Primary antibodies against ABCB1 (cat. no. 13342), PARP (cat. no. 9532) and LC3-I/II (cat. no. 12741) were obtained from Cell Signaling Technology, Inc. Primary antibodies against ABCC1 (cat. no. ab24102), ABCC2 (cat. no. ab203397), and ABCG2 (cat. no. ab24115) were obtained from Abcam. Primary antibodies against E-cadherin (cat. no. 20874-1-AP), vimentin (cat. no. 60330-1-Ig), Bcl-2 (cat. no. 12789-1-AP), p53 (cat. no. 10442-1-AP), ERCC1 (cat. no. 14586-1-AP), CA9 (cat. no. 11071-1-AP), DHRS2 (cat. no. 15735-1-AP), GAPDH (cat. no. 60004-1-Ig) and β-actin (cat. no. 60008-1-Ig) were obtained from Proteintech Group. The PrimeScripts RT reagent kit and SYBRs Premix Ex Taq™ were obtained from Takara (Takara Biotechnology Co., Ltd.).

The HCT116 colorectal carcinoma cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences. HCT116 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 culture medium (Gibco-BRL; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The Oxa-resistant CRC cell line HCT116/Oxa was established in our laboratory. Briefly, HCT116 cells were exposed to a series of stepwise-increased concentrations of Oxa to develop an HCT116/Oxa-resistant cell line. Both cell lines were cultured in a 5% CO₂ atmosphere at 37°C.

Cells were sonicated three times on ice using a high intensity ultrasonic processor (Ningbo Scientz Biotechnology Co., Ltd.) in lysis buffer (8 M urea, 1% protease inhibitor cocktail). After 12,000 × g at 4°C for 10 min, protein concentration was determined with a BCA kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. For digestion, the protein solution was reduced with 5 mM dithiothreitol at 56°C for 30 min and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. Triethylammonium bicarbonate buffer (TEAB) was added to dilute samples. Finally, trypsin was added at a 1:50 trypsin: Protein mass ratio for the first digestion overnight and a 1:100 ratio for a second 4 h-digestion. Subsequently, peptides were then desalted by a Strata X C18 SPE column (Phenomenex), vacuum-dried, dissolved in 0.5 M TEAB, and labeled based on the tandem mass tag (TMT) kit. Briefly, 1 U of TMT reagent was dissolved in acetonitrile and added to peptides for incubation for 2 h at room temperature, followed by pooling, desalting, and drying by vacuum centrifugation. Agilent 300Extend C18 column (5 µm particles, 4.6 mm ID, 250 mm length) was used to fractionate peptides. Briefly, peptides were first separated with a gradient of 8 to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, they were combined into 18 fractions and subjected to vacuum-drying.

The peptides were dissolved in 0.1% formic acid (solvent A), and separated using EASY-nLC 1000 ultra-performance liquid chromatography UPLC (Thermo Fisher Scientific, Inc.). The gradient was an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23–35% in 8 min, climbing to 80% in 3 min, and holding at 80% for the last 3 min at a constant flow rate of 400 nl/min.

The peptides were subjected to a nano electrospray ionization source followed by tandem mass spectrometry (MS/MS) in Q Exactive™ Plus (Thermo Fisher Scientific, Inc.) coupled online to the UPLC. The MS scan was set as 350–1,800 m/z and 70,000 resolution; the MS/MS scan was set as 100 m/z and 17,500 resolution. The automatic gain control was set at 5E4, and the data-dependent acquisition procedure was applied to data acquisition.

The resulting MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). Searches were performed against SwissProt human database (20,130 sequences) and reverse decoy database. Trypsin/P was specified as a cleavage enzyme allowing up to two missing cleavages. For precursor ions, the mass tolerances of the first search and main search were 20 and 5 ppm, respectively. The mass tolerance of fragment ions was 0.02 Da. The false discovery rate was adjusted to <1% and the minimum score for peptides was set as >40.

MS analysis of TMT-labeled samples was performed on Q Exactive Protein intensities resulting from average single TMT reporter ion intensities obtained for each peptide associated with a specific protein. The average ratio of differential TMT 127/126 expression (1.5-fold increase or decrease) represents the ratio of two samples. Specifically, differentially expressed proteins were identified in our TMT experiment using 1.5 and 0.67 as upregulation and downregulation cutoff points, respectively.

Gene Ontology (GO) annotation and enrichment analyses were performed using DAVID (the Database for Annotation, Visualization and Integrated Discovery). GO with a corrected P<0.05 was considered to indicate a statistically significant difference. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher's exact test to assess the enrichment of differentially expressed proteins against all identified proteins. Pathways with a corrected P<0.05 were considered significant. These pathways were classified into hierarchical categories according to the KEGG website.

The viability of HCT116/Oxa cells and parental cell was measured by the CCK-8 assay (Dojindo Molecular Technologies, Inc.). Briefly, cells were seeded in 96-well plates at a density of 1.0×10⁴ cells and treated with different concentrations of Oxa for 48 h. Then 10 µl CCK-8 reagent was added to each well and incubated at 37°C for another 1 h. The absorbance was measured for each well at a wavelength of 450 nm. All assays were performed in triplicate.

