Cell culture and transfection

Normal human colon mucosal epithelial cell line (NCM460), colon cancer cell lines (HCT116, DLD1, SW620, HT29, and SW480), and human embryonic kidney cell line (HEK293T) were purchased from the Shanghai Institute of Cell Research, Chinese Academy of Sciences. NCM460, DLD1, SW620, HT29, and SW480 were cultured in DMEM medium (Solarbio Science & Technology Co., Ltd, Beijing, China) contained 10% fetal bovine serum (FBS, ExCell Bio, Suzhou, China), 100 U/ml streptomycin and 100 U/ml penicillin (Solarbio Science & Technology Co., Ltd, Beijing, China). HCT116 and HEK293T were cultured in RPMI 1640 medium (Solarbio Science & Technology Co., Ltd, Beijing, China) contained 10% FBS, 100 U/ml streptomycin and 100 U/ml penicillin. All cell lines were incubated in a constant temperature incubator set at 37℃ and with a CO2 concentration of 5%.

Lentiviral vectors for knockdown (sh-OTUB2#1, sh-OTUB2#2, sh-SP1, and sh-GINS1) and overexpression (OTUB2, SP1, and GINS1) were purchased from Hanbio (Shanghai, China). FLAG-OTUB2, HA-SP1, and MYC-ubiquitin (wild type, K6R, K11R, K27R, K29R, K33R, K48R, K63R, and K48) plasmids were purchased from Genechem (Shanghai, China). The transfection procedures for all lentiviral vectors and plasmids were conducted in strict adherence to the manufacturer’s prescribed protocols. And the sequences of all shRNAs were presented in the Table S1.

Establishment of tumor stem cells and oxaliplatin-resistant cells

For the establishment of tumor stem cells, colon cancer cells (HCT-116 and DLD1) were inoculated onto ultra-low adhesion 6-well culture plates (Corning, Glendale, Arizona, USA) at a density of 5 × 103 cells per well. The cells were cultured in serum-free DMEM/F12 medium (Gibco, Grand Island, New York, USA) supplemented with B27(2%, Gibco, Grand Island, New York, USA), EGF (20 ng/ml, PEPROTECH, Rocky Hill, USA), and bFGF (20 ng/ml, EPROTECH, Rocky Hill, USA). After 7–14 days of culture under these conditions, tumor stem-like cell spheres formed uniformly and densely. These spheres were collected for further culturing to obtain a large number of tumor stem cells.

For the construction of oxaliplatin-resistant cells, the colon cancer cells were exposed to oxaliplatin at initial concentrations equivalent to 1/5 of the IC50 value in parent cells (HCT-116 and DLD1) for a duration of 48 h. Subsequently, the medium was discarded, and the cells were washed twice with PBS before being replenished with drug-free normal medium. Once the cells resumed normal growth, this process was repeated eight more times. Following stable cell growth at this particular drug concentration, the oxaliplatin dosage was gradually increased for further culturing. The drug concentration of oxaliplatin gradually increased until the resistance index (RI) exceeded 5 (RI = IC50 value in oxaliplatin-resistant cells / IC50 value in parent cells), signifying the successful establishment of oxaliplatin-resistant colon cancer cell lines.

Quantitative real-time PCR (qRT-PCR)

The extraction of total RNA from cells or tissues was performed using the Trizol method, followed by reverse transcription into cDNA (TransGen Biotech, Beijing, China). The cDNA was utilized for real-time quantitative PCR analysis (TaKaRa, Biomedical Technology Co., Ltd, Beijing, China). The 2− ρρCt method was employed for data analysis. The primer sequence is provided in Table S2.

