We obtained RKO, HCT116 parental, and Oxaliplatin-resistant (HCT116/OXA) cell lines from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Eggenstein, Germany) with 10% fetal bovine serum (FBS; Gibco, Eggenstein, Germany) and 100 U/ml penicillin-streptomycin (Solarbio, Beijing, China) at 37°C and 5% CO₂.

Oxaliplatin was purchased from TargetMol (Shanghai, China), stored at −80°C, and dissolved in phosphate-buffered saline (PBS) (Solarbio, Beijing, China). HOXB8 antibody was obtained from Biorbyt (San Francisco, California, USA). The antibodies against phospho-STAT3 (p-STAT3) and STAT3 were purchased from Cell Signaling Technology (Danvers, USA). A quantitative real-time polymerase chain reaction (qRT-PCR) kit was purchased from Takara (Shiga, Japan). TRIzol reagent and PCR primers were obtained from Invitrogen (Carlsbad, USA).

Cell proliferation was assessed by the MTT assay (Solarbio, Beijing, China). The cells (3–4 × 10³ cells/100 μl per well) were seeded in a 96-well plate overnight and then exposed to different concentrations of OXA (0.5, 1, 2.5, 5, 10, 20, 40, 60, and 80 µM) for 48 h. Subsequently, the MTT reagent was added to each well and incubated in a 5% CO₂ atmosphere in an incubator at 37°C. The culture medium was removed after 3 h, and 150 μL dimethyl sulfoxide (DMSO) was added into each well to dissolve the formazan. The absorbance (OD 490 nm) was measured using the SpectraMax iD3 Multi-Mode Microplate Reader (MD, USA).

The colony formation assay was done to assess the cell colony-forming ability. The cells (1500 cells per well) were incubated in a 6-well plate overnight. After treatment with various concentrations of OXA (0.1, 0.5, and 1 µM) for 24 h, the cells were continued to be cultured for 7–10 days at 37°C and 5% CO₂ in an incubator. Finally, the colonies were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies were counted using Image J software.

Negative control siRNA and siRNA against HOXB8 were synthesized from GenePharma (Shanghai, China). To knockdown HOXB8 in RKO, HCT116, and HCT116/OXA cell lines, HOXB8 siRNA was transfected into cells using Lipofectamine^TM 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Transfection efficiency was evaluated by qRT-PCR and western blot analyses. The negative control siRNA and HOXB8 siRNA sequences have been listed as follows: siHOXB8-1 (sense: 5′-CCUAUUUAAUCCCUAUCUGTT-3′ and antisense: 5′-CAGAUAGGGAUUAAAUAGGTT-3′); siHOXB8-2 (sense: 5′-UCAACUCACUGUUCUCCAATT-3′ and antisense: 5′-UUGGAGAACAGUGAGUUGATT-3′); negative control (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense: 5′-ACGUGACACGUUCGGAGAATT-3′).

The HOXB8 overexpressing vector and empty lentiviral vector were used to establish HOXB8 overexpressing colon cancer cells as described previously (Lin et al., 2015). The HOXB8 overexpressing vector, pVSV-G vector, and pGag/Pol vector were transfected into 293T cells to produce lentiviral particles using Lipofectamine^TM 3000 reagent (Invitrogen) according to the manufacturer’s instruction. After 48 h, the viruses were collected and the target cells were infected with 10 μg/mL Polybrene to establish stable HOXB8 overexpressing cell lines. The culture media was replaced after 8 h. The transfection efficiency was evaluated by western blot and qRT-PCR analyses.

Total RNA was isolated from cells using the TRIzol reagent. The RNA integrity and quantity were evaluated in the NanoDrop One Spectrophotometer (Thermo Fisher Scientific, USA). The total RNA was reverse transcribed into the cDNA using the PrimeScript^TM RT Master Mix (Takara, Shiga, Japan). Subsequently, RT-PCR was performed using the Mastercycler ep realplex Real-time PCR system (Eppendorf, Germany) with the TB Green Premix Ex Taq^TM II reagent (Takara, Shiga, Japan). The relative expressions of the mRNA molecules were normalized using the 2^−ΔΔCT method (Livak and Schmittgen, 2002). The primers used for qRT-PCR analysis have been listed as follows: HOXB8 (Forward: 5′-TAAGCGGCGATTCGAGGTAT-3′, Reverse: 5′-TGTTTCTCCAGCTCCTCCTG-3′); GAPDH (Forward: 5′-TCAAGGCTGAGAACGGGAAG-3′, Reverse: 5′-GACTCCACGACGTACTCAGC-3′).