The cell migration assay was performed using a Transwell system (8 µm; Corning Inc.) according to the manufacturer's protocol. Approximately 5×10⁴ cells were re-suspended in serum-free medium in the top chamber of the insert, and the bottom chamber was filled with DMEM/F12 containing 10% FBS. After 24 h of incubation, the cells that had migrated to the lower side of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 20 min at room temperature. The images were captured using a fluorescence microscope (E1000M Eclipse; Nikon Corp., Tokyo, japan) and the migrated cells was counted.

The siRNA against human ERCC1, DHRS2, p53 and control siRNA were synthesized by Shanghai GenePharma Co., Ltd. The sequence of the control siRNA was: 5′-UUCUCCGAACGUGUCACGUTT-3′. The target sequences for ERCC1 siRNA were: siRNA1, 5′-GCCAAGCCCUUAUUCCGAUTT-3′; and siRNA2, 5′-GCGACGUAAUUCCCGACUATT-3′. The target sequences for DHRS2 siRNA were: siRNA1, 5′-GCGUGGUUCCAGGAAUUAUTT-3′; and siRNA2, 5′-GCUGUCAUCCUGGUCUCUUTT-3′. The target sequences for p53 siRNA were: siRNA1, 5′-CCACCAUCCACUACAACUATT-3′; and siRNA2, 5′-CCACUGGAUGGAGAAUAUUTT-3′. Cells (1×10⁶/well) were seeded on a 6-well plate and cultured until the next day. They were then transfected with 100 pmol siRNA oligomer mixed with Lipofectamine 2000 reagent (Life Technologies; Thermo Fisher Scientific, Inc.) in serum-reduced medium according to the manufacturer's instructions. After 6 h, the medium was changed to complete culture medium, and the cells were incubated at 37°C in a CO₂ incubator for another 24–48 h before harvest.

Total RNA from cell lines was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's guidelines. A total of 500 ng RNA was converted into cDNA using the PrimeScript™ kit (Takara Bio, Inc.) under the following conditions: 37°C for 15 min, 85°C for 5 sec, and were held at 4°C. Quantification of target genes and the reference gene (GAPDH) was studied in triplicate on the ABI-7500 system (Applied Biosystems, Inc.; Thermo Fisher Scientific, Inc.) using SYBR-Green fluorescent-based assay (Takara Bio Inc.). The reaction conditions were as follows: Pre-denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 34 sec. The primers used were as follows: DHRS2 forward, 5′-TCACAGAAAGCCTAGCACAG-3′ and reverse, 5′-TGAGACCATCACCAAGCG-3′; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′; ERCC1 forward, 5′-CTCAAGGAGCTGGCTAAGATGT-3′ and reverse, 5′-CATAGGCCTTGTAGGTCTCCAG-3′; vimentin forward, 5′-TGAGTACCGGAGACAGGTGCAG-3′ and reverse, 5′-TAGCAGCTTCAACGGCAAAGTTC-3′; and E-cadherin forward, 5′-TACACTGCCCAGGAGCCAGA-3′ and reverse, 5′-TGGCACCAGTGTCCGGATTA-3′. The quantification was based on ΔΔCq calculations (24) and was normalized to GAPDH as a loading control.

Treated cells were washed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 2% bovine serum albumin for 30 min at room temperature, cells were incubated with primary antibodies against E-cadherin or vimentin (1:100 dilution) at 4°C overnight. Slides were then washed three times with PBS and incubated with Alexa Fluor 488 (1:1,000 dilution; cat. no Ab150077) or Alexa Fluor 594-conjugated secondary antibodies (1:1,000 dilution; cat. no. ab150120; both Abcam) for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (10 µg/ml) for 10 min. Samples were examined to analyze the expression of E-cadherin and vimentin.

After washing three times with ice-cold PBS, cells were lysed by western blot lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 1mM phenylmethylsulfonyl fluoride on ice. Lysates were centrifuged at 12,000 × g for 20 min at 4°C and denatured at 100°C for 10 min. Equal amounts of protein samples (30 µg/sample) were loaded in each well of a 10% sodium dodecyl sulfate polyacrylamide gel, then electrophoretically transferred onto 0.45 µm polyvinylidene difluoride membranes. Following blocking with 5% non-fat milk at room temperature for 2 h, the membranes were incubated with primary antibodies (1:1,000 dilution) at 4°C overnight, then with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000 dilution; cat. no. SA00001-1) or horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000 dilution; cat. no. SA00001-2; both Wuhan Sanying Biotechnology) for 2 h at room temperature. Chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.) was used to detect specific immune complexes. Protein gray value detection was performed using ImageJ software (version 1.4.3.67; National Institutes of Health, Bethesda, MD, USA).

Results were expressed as the means ± SD of three independent experiments unless otherwise specified. Data from two groups were compared using two-tailed unpaired Student's t-test. One-way analysis of variance (ANOVA) with Tukey's post hoc test was used to assess differences among groups. These analyses were performed using GraphPad Prism Software version 5.0 (GraphPad Software Inc.). P<0.05 was considered to indicate a statistically significant difference.

