cell culture
Human-derived colorectal cancer cell lines HCT-116, CACO-2, and SW48 were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai,China).HCT-116 was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, Eggenstein, Eggenstein) and 100 U/ml penicillin/streptomycin ( Solarbio, Beijing, China) in RPMI-1640 medium. both SW48 and CACO-2 cells were maintained in DMEM with the same supplements. All cells were kept in a humidified incubator at 37°C containing 5% CO2. When cell fusion reached approximately 80–100%, cells were collected and passaged with 0.25% trypsin for the following experiments.
Reagents and antibodies
Cetuximab was obtained from Merck KGaA, Germany. HOXB8 antibody was obtained from biorbyt (San Francisco, CA, USA). Phosphorylated STAT3 (p-STAT3), STAT3 antibody, and GAPDH antibody were purchased from Cell Signaling Technology (Danvers, USA).Quantitative immediate polymerase chain reaction (qRT-PCR) kit was purchased from Takara (Shiga, Japan).TRIzol reagent and PCR primers obtained from Invitrogen (Carlsbad, USA).
Cell proliferation assay
Cell proliferation was assessed by MTT assay (Solarbio, Beijing, China). According to pharmacokinetics, the maximum in vivo plasma concentration Cmax Mean (SD) = 306 (63) µg/ml when cetuximab was administered at a dose of 500 mg/m2 every 2 weeks [21]. HCT-116, CACO-2, and SW48 cells were inoculated in 96-well plates (6000 cells per well for HCT-116, CACO-2, and 4000 cells per well for SW48) overnight, and then exposed to different concentrations of cetuximab (cetuximab concentrations for HCT-116, CACO-2 cell lines were: 0, 10, 50, 100, 200, 400, 800, 1000, 2000 µg/ml, and cetuximab concentration for SW48 cell line: 0, 10, 20, 40, 80, 100, 150, 200, 300 µg/ml) for 48 hours. Subsequently, MTT reagent was added to each well and incubated in a 5% CO 2 incubator at 37°C. After 4 h, the medium was removed and 150 µl dimethyl sulfoxide (DMSO) was added to each well to break down the formaldehyde dimethyl sulfoxide. The absorbance of OD 490 nm was measured using a Multi-function Enzyme Labeler iD3 (MD, USA).
colony formation assay
Cell colony forming ability was detected by colony formation assay. HCT-116, CACO-2, and SW48 were spread on 6-well plates (1000 cells per well) and then incubated overnight. After treatment with different concentrations of cetuximab (0, 10, 20, 40, 80, 100 µg/ml) for 48 h, the cells were continued to be incubated at 37°C and 5% CO2 incubator for 7–10 days. Finally, colonies were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The colonies were counted using Image J software.
Wound healing test
Cell migration motility was tested by wound healing assay. HCT-116, CACO-2, and SW48 were grown in 6-well plates and cultured as confluent monolayers. Cells were carefully scraped using a 10 µl pipette tip and debris was removed by washing with 1× PBS. Cell migration was assessed after treatment with different concentrations of cetuximab (0, 10, 50, 100, 200, 300 µg/ml) for 48 h and changes in migration size were observed with an inverted microscope.
HOXB8 small interfering (si)RNA transfection
Negative control siRNA (NC-siRNA) and siRNA against HOXB8 were synthesized from GenePharma (Shanghai, China).To knock down HOXB8 in the HCT-116, CACO-2 cell line, LipoRNAi (Beyotime, China) was used to transduce the HOXB8 siRNA was transfected into cells. Transfection efficiency was evaluated by qRT-PCR and protein blotting analysis. The negative control siRNA and HOXB8 siRNA sequences were as follows: siHOXB8-1 (positive sense 5'-GUUCCUAUUUAAUCCCUAUTT-3', negative sense 5'-AUAGGGAUUAAAUAGGAACTT-3 '); siHOXB8-2 (justice 5'-GCAAUUUCUACGGCUACGATT-3', antisense 5'-UCGUAGCCGUAGAAAUUGCTT − 3 '); negative control siRNA (justice 5'-UCUUUCCGAACGUGUCACGUTT-3', antisense 5'-ACGUGACACGUUCGGAGAATT − 3 ').
Establishment of HOXB8 overexpressing cell lines
The SW48 cell line overexpressing HOXB8 was established using HOXB8 overexpression vector and empty lentiviral vector. To generate lentiviral particles, HOXB8 overexpression vector, pVSV-G vector and pGag/Pol vector were transfected into 293T cells with LipofectamineTM 3000 reagent according to the manufacturer's instructions (Invitrogen). virus was collected after 48 h, and the stable HOXB8 overexpression cell line was established by infecting the SW48 cells with 10 µg/mL polybrene. The culture medium was changed after 8 h. The transfection efficiency was evaluated by western blot and qRT-PCR analysis.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from cells using TRIzol reagent. the integrity and amount of RNA was assessed using NanoDrop One (Thermo Fisher Scientific, USA). Total RNA was reverse transcribed into cDNA using PrimeScriptTM RT Master Mix (Takara, Shiga, Japan). RT-PCR was then performed on a Mastercyker ep realplex from Eppendorf, Germany using TB Green Premix Ex taq™ II reagent (Takara, Shiga, Japan). 2−ΔΔCT method (Livak and Schmitgen 2002) was used to standardize the Relative expression of mRNA levels. Primers used for qRT-PCR analysis are shown below: HOXB8 (forward: 5'-TAAGCGGCGATTCGAGGTAT-3', reverse: 5'TGTTTCTCCAGCTCCTG-3') ; GAPDH (forward: 5'-TAAGCGGCGATTCGAGGTAT-3', reverse: 5'TGTTTCTCCAGCTCCTG-3') ; and GAPDH (forward:5'-TCAAGGCTGAGAACGGGAAG-3' reverse:5'-GACTCCACGACGTACTCAGC − 3').
Western blot analysis
Total proteins were extracted from the cells with RIPA lysis buffer, and protein concentration was determined by the Bradford (Bio-Rad, Hercules, CA, USA) method. Equal amounts of protein (80 µg) were obtained by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were closed with 5% skimmed milk for 1.5 h at room temperature and incubated with primary antibodies (HOXB8 (1:1000), p-STAT3 (1:1000), STAT3 (1:1000)) at 4°C overnight. The membranes were then washed with TBST and incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse or anti-rabbit (1:4000) secondary antibodies for 1 h at room temperature. Enhanced chemiluminescence (ECL) development solution (Bio-Rad, Hercules, CA, USA) was used to visualize protein bands.
Statistical analysis
The data were presented as mean ± standard deviation (SD) from three independent assays. All results were analyzed using GraphPad Prism 8.0 and SPSS 21.0 software via the Student’s t-test or one-way analysis of variance (ANOVA) between different groups. Statistically significant differences were considered to exist at a p value < 0.05.