Western blotting

The total protein was extracted from cells or tissues using RIPA supplemented with 1% PMSF and 1% phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). The protein samples were subjected to electrophoresis on a 10% SDS-PAGE gel, followed by transfer onto a PVDF membrane (Merck Millipore Ltd, Germany). The PVDF membrane was blocked with 5% skim milk for 2 h. Subsequently, the PVDF membrane was incubated with the specific primary antibody at 4℃ for a duration of 12 h. Finally, the PVDF membrane was incubated with the secondary antibody (1:10000, Proteintech, Wuhan, China) for 1 h and then subjected to exposure using BIO-RAD gel electrophoresis imaging system. All primary antibodies employed in Western blotting were as follows: anti-OTUB2(1:1000,Affinity), anti-SP1(1:2000, Proteintech), anti-GINS1(1:10000,Abcam), anti-CD44 (1:1000, Affinity), anti-CD133 (1:1000, Affinity), anti-NANOG (1:1000, Affinity), anti-OCT4 (1:2000, Proteintech), anti-SOX2 (1:5000, Proteintech), N-cadherin (1:2000, Proteintech), E-cadherin (1:10000, Proteintech), Vimentin (1:2000, Proteintech), anti-GAPDH (1:10000, Proteintech), anti-β-tubulin (1:5000, Proteintech).

Immunohistochemistry (IHC)

The tissue was embedded in paraffin blocks and sectioned into 4-micron-thick slices. The tissue sections were incubated with primary specific antibodies followed by incubation with an HRP-conjugated secondary antibody. The specimen was subsequently subjected to DAB staining followed by hematoxylin restaining, and ultimately captured under a microscope. All primary antibodies employed in immunohistochemistry were as follows: anti-OTUB2(1:50, Affinity), anti-CD44 (1:50, Affinity), anti-CD133 (1:50, Affinity), anti-Ki67 (1:2000, Proteintech), anti-PCNA (1:1500, Proteintech), anti-ubiquitin (1:200, Santa Cruz).

Immunofluorescence staining assay

The 24-well culture plate was inoculated with 2 × 104 colon cancer cells that were treated differently, and then incubated at 37 °C for 24–36 h. Then, the adherent cells were washed three times with PBS solution and subsequently fixed with a 4% paraformaldehyde for a duration of 20 min. Subsequently, at room temperature, the attached cells were immersed in 0.5% Triton X-100 solution for 20 min and then incubated with 5% goat serum for 30 min to mitigate non-specific binding. Then, anti-OTUB2(1:200, Affinity), anti-CD44(1:200, Affinity), anti-CD133(1:200, Affinity), anti-N-cadherin (1:100, Proteintech), anti-E-cadherin (1:100, Proteintech), anti-Vimentin (1:100, Proteintech) were added into each well and incubated at 4°C for 12 h. The labeled antibodies of various hues were added and incubated at ambient temperature for a period of 1 h, followed by staining the nucleus with DAPI for 5 min. Ultimately, fluorescence microscopy was employed for the purpose of imaging.

Subcutaneous xenograft assay

Forty female BALB/c nude mice that were 4 weeks old were purchased from Nanjing Kris Biotechnology Co., LTD, the forty BALB/c nude mice were randomly divided into four groups (NC-OTUB2, sh-OTUB2#1, Vector-OTUB2, and OTUB2). HCT116 cells (1 × 107) stably transfected with NC-OTUB2, sh-OTUB2#1, Vector-OTUB2, and OTUB2 were resuspended in 200µL PBS and subcutaneously injected into the armpit of BALB/c nude mice. After tumors reached approximately 70 mm3, each stable transfection group was subsequently randomly allocated into two subgroups: chemotherapy group (oxaliplatin injection) and placebo group (DMF injection). The chemotherapy group received intraperitoneal injections of oxaliplatin (5 mg/kg, every 3 days) for a duration of 3 weeks, while the placebo group received intraperitoneal injections of an equivalent volume of DMF as oxaliplatin within the same time frame. The tumor’s length and width were measured using a vernier caliper at 5-day intervals, and the tumor volume (V) was calculated using the following formula: V = 0.52 × length × width². After a period of 35 days following the inoculation of HCT116 cells, all nude mice were euthanized and the tumor mass was extracted for photographic analysis, H&E staining, and immunohistochemistry. The Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co,. Ltd has granted approval for all animal experiments in our research.

