Human CRC HCT116 and HT29 cell lines were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cell lines were authenticated by STR analysis (Shanghai Biotechnology Co., Ltd., http://www.cellresource.cn/index.aspx). Cells were grown in Dulbecco's minimal essential medium (DMEM, Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin streptomycin-glutamine (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂.

Human CRMP5-overexpression or -knockdown plasmids were constructed using pCDH and pLKO.1 vectors (Genomeditech), respectively. Recombinant plasmids and packaging plasmids, including VSVG (0.75 µg, Addgene) and psPAX2 (1.72 µg, Addgene), were transiently transfected into 293 cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) to produce lentivirus using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cultured CRC cells were infected with the appropriate lentivirus in the presence of polybrene (5 µg/ml; Sigma-Aldrich; Merck KGaA) at 37°C for 12 h, prior to being selected using puromycin (2 µg/ml; Sigma-Aldrich; Merck KGaA). An empty vector or a nonsense scrambled oligonucleotide was used as a negative control. Following selection with puromycin (2 µg/ml; Sigma-Aldrich; Merck KGaA) for 2 week at 37°C, transfected cells were harvested for subsequent experimentation. CRMP5 gene expression of the transfected cells was evaluated via reverse transcription-quantitative (RT-q)PCR and western blot analyses.

Cultured CRC cells treated without or with selumetinib (500 nM) at 37°C for 24 h were collected and lysed with 1X RIPA Lysis Buffer (Sigma-Aldrich; Merck KGaA) containing 1X Protease Inhibitor Cocktail (Sigma-Aldrich; Merck KGaA). The concentration of total protein was measured with a BCA protein assay kit (Beyotime Institute of Biotechnology). Total protein (30 µg/lane) was separated using 6–12.5% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and blocked with 5% skimmed milk at room temperature for 1 h. Next, membranes were incubated with primary antibodies against CRMP5 (Abcam; cat. no. ab36203; 1:2,000), Ki67 (Abcam; cat. no. ab15580; 1:1,000), Caspase-3 (CST; cat. no. 96659665; 1:1,000), E-cadherin (CST; cat. no. 3195; 1:1,000), MMP-2 (CST; cat. no. 4022; 1:1,000), MMP-9 (CST; cat. no. 13667; 1:1,000) and vimentin (CST; cat. no. 5741; 1:1,000) overnight at 4°C, followed by incubation with fluorescence-conjugated goat ant-mouse IgG H&L (Abcam; cat. no. ab216772) or goat an-rabbit IgG H&L (Abcam; cat. no. ab216773) secondary antibodies (dilution, 1:1,000) at room temperature for 1 h. Finally, bands were detected using a two-color infrared laser imaging system (Odyssey; LI-COR Biosciences).

For colony formation assay, cells (1,000/well) were seeded into 12-well plates and cultured in fresh DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) with or without selumetinib (500 nM) for approximately two weeks to allow colony formation. Cells were then fixed with 4% paraformaldehyde at room temperature for 10 min, and stained with 1% crystal violet at room temperature for 20 min. The number of colonies was counted using ImageJ software (v1.51; National Institutes of Health). Cell viability was measured using the Cell Counting kit-8 (CCK-8) assay. Cultured CRC cells (2,000 cells/well) were seeded into 96-well plates for 24 h. The next day, cells were treated with increasing doses (0, 1, 5, 10, 50, 100 and 200 µM) of 5-FU (Sigma-Aldrich; Merck KGaA) at 37°C for 48 h. Next, CCK-8 solution (10 µl/well; Dojindo Molecular Technologies, Inc.) was added to each well and absorbance was measured at 450 nm using a microplate reader.

CRC cells (2×10⁵ cells/well) in 200 µl complete DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) were seeded into the upper Transwell chamber (12-µM pore size; BD Biosciences) and DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) with 15% FBS was plated into the lower chamber. After 48 h at 37°C in culture, cells adhering to the underside of the transmembrane were fixed with 20% methanol for 10 min at room temperature, and then stained with 0.1% crystal violet for 1 h at room temperature. Finally, the images were captured using an inverted microscope (Carl Zeiss AG; magnification, ×100).

Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen GmbH) and reverse transcribed into cDNA with specific oligo (dT) primers using the Prime Script RT Reagent kit (Takara Bio, Inc.). The temperature and duration of RT were: 37°C for 15 min, followed by 85°C for 5 sec. RT-qPCR was performed in 96-well plates using SYBR Premix Ex Taq II kit (Takara Biotechnology Co., Ltd.), on an Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific, Inc.) to measure mRNA levels. The thermocycling conditions used were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The 2^−ΔΔCq method (18) was used to calculate relative mRNA levels and GAPDH was used for normalization of specific mRNA expression. Primers used in RT-qPCR are listed in Table SI.

The Colorectal adenocarcinoma dataset (dataset no. TCGA-COADREAD) from TCGA (https://portal.gdc.cancer.gov) was downloaded to determine the correlation between selected gene (DPYSL5) and patient survival (19,20). A total of 376 patients with transcriptional profiles and basic clinical information, including age, sex, stage, overall survival and survival status were enrolled in the present study. RNaseq v2 mRNA expression data and clinical information were retrieved from the cBioportal (21). Survival analysis was performed using the R package survival (v3.5.3; http://www.bioconductor.org/packages), and survival curves were fit using the survfit function. The difference between high- and low-expression groups was tested by survdiff using the median as the cut-off.

