Tissue samples were obtained from 6 Chinese patients who underwent surgical resection for primary EC and para-tumor normal endometrial tissues at The Second Affiliated Hospital of Zhejiang University (Hangzhou, China) between 2014 and 2016. None of the patients had received preoperative treatments such as irradiation or chemotherapy. Written informed consent was obtained from all patients and the study was approved by The Second Affiliated Hospital of Zhejiang University ethics committee. Small pieces (~0.5 cm³) were cut and washed briefly in sterile PBS to remove blood contamination. All the samples were frozen within 20 min of delivery and stored in liquid nitrogen for western blotting and quantitative PCR analysis. The clinical pathological data of all patients are summarized in Table I.

Δ⁹-tetrahydrocannabinol (THC, 10 mg/ml in ethanol) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and store at −20°C.

Briefly, total cellular RNA was extracted using TRIzol^® (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the supplier's instructions. cDNA was generated using 1 mg total RNA and a QuantiTect Reverse Transcription kit (Qiagen, Berlin, Germany). qPCR was performed using SYBR-Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA) methods. The primer sequences for qPCR analysis are shown in Table II.

For analysis of CB₁R, CB₂R, E-cadherin, N-cadherin, Vimentin (VIM), MMP-2, MMP-9 expressions, tissues and cells were lysed in RIPA Lysis buffer, then homogenized by vigorous mixing for 30 min on ice, and centrifuged at 12,000 × g for 30 min. Total protein concentration was measured using the bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc., Bonn, Germany). Proteins were separated on a 10% sodium dodecyl sulfate polyacylamide gel. Following transfer to PVDF membrane and blocking with 5% non-fat milk powder, blots were probed with specific antibodies CB₁R (1:1,000; Abcam, Cambridge, UK), CB₂R (1:1,000; Abcam), E-cadherin (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), N-cadherin (1:1,000; Cell Signaling Technology, Inc.), VIM (1:1,000; Cell Signaling Technology, Inc.), MMP-2 (1:1,000; Abcam), MMP-9 (1:1,000; Abcam) and GAPDH (1:5,000; Abcam) at 4°C overnight. Subsequently, membranes were washed and incubated with anti-rabbit IgG (1:5,000; Cell Signaling Technology, Inc.) or anti-mouse IgG (1:5,000; Cell Signaling Technology, Inc.). Ultimately, proteins were visualized using the enhanced chemiluminescence reagents (Thermo Fisher Scientific, Inc.), and the relative expression of CB₁R and CB₂R, protein levels were analyzed by densitometry using the Image-J imaging analysis software (National Institutes of Health, Bethesda, MD, USA).

MMP-9 level was measured in supernatants from cells treated with for 24 h. Protein levels in the supernatants were assayed using a MMP-9 ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) following the manufacturer's instruction. Optical density was measured at 450 nm. MMP-9 concentration was calculated by comparing the data to the known standards for MMP-9 proteins.

The EC cell lines HEC-1B, and An3ca were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM)-F12 (Gibco; Thermo Fisher Scientific, Inc., Auckland, New Zealand) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO₂.

Cells were seeded in 96-well plates (2,000 cells/well) in 100 µl of DMEM-F12 medium. Then the medium was changed to one that contained different dose of 0.1–20 µM THC for 24 h, and 20 µl MTS reagent (Promega Corporation, Madison, WI, USA) was added to each well before incubation at 37°C for 2 h. The absorbance at 450 nm was measured using a SpectraMax 190 microplate reader (Bio-Rad Laboratories, Inc.).

Cells were suspended in serum-free DMEM-F12 medium and plated at a density of 5×10⁴ cells/well in transwell chambers equipped with 8.0 µm pore polycarbonate membranes (Corning Incorporated, Corning, NY, USA). Complete medium (800 µl) was added to the lower chamber. After incubation for 16 h, fluorescent stain (calcein-AM) was added to each chamber and incubated for 30 min. Then, the cells that migrated to the basal side of the membrane were counted at 100× magnification by fluorescence analysis (Nikon Corporation, Tokyo, Japan).

Oligonucleotides for human MMP-9, CB₁R, CB₂R siRNA kit were purchased from GenePharma (Shanghai, China). The kit contains three predesigned duplexes targeting a specific MMP-9 gene. Cells were transfected with MMP-9, CB₁R, CB₂R siRNA or NC using the opti-MEM plus X-treme GENE siRNA transfection reagent (Roche, Mannheim, Germany) according to the instruction. Stably infected cells were selected and processed for further analysis by western blotting. After 48 h of post-transfection, western blot analyses were further performed. For gene overexpression, the recombinant lentiviruses carrying MMP-9 or control were obtained as gifts from colleague and used according to the manufacturer's protocol. Briefly, HEC-1B cells were seeded at 2×10⁵ cells/well in a 6-well plate. After adherent cells reached ~40% confluence, they were infected with an LV-MMP-9 or a control supplemented with 8 µg/ml polybrene (Sigma-Aldrich; Merck KGaA). Treated cells were selected with puromycin to generate puromycin-resistant clones, which were assayed by qPCR and western blotting.