Tumor lung metastasis assay

Twenty female BALB/c nude mice that were 4 weeks old were purchased from Nanjing Kris Biotechnology Co., LTD, the twenty BALB/c nude mice were randomly divided into four groups (NC-OTUB2, sh-OTUB2#1, Vector-OTUB2, and OTUB2). HCT116 cells (2 × 106) stably transfected with NC-OTUB2, sh-OTUB2#1, Vector-OTUB2, and OTUB2 were resuspended in 100µL PBS injected into the caudal vein of BALB/c nude mice. After a period of 35 days following the inoculation of HCT116 cells, all nude mice were euthanized and the lungs of mice was extracted for H&E staining. The Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co,. Ltd has granted approval for all animal experiments in our research.

Sphere-formation assay

The colon cancer cells (1 × 103) were implanted in ultra-low adhesion 6-well culture plates (Corning, Glendale, Arizona, USA). The colon cancer cells were cultured in serum-free DMEM/F12 medium (Gibco, Grand Island, New York, USA) supplemented with B27(2%, Gibco, Grand Island, New York, USA), EGF (20 ng/ml, PEPROTECH, Rocky Hill, USA), and bFGF (20 ng/ml, EPROTECH, Rocky Hill, USA). After a duration of 14 days, microscopic photographs were captured and the quantity and size of the spheres were quantified.

Cell viability assay

The colon cancer cells (1 × 104) were seeded in 96-well culture plate and incubated at 37 °C for 8–12 h. Subsequently, the 96-well plates were supplemented with medium containing varying concentrations of oxaliplatin and incubated for 48 h. The medium was supplemented with 10 µL of cell counting kit 8 (UElandy, Suzhou, China) per 100 µl, followed by incubation at 37℃ for 2 h. Finally, the absorbance at 450 nm was measured using the microplate reader (Thermo Fisher Scientific, Waltham, USA). The cell viability rate (%) was determined using the formula: [(OD treatment - OD blank)/ (OD control - OD blank)] × 100%.

EdU assay

The 96-well plates were inoculated with 2 × 104 colon cancer cells and incubated at 37 °C for a duration of 8–12 h. Then, the culture was supplemented with EDU (UElandy, Suzhou, China) at a concentration of 50 µM and incubated for 2 h. The cells were subsequently fixed using a 4% paraformaldehyde solution and then neutralized with a glycine solution at a concentration of 2 mg/mL. Then, the cells were treated with a 3% BSA solution as a cell cleanser and stimulated with a 0.5% Triton X-100 solution as an osmotic enhancer. Ultimately, the cells were incubated in the dark with Click-iT working solution for 30 min and with 1×Hoechst 33,342 solution for 15 min respectively, followed by fluorescence microscopy imaging.

Flow cytometry

For apoptosis detection, the colon cancer cells were inoculated into 6-well culture plate. Once the cell density in each well exceeded 80%, the cells were gently washed with pre-cooled PBS and subsequently collected in flow cell tubes. Subsequently, the cells were treated with FITC-Annexin V and PI dyes (UElandy, Suzhou, China), followed by a 15-minute incubation in the dark. Finally, flow cytometry was employed for detection. The positive rate of FITC-Annexin V staining indicates the percentage of cells undergoing early apoptosis, while the positive rate of PI staining represents the proportion of cells in late-stage apoptosis.

For cell cycle detection, the colon cancer cells were collected, centrifuged, washed with pre-cooled PBS, fixed with pre-cooled 70% ethanol, and stored overnight in a -20℃ refrigerator. On the following day, the fixed cells were rinsed with pre-cooled PBS and subsequently treated with PI (UElandy, Shanghai, China). Finally, flow cytometry was employed for detection.

Cell counting kit-8 (CCK8) assay

The colon cancer cells (5000 cells/well) were inoculated into a 96-well plate. Subsequently, the medium containing 10% CCK-8 (Biosharp, Beijing, China) was added to each well at the specified time points (6 h, 24 h, 48 h, and 96 h). Following incubation at 37℃ for a duration of 2 h, the absorbance of each well was measured at a wavelength of 450 nm. The obtained data were then analyzed to assess cell proliferation capacity.

Colony formation assay

The colon cancer cells (1000 cells/well) were seeded into 6-well plates. Following a two-week incubation period, the cells were fixed using 4% paraformaldehyde, stained with crystal violet, and subsequently photographed to determine the number of spherical cells for assessing cellular proliferation ability.

