 rpm for 10 min. The cell pellets were washed twice with PBS. PI/RNase staining solution (500 µl, BD Biosciences, Franklin Lakes, New Jersey, USA) was added to each tube, mixed gently by pipetting, and then kept at room temperature in the absence of light for a duration of 15 min. The cells were subsequently examined with a flow cytometer (FACSCanto, BD Biosciences).

Cells were seeded in a 6-well plate in 10% FBS medium and grown to reach an 80–90% confluence. A scratch was made on the cell monolayer using a pipette tip, followed by rinsing with sterile PBS to remove debris. Reference points were then made on the plate’s outer bottom of the plate using a tip marker. Images were captured at 0 and 72 h using an Olympus microscope in bright field microscopy. The scratched area was measured with ImageJ [25], and wound-healing ability was assessed using the formula [(initial wound area) − (wound area at specified time)] / (initial wound area).

Cell migration and invasion were evaluated by performing Transwell assays. Transwell chambers (8.0 μm pore size, Merck) were prepared in 24-well plates with or without Matrigel matrix (Corning, Steuben County, New York, USA). Cells were placed in serum-free medium in the upper chambers, while 10% FBS medium served as a chemoattractant in the lower chambers. After 24 h of culture, cells were fixed with ethanol for 20 min, stained with crystal violet (Beyotime) for 30 min, and analyzed using an Olympus microscope. The number of cells in five random fields was counted.

2.10 Analysis of RNA-seq and transcriptome

SK-UT-1 cells treated with Vector or TRPV4-OE lentivirus were used as control and treatment group, respectively. Three sets of samples from each group were obtained for both RNA-seq and proteomics analyses. For transcriptome analysis, total RNA was extracted from the cells and sent to OE Biotech Co., Ltd (Shanghai, China) for sequencing using the Illumina Novaseq 6000 sequencer. Data analysis was conducted using the OE Cloud Platform. For proteomics analysis, the same cells were lysed with buffer and kept on ice for 30 min. After centrifugation at 12,000 rpm and 4 °C for 20 min, the supernatant was collected, and protein concentration was measured using a BCA kit. SDS-PAGE analysis was performed, followed by trypsin digestion and labeling of the proteins. The labeled samples were then mixed in equal amounts for chromatographic separation, and subsequently analyzed by LC-MS/MS for proteomics data analysis.

2.11 Animal experiments

BALB/c nude mice (male, 6–8 weeks old, 20–25 g, SPF) were obtained from SLAC Laboratory Animal Co (Shanghai, China). Matrigel and LMS cells suspension in PBS were mixed to form 200 µl of cell-embedded matrix gels, and animals were subjected to intraperitoneal injection experiments. Following the injection of transfected SK-UT-1 cells (1 × 107), the mice were divided into three groups. Following a period of 23 days, the mice were euthanized and the tumors were removed for monitoring of metastasis. Animal studies were carried out following applicable guidelines and regulations.

2.12 Enzyme-linked immunosorbent assay (ELISA) and conditioned medium (CM) collection

LMS cells were cultured for 24 h. Then after centrifugation at 3000 rpm for 10 min the supernatant was collected. ECM1 levels in the supernatant were measured by ECM1 ELISA kit (BYabscience, Nanjing, China). Each sample (50 µl) was incubated with 100 µL ECM1 antibody-HRP for 60 min at 37 °C. After washing five times, substrates A and B (50 µl each) were added and incubated at 37 °C in the dark for 15 min. 50 µl stop solution was added to the sample. OD values was acquired utilizing a multimode reader set at a wavelength of 450 nm.

Cells at 80% confluence were fed with Opti-MEM. After 24 h, the supernatant was collected into a tube as CM. The CM then mixed with the same volume of methanol and a quarter volume of chloroform and vortexed. After centrifugation at 13,000 rpm for 5 min, the aqueous phase was removed without disturbing the protein layer. With an additional volume of methanol added into the tube, a protein pellet forms at the site of maximal centrifugation force. Then 5× protein loading dye was added to the protein pellet for ECM1 detection by WB.

2.13 Statistical analysis

The data were presented as the average plus or minus the standard deviation (SD). The unpaired t-test was used to determine statistical significance in two-group experiments. To compare three or more groups, we utilized one-way analysis of variance (ANOVA). The correlation between two variables was measured by Pearson’s or Spearman’s rank correlation coefficient. Statistically significant results with a P value less than 0.05 were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001.