All data were expressed as the mean ± the standard error (SEM) and analyzed using the SPSS 19.0 statistical analysis software (SPSS, Inc., Chicago, IL, USA). Statistical significance was determined using an unpaired Student's t-test and one-way ANOVA followed by Dunett's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Cannabinoid receptors are overexpressed in different cancers, including skin, breast and lung cancers (11–13). Nevertheless, so far as we are concerned that none work has been indicated to present the expression of cannabinoid receptors in EC. Therefore, we first investigated CB₁R and CB₂R expressions in normal endometrium (6 samples) and paired adjacent normal tissues (6 samples) using PCR and western blot. These data indicated both CB₁R and CB₂R were overexpressed in EC tissues compared with the normal endometrium (Fig. 1A and B). Moreover, the expressions of CB₁R and CB₂R were positively correlated with VIM, the marker of mesenchymal cells (Fig. 1A and C).

In the present study, the effect of THC on cell proliferation was analyzed in EC cell lines HEC-1B and An3ca cells. Fig. 2A showed that THC acted as a concentration-dependent inhibitor of cell growth. Tumor growth and metastasis are the leading causes of cancer-related mortality, specifically, tumor cells can migrate from the site of primary tumor and then invade neighboring tissues. To determine the role of THC in EC progression, the transwell assay was designed to test whether the treatment of THC altered the locomotive potential of tumor cells. After 16 h of incubation, THC resulted in a significant decrease in cell migration (Fig. 2B and C). In conclusion, THC obviously suppressed the proliferation and migration capabilities of HEC-1B and An3ca cells.

Tumor EMT, a key step for tumor progress, is always defined as specific phenotypic and morphological alterations in epithelial cancer cells, causing them to transform into mesenchymal type cells during metastasis in many cancers (14). In tumor EMT, epithelial molecular markers, including E-cadherin, β-catenin are downregulated, whereas the levels of mesenchymal molecular markers, including N-cadherin, VIM are upregulated (15), we also get the similar result that VIM was elevated in EC tissues (Fig. 1B). In our study, the expressions of EMT protein markers were evaluated to explore the potential relationship between THC and EMT. In the THC-treated HEC-1B and An3ca cells, the level of epithelial cell marker (E-cadherin) was increased, while the mesenchymal cell markers (N-cadherin, and VIM) were both decreased, as determined by western blot analysis and qPCR (Fig. 3A and B) assay. This evidence suggested that THC plays crucial roles in tumor EMT.

To elucidate the mechanisms by which THC is engaged in the metastasis of EC cells, we detected the level of MMPs after treated with THC. MMPs are functionally related to tissue remodeling processes, where MMP-2 and MMP-9 can be utilized as catalytic in these processes in vivo. Recent researches have been tried to demonstrate the molecular basis and pathophysiology of EC, MMP-9 plays important roles in invasion and metastasis by regulating the signaling pathways that control cell growth and invasion (16). In our study, we also observed THC significantly inhibited the secretion of MMP-9 in HEC-1B and An3ca cells by qPCR, western blot and ELISA assay, while the expression of MMP-2 was not significantly altered (Fig. 4A-C). This result supports the hypothesis that THC regulates EMT and the EC cell metastasis is mediated by MMP-9.

To further confirm that THC decreased EC cell mobility through MMP-9 signaling pathway, siRNA was used to silence the MMP-9 expression in HEC-1B and An3ca cells. The presences of HEC-1B and An3ca cells with MMP-9 silencing was verified by qPCR (Fig. 5A) and western blot analyses (Fig. 5B). Furthermore, the transfection with MMP-9 siRNA decreased the migration capacity of HEC-1B and An3ca cells, as shown by the transwell assay (Fig. 5C and D). In addition, the lentivirus of MMP-9 gene overexpression vector and control vector were infected into HEC-1B cells. Stably infected cells were used to perform cell migration assay (Fig. 5E and F). HEC-1B cells overexpress MMP-9 exhibited significantly higher migratory ability than the control group and also slightly reverse the THC-reduced cell migration (Fig. 5G). Furthermore, MMP-9 overexpression in HEC-1B cells can reverse the EMT protein markers which were changed by THC treatment (Fig. 5H). Overall, these data further indicated that MMP-9 mediates THC-reduced EMT and cell mobility in EC cells.

Previous studies (17,18) have shown that chronic THC administration causes downregulation of CBRs. Next, siRNA was used to decrease the active level of CB₁R and CB₂R, which may be the result of THC exposure (Fig. 6A). In addition, downregulation of CB₁R and CB₂R attenuated the MMP-9 expression in HEC-1B cell (Fig. 6B) and also inhibited the migration of HEC-1B cells (Fig. 6C). The data potentially revealed that THC signal transfer into cells through CB₁R and CB₂R to develop anti-tumor ability.