The total protein was extracted from cells by using RIPA lysis buffer and the protein concentration was measured by using the Bradford method (Bio-Rad, Hercules, CA, USA). Equivalent amounts of proteins (80 μg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk for 1.5 h at room temperature and incubated with the primary antibodies HOXB8 (1:1000), p-STAT3 (1:1000), STAT3 (1:1000) overnight at 4°C. Subsequently the membranes were washed with Tris-buffered saline with 0.1% Tween® 20 detergent (TBST), and incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse or anti-rabbit (1:4000) secondary antibodies at room temperature for 1 h. The enhanced chemiluminescence (ECL) developing solution (Bio-Rad, Hercules, CA, USA) was used to visualize the protein bands.

All 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. The differences between different groups were assessed using the Student’s t-test or one-way analysis of variance (ANOVA). Statistical significance was fixed at a p-value < 0.05.

In order to confirm that HCT116/OXA was indeed resistant to oxaliplatin, we performed MTT assays for both HCT116 parent and HCT116/OXA cells after treatment with various concentrations of oxaliplatin. The results showed that oxaliplatin inhibited HCT116 cell growth in a dose-dependent manner with a half-maximal inhibitory concentration (IC₅₀) value of 6.45 µM. As expected, HCT116/OXA cells exhibited significant resistance to oxaliplatin (IC₅₀ > 80 µM) (Fig. 1A). Colony formation assays further showed that treatment with 1.5 µM oxaliplatin markedly inhibited the colony-forming ability of HCT116 cells, however, it almost had no effect on HCT116/OXA cells (Figs. 1B and 1C). These results suggested that HCT116/OXA cells are specifically and significantly resistant to oxaliplatin. Accumulating evidence has suggested that both HOXB8 and STAT3 were involved in drug resistance documented in several cancers like osteosarcoma, colorectal cancer, and non-small lung cancer (Lu et al., 2021; Gao et al., 2022; Ochi et al., 2022). Hence, we checked and compared the expression of HOXB8 and p-STAT3 in both HCT116 and HCT116/OXA cells. The expression of HOXB8 and p-STAT3 was markedly upregulated in HCT116/OXA cells compared with those in HCT116 cells (Figs. 1D and 1E). This suggested that HOXB8 and p-STAT3 were involved in oxaliplatin resistance in colon cancer cells.

To explore the effect of HOXB8 on oxaliplatin-mediated colon cancer cell growth inhibition, we knocked down HOXB8 by using two independent siRNAs in HCT116 and RKO cells. Both qRT-PCR and western blot analyses confirmed the knockdown efficiency. This was deduced as HOXB8 mRNA and protein levels were significantly decreased after transfection of HOXB8 siRNAs (Figs. 2A–2C). Further, HOXB8 knockdown markedly inhibited p-STAT3 expression in both HCT116 and RKO cells (Fig. 2C). HOXB8 knockdown inhibited colon cancer cell growth and potentiated oxaliplatin-mediated colon cancer cell growth inhibition (Figs. 2D and 2E). This suggested that the HOXB8-STAT3 axis is closely involved in oxaliplatin sensitivity in colon cancer cells.

To further investigate whether HOXB8 plays an important role in oxaliplatin resistance in colon cancer cells, we established stable HOXB8 overexpressing HCT116 (HCT116/HOXB8) and RKO (RKO/HOXB8) cells. We found that the expression of HOXB8 mRNA and protein was significantly upregulated in HCT116/HOXB8 and RKO/HOXB8 compared to corresponding control cells (Figs. 3A–3D). Further analysis showed that HOXB8 overexpression promoted colon cancer cell growth and significantly attenuated the growth inhibitory effect of oxaliplatin in both HCT116 and RKO cells (Figs. 3E and 3F). Similarly, HOXB8 overexpression increased the colony-forming ability of both HCT116 and RKO cells and impaired the inhibition of the colony-forming ability induced by oxaliplatin (Figs. 4A and 4B). As we hypothesized, HOXB8 overexpression significantly induced p-STAT3 expression (Figs. 4C and 4D), indicating the pivotal role of the HOXB8-STAT3 axis in oxaliplatin resistance in colon cancer cells.

We demonstrated in the previous sections that HOXB8 was involved in oxaliplatin resistance in colon cancer cells. To further confirm the critical function of HOXB8 in OXA-resistant colon cancer cells, we knocked down HOXB8 in HCT116/OXA cells. We observed downregulated levels of HOXB8 mRNA and protein by qRT-PCR and western blot analyses, respectively (Figs. 5A–5C). In lieu of our hypothesis, HOXB8 knockdown significantly reduced p-STAT3 expression in HCT116/OXA cells (Figs. 5B and 5C). While OXA treatment (60 µM) did not inhibit HCT116/OXA cell proliferation, HOXB8 knockdown markedly suppressed HCT116/OXA cell proliferation (Fig. 5D). Importantly, the growth inhibitory effect of HOXB8 knockdown is similar between HCT116 and HCT116/OXA cells, suggesting that targeting HOXB8 might be effective not only for CRC cells but also for OXA-resistant CRC cells.