As revealed in Fig. 1A, HCT116/Oxa cells revealed increased resistance to Oxa compared with their parental cells. To explore which protein was responsible for Oxa resistance in HCT116/Oxa cells, western blot analysis was used. Elevated levels of ERCC1, a major protein involved in the nucleotide excision repair pathway, were observed in HCT116/Oxa cells compared with HCT116 cells (Fig. 1B). To confirm whether ERCC1 expression is involved in Oxa-resistance, the HCT116/Oxa cell sensitivity to Oxa was analyzed after ERCC1 knockdown. ERCC1 mRNA and protein expression levels were significantly suppressed by si-ERCC1 transfection in HCT116/Oxa cells (Fig. 1C and D), and ERCC1 knockdown effectively attenuated Oxa-resistance in HCT116/Oxa cells (Fig. 1E).

EMT plays a critical role in drug resistance. Morphological analysis under an inverted phase contrast microscope revealed that HCT116 cells had a round morphology, while HCT116/Oxa cells were of irregular shapes including long strips, fusiform, and polygonal (Fig. 2A). These morphological differences indicated that HCT116/Oxa cells have a mesenchymal-like phenotype as evidenced by the relative downregulation of the epithelial biomarker E-cadherin and upregulation of the mesenchymal biomarker vimentin (Fig. 2B and C). The EMT phenotype of HCT116/Oxa cells was confirmed by immunofluorescence staining (Fig. 2D). The Transwell assay revealed that HCT116/Oxa cells had a higher capacity for migration relative to HCT116 cells (Fig. 2E).

To gain further understanding of the molecular mechanisms involved in Oxa-resistance in HCT116/Oxa cells, TMT-based quantitative proteomic analysis was performed. A total of 5,345 proteins were identified in this study, of which 4,599 had available quantitative information. A full list of proteins and relevant MS data are presented in Supplementary Data I. Of the 4,599 commonly expressed proteins, 64 were upregulated (fold ratio >1.5) and 50 were downregulated (fold ratio <0.67) in HCT116/Oxa cells compared with HCT116 cells (a full list of proteins is presented in Supplementary Data II). The differential expression of proteins commonly expressed in parental and Oxa-resistant cell lines was further visualized using an expression-based heatmap (Fig. 3A).

To classify categories of commonly expressed proteins and canonical pathways affected by the up- or downregulation of these proteins, GO enrichment analysis was performed using DAVID and KEGG. The enrichment analysis of dysregulated proteins in cellular component, biological process, and molecular function is presented in Fig. 3B. In cellular component analysis, the majority of identified proteins were classified into the extracellular region, extracellular region part, extracellular space, and extracellular exosome. Molecular functional classification revealed that most of these proteins were involved in oxidoreductase activity, aldehyde dehydrogenase [NAD(P)⁺] activity, oxidoreductase activity, acting on the CH-OH group of donors, and virus receptor activity. Regarding biological processes, most of the identified proteins were enriched in small molecule metabolic processes, single organism metabolic processes, oxidation-reduction processes, and organic acid metabolic processes. KEGG analysis identified nine significant pathways (P<0.05; Fig. 3C), with lysosome and central carbon metabolism in cancer being the most significantly enriched. Notably, there was a good agreement of selected validation immunoblots with MS data (Fig. 3D).

To explore the role of DHRS2 in CRC cell Oxa-resistance, transient knockdown experiments were next performed. HCT116/Oxa cells transfected with control or DHRS2 siRNAs were treated with different concentrations of Oxa. As revealed in Fig. 4A and B, DHRS2-specific siRNA significantly reduced DHRS2 mRNA and protein expression. The CCK-8 assay revealed that the suppression of DHRS2 decreased the viability of HCT116/Oxa cells compared with cells transfected with control siRNA (Fig. 4C). Transwell assays revealed that the migration capacities of HCT116/Oxa cells were reduced in the si-DHRS2 group relative to the si-NC group (Fig. 4D).

To investigate the function(s) of DHRS2 in Oxa-resistance in HCT116/Oxa cells with an EMT phenotype, the protein expression of ERCC1 and E-cadherin was assessed. Western blotting demonstrated reduced expression of ERCC1 and increased expression of E-cadherin in the si-DHRS2 group compared with the si-control group (Fig. 4E). Collectively, these data revealed that DHRS2 is an important factor in Oxa resistance and EMT in CRC cells.

Next, the mechanism underlying the involvement of DHRS2 in Oxa-resistance in CRC cells was explored. Because DHRS2 has been reported to stabilize p53 in an osteosarcoma cell line (25), it was examined whether it was also stabilized in HCT116/Oxa cells. Our previous results revealed that DHRS2 and p53 were markedly increased in HCT116/Oxa cells compared with the parental cell line. Silencing of DHRS2 in HCT116/Oxa cells by siRNA decreased p53 compared with control cells (Fig. 5A). To assess whether p53 regulates ERCC1 expression, p53 siRNA was used and the results revealed a significant decrease of ERCC1 protein expression (Fig. 5B).