RNA was extracted using Direct-zol™ RNA kit (Zymo Research Corp.), purified with RNeasy Plus Mini kit (Qiagen GmbH). One hundred nanograms of mRNA was used as input to the TruSeq mRNA Sample Prep v2 kit (Illumina, Inc.), with the poly-A pulldown step. Sample preparation was performed according to the manufacturer's protocols. The libraries were sequenced on HiSeq X ten (Illumina, Inc.) using 150 bp paired-end reads. The sequencing data termed raw data (raw reads) were subjected to quality control (QC). Following QC, raw data were filtered into clean data, and clean reads were aligned to the reference genome (February 2009, GRCh37/hg19) using BWA (v0.7.12). Genes with an adjusted P-value of <0.05 identified by DESeq2 (v1.12.3) were defined as differentially expressed. The statistical enrichment of differentially expressed genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was determined by Cluster Profiler (v3.0.4).

Data are presented as the mean ± standard deviation of three independent experiments. Student's t-test or one-way analysis of variance, followed by the Bonferroni post hoc test, was used to compare the treated and corresponding control groups. Survival curves were estimated using the Kaplan-Meier method and analyzed by the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the clinical significance of CRMP5 in CRC, survival analysis was conducted using TCGA COADREAD data. Notably, high expression of CRMP5 was significantly associated with poor survival in 367 patients from COADREAD (Fig. 1A). Furthermore, co-expression analysis by Pearson correlation demonstrated that the majority of genes co-expressed with CRMP5 were mainly associated with the immune response (Fig. 1B and C). These data suggested that CRMP5 may serve an important role in tumor inflammation, thereby promoting cancer development and subsequently causing poor survival.

To investigate the potential role of CRMP5 in CRC, CRMP5 was overexpressed in HT29 cells using lentivirus-mediated human CRMP5 cDNA. CRMP5-overexpression was confirmed by RT-qPCR and Western blotting (Fig. 2A). HT29 cells overexpressing CRMP5 had an increase in cell number compared with the wild-type (WT) cells and cells transfected with empty plasmid (NC), indicating that high levels of CRMP5 promoted CRC cell proliferation (Fig. 2B). This result was further confirmed by a colony formation assay which demonstrated that CRMP5-overexpression significantly increased the colony forming ability of HT29 cells (Fig. 2C). Additionally, Transwell migration assays demonstrated that CRMP5-overexpression promoted HT29 cell migration (Fig. 2D). Additionally, the expression levels of common markers for cell proliferation, migration and invasion were measured. As expected, an increase in the expression of Ki67, a common marker for cellular proliferation, was observed and commonly used markers of migration and invasion, including E-cadherin, vimentin, MMP-2 and MMP-9, while expression of caspase-3, an apoptosis-related protein, was decreased in CRMP5-OE HT29 cells compared with WT or NC as detected by RT-qPCR and Western blotting (Fig. 2E and F).

CRMP5 expression was downregulated in HCT116 cells using shRNA-encoding lentiviruses. RT-qPCR and Western blotting confirmed CRMP5-knockdown in HCT116 cells (Fig. 3A). Additionally, the results of the present study demonstrated that CRMP5-knockdown markedly inhibited cell proliferation and migration (Fig. 3B-D). Similarly, CRMP5-knockdown also increased caspase-3 expression and decreased the expression of Ki67, E-cadherin, vimentin, MMP-2 and MMP-9 (Fig. 3E and F). Taken together, these data indicated that CRMP5 regulates CRC cell proliferation and serves a critical role in regulating CRC development.

To further investigate the molecular mechanisms of CRMP5 in regulating CRC development, RNA-Seq analysis was performed in CRMP5-overexpressed and -knockdown cell lines. KEGG pathway enrichment analysis demonstrated that differentially expressed genes in CRMP5-overexpression or -knockdown cells were significantly enriched in the MAPK and metabolic signaling pathways (Fig. 4A and B). To further investigate the regulatory mechanisms of CRMP5 in CRC, MAPK signaling was selectively blocked by using Selumetinib (AZD6244), a potent, selective and ATP-uncompetitive inhibitor of MAPK/ERK1/2 to determine their effect on cell proliferation. Notably, it was observed that inhibiting ERK1/2 activity with selumetinib markedly alleviated the change in colony formation, as well as the changes in Ki67, E-cadherin, vimentin, MMP-2, MMP-9 and caspase-3 expression levels in HCT116 and HT29 cell lines induced by CRMP5 (Fig. 4C and D). These data suggested that CRMP5 may regulate CRC cell proliferation and development through MAPK signaling.

Therefore, the role of CRMP5 in chemotherapeutic drug resistance was examined. CRMP5-overexpression and control CRC cells were treated with increasing doses of 5-fluorouracil (5-FU), a chemotherapeutic drug commonly used in CRC (22). Results of CCK-8 assays demonstrated that 5-FU treatment significantly decreased the viability of CRC cells while CRMP5 overexpression significantly reversed the inhibitory effects induced by the drug (Fig. 5A). Additionally, a retrospective study was performed using 220 clinical samples from patients with CRC in a well-established TCGA-COAD cohort to determine the role of CRMP5 in CRC recurrence. The results of the present study demonstrated that high expression of CRMP5 significantly indicated poor disease-free survival of patients with CRC (Fig. 5B). Taken together, these data suggested that CRMP5-overexpression induces chemotherapy resistance and tumor recurrence of CRC.