Transwell migration assay

The transwell chamber (JET BIOFIL, Guangzhou, China) was positioned within 24-well plates. The colon cancer cells were resuspended in serum-free medium at a concentration of 10,000 cells per 100 µl. Subsequently, these cells were inoculated into the upper chambers (200 µl/upper chamber), while each lower chamber was filled with 600 µl of complete media containing 10% fetal bovine serum. After 24 to 72 h of incubation, the cells that migrated from the upper chamber to the lower chamber were fixed using a 4% paraformaldehyde solution. Subsequently, they were stained with crystal violet and ultimately visualized and quantified under a microscope.

Wound healing assay

The colon cancer cells (5 × 105) were inoculated into 6-well culture plate. After the cell monolayer was attached to the 6-well plate, we utilized a 200ul pipette tip to create a wound in the center of the adherent cells. Next, the medium in the 6-well culture plate was replaced with serum-free medium to suppress cell proliferation. Finally, the wound was documented at 0 h and 24 h through photography. The migratory capacity of cells was assessed by quantifying the extent of wound healing.

Immunoprecipitation (IP)

Colon cancer cells or 293T cell were lysed using pre-cooled RIPA lysis supplemented with 1% PMSF and 1% phosphatase inhibitor for 30 min, followed by centrifugation at 12,000 rpm for 10 min to collect the cell-containing supernatant. The supernatant was then incubated with a primary antibody (2 µg) at room temperature for 1 h and subsequently mixed with protein A/G plus-agarose (40µL) from Santa Cruz, which was incubated in a shaker at 4℃ for 8–12 h. After incubation, centrifuge at 2500 RPM for 5 min, collect the sediment, and perform a wash with pre-cooled 10% RIPA lysis. Finally, the sediment was dissolved in electrophoresis sample buffer (2×) and boiled for 10 min before Western blotting.

Mass spectrometry analysis

The technique of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed in our study for the purpose of conducting mass spectrometry analysis. The anti-OTUB2 or anti-IgG immunoprecipitate was isolated from HCT116 cell lysates using the IP method as described above. Subsequently, the immunoprecipitated proteins were subsequently subjected to digestion using trypsin of modified sequence-grade. Finally, the fragmented peptides were subjected to LC-MS/MS analysis in order to identify the interacting proteins of OTUB2.

Ubiquitination assays

The colon cancer cells or HEK 293T cells were initially treated with 15µM MG-132 at 37 °C for a duration of 8 h. Subsequently, immunoprecipitates targeting anti-sp1, anti-GINS1, anti-ubiquitin, or anti-HA were isolated from the lysates of either colon cancer cells or 293T cells using the aforementioned IP method. Finally, Western blotting was performed to analyze the obtained immunoprecipitates.

Chromatin immunoprecipitation (ChIP-qPCR)

The digested samples of colon cancer cells were crosslinked using 1% formaldehyde, and the crosslinking reaction was terminated with glycine. Subsequently, ultrasound was employed to disrupt the DNA-protein complex formed through cross-linking. Then, anti-SP1 or anti-IgG antibodies were added to samples form an antibody-target protein-DNA complex (with a suitable number of samples reserved as Input DNA samples). Next, Protein A beads were utilized for precipitation in order to selectively enrich DNA fragments that bind to the target protein. Subsequently, the samples were subjected to multiple washes with a wash buffer to eliminate non-specifically bound chromatin and achieve purification. The washed sample was eluted using ChIP eluting buffer. Following this, the decrosslinking buffer (0.2 M NaCl) was added to samples overnight at 65℃ for reversing the crosslinking process. Subsequently, the samples were fixed with RNaseA at 37℃ for 1 h and treated with 0.5 M EDTA, 1 M Tris HCl, and protease K at 45℃ for 2 h. Finally, the sample was dissolved in ddH2O for conducting subsequent quantitative PCR (q-PCR) experiments. Primers for all regions were presented in Table S3. And the data analysis involved employing the “ΔΔCt” method.

