2.1 Cell culture and treatment

CRC cell lines SW620 and HCT116 accessed from BeNa Culture Collection (BNCC, Beijing, China) were severally nourished by Dulbecco's modified Eagle medium (DMEM; Sangon Biotech, Shanghai, China) and Roswell Park Memorial Institute (RPMI)-1640 medium (Sangon Biotech, Shanghai, China) enhanced with 10% fetal bovine serum (FBS; PAA Clone, Coelbe, Germany) in a water-saturated atmosphere equipped with 5% CO2 at 37 °C. Additionally, both cells were treated by butorphanol (Cayman Chemical, Ann Arbor, MI, USA) at varying doses (0, 2.5, 5, 10, 20, 40 and 80 μg/ml).

2.2 Gene overexpression

With reference to Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), cells were treated with the SIGMAR1 adenovirus (Ov-SIGMAR1) and control adenovirus (Ov-NC) gained from Vigenebio (Shandong, China), abiding by the kit recommendations.

2.3 Cell counting kit-8 (CCK-8)

Cells sowed into the 96-well flat-bottomed plate (5 × 103 cells/well) were administrated with ascending doses of butorphanol (0, 2.5, 5, 10, 20, 40 and 80 μg/ml), each well of which was subsequently given 10 μl CCK8 working liquid (Abmole Bioscience, USA) and left for 2 h. Under a microplate reader, the absorbance reading was implemented at 450 nm.

2.4 5-Ethynyl-2′-deoxyuridine (EDU) staining

Cell proliferation was assayed sticking to the protocol of the EdU Labeling Kit (Sangon Biotech, Shanghai, China). After the treatment of cells distributed into 96-well plates (1.5 × 104 cells/well) with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), they were processed with pre-warmed complete medium nourished with 10 µM EDU working solution for 1 h. Afterwards, cells were exposed to 4% paraformaldehyde for half an hour and 0.5% Triton X-100 for 10 min for separate immobilization and perforation. Following being covered with reaction buffer and dyed by DAPI, the viable cells were observed by employing a fluorescence microscope.

2.5 Wound healing assay

Following cells dispensed into 6-well plates (5 × 105 cells/well) were treated with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), the longitudinal cell-free regions were formed in the confluent monolayer by means of a micropipette tip. The wound closure extent was recorded at predetermined time intervals under a light microscope.

2.6 Transwell assay

Transwell compartments were paved with Matrigel for invasion assay. After being treated with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), a total of 5 × 104/well cells suspended in FBS-deprived media and 500 μl medium were severally drawn into the upper and bottom compartments. The membrane of the upper level was exposed to methanol and dyed by 0.5% crystal purple. The transmigrated cells were photographed under a light microscope.

2.7 Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL)

After the treatment of cells distributed into 96-well plates (4 × 104 cells/well) with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), they were exposed to 4% paraformaldehyde for immobilization. Following being mixed with 50 μl TUNEL working buffer (Yeasen, Shanghai, China) for 1 h shielded from light, cells were processed with 100 μl reaction buffer. After DAPI nuclear staining, the stained apoptotic cells were visible under a fluorescence microscope.

2.8 Western blot

Through RIPA buffer (Real Times, Beijing, China), the protein samples were prepared for quantification based on the BCA method (Real Times, Beijing, China). Then, PVDF membranes that were to transfer the proteins resolved by 10% SDS-PAGE were closed with 5% BSA and treated by primary antibodies overnight at 4 °C and goat anti-rabbit HRP antibody for 1 h. The interaction was monitored with the RealECL reagent (Real Times, Beijing, China).

2.9 Statistical analyses

The statistical data plotted utilizing GraphPad Prism 8 software (GraphPad Software, Inc.) were denoted as the mean ± standard deviation. The statistical significance of the difference among means were determined by analysis of variance (one-way) with Tukey’s test. The normality of the data was tested using the Shapiro–Wilk test. The mean values were acknowledged to possess significance in statistics when p ≤ 0.05.