Agarose gel electrophoresis

Weigh 2 g of dry agarose powder and dissolve it in 50 ml of TBE electrophoresis solution (Sangon Biotech, Shanghai, China). Heat the mixture in a microwave oven on medium heat. Once boiling, remove from heat and allow it to cool to 60℃ at room temperature. Repeat the heating and cooling process twice, removing it from heat and allowing it to cool to approximately 50℃ each time. Immediately add 1 µl of GoldView solution (Solarbio Science & Technology Co., Ltd, Beijing, China), thoroughly mix, and transfer the mixture into a rubber plate. Insert a comb into the plate and let it completely cool before proceeding with electrophoresis. The amplified binding region products of each decross-linked purified DNA were sequentially sampled as follows: input, IgG (negative control), Histone E3(positive control), and IP: SP1. After completing electrophoresis, perform exposure photograph analysis using a multi-functional gel analyzer.

Luciferase reporter assay

The 24-well plates were seeded with 1 × 105 colon cancer cells and incubated at 37℃ for 8–12 h. Next, plasmid co-transfection was performed using lipofectamine 2000 (Invitrogen, USA) as the transfection reagent. Finally, the relative luciferase reporter activity was measured 48 h post-transfection using the Dual-Luciferase Reporter kit (Promega, USA) in accordance with the manufacturer’s instructions.

Transcriptome sequencing

Large-scale expression profiling was conducted by performing transcriptomic analysis on stable HCT116 cells (NC and sh-OTUB2#1). Total RNA was extracted from HCT116 cells using the Trizol method, followed by DNase I treatment (TaKaRa, Biomedical Technology Co., Ltd, Beijing, China) to remove DNA. The Illumina TruSeqTM RNA Sample Preparation Kit (San Diego, CA) was utilized to prepare an RNA-SEQ transcriptome library with 1 µg of total RNA, following the provided instructions. After quantification using TBS380, the RNA-seq sequencing library was sequenced on the Illumina HiSeq xten sequencer. The original reads were subjected to SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) for trimming and quality control purposes using default parameters. Data analysis was performed online on the Majorbio Cloud Platform (www.majorbio.com ).

Clinical samples collection

We collected a total of 124 pairs of tumor and adjacent tissue samples from the Gastrointestinal Surgery Department of the Second Affiliated Hospital of Nanchang University, all pathologically diagnosed with colon cancer. The clinical and pathological characteristics of the patients are summarized in Table 1. The study has received approval from the Ethics Committee of the Second Affiliated Hospital of Nanchang University.

Bioinformatics analysis

The TCGA-COAD dataset (https://portal.gdc.cancer.gov/) was utilized for the analysis of mRNA expression levels of OTUB2, CD44, and CD133 in colon cancer and adjacent normal colon tissues. The Human Protein Atlas (HPA) database was employed to investigate the protein expression level of OTUB2 in colon cancer tissues compared to normal colon tissues. AlphaFold (https://alphafold.ebi.ac.uk/), a structural prediction database, was used to predict the protein structure of OTUB2. Additionally, the UCSC database (https://genome.ucsc.edu/) and PROMO usage database (https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) were utilized to predict SP1 as a potential transcription factor for GINS1. GSE10950, GSE39582, GSE41258, and GSE44861 datasets from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) database were used to validate the correlation between GINS1 and transcription factors. Furthermore, the UbiBrowser database (http://ubibrowser.ncpsb.org/) predicted OTUB2 was the deubiquitinating enzymes of SP1. Lastly, the JASPAR database (https://jaspar.elixir.no/) was employed to identify potential SP1 binding sites within the promoter region of GINS1.

Statistical analysis

All data were analyzed using GraphPad Prism 9.0 software (GraphPad, La Jolla, USA) or R software (version:4.3.2). Student t-test was employed to compare the mean differences between two groups. One-way ANOVA test was employed to assess the mean differences among multiple groups. Kaplan-Meier survival curves were assessed using the Log-rank test. Nonlinear regression was utilized to fit the inhibition curve, and the IC50 value of oxaliplatin was calculated using GraphPad Prism 9.0 software. Spearman correlation analysis was employed to examine the relationship between two variables. All experiments were independently repeated three times. Statistical significance was considered at p < 0.05.