In total, 58 patients with CRC were included in the present study, who had been diagnosed and subjected to total surgical resection prior to radiotherapy at The Fourth Hospital of Hebei Medical University (Shijiazhuang, China) between January 2014 and December 2014. Disease diagnosis was based on the clinical presentation, medical history and family history, physical examination, laboratory tests, endoscopy, imaging detection, and histopathological examination (14). Of these patients, there were 5 cases of tubulovillous adenoma, 11 cases of papillary carcinoma, 21 cases of tubular adenocarcinoma, 13 cases of mucinous adenocarcinoma, 6 cases of signet ring cell carcinoma and 2 cases of undifferentiated carcinoma. The occurrence of lymph node metastasis was confirmed in 26 cases by postoperative pathological examination on biopsy, and the remaining 32 cases were free from lymph node metastasis. All patients were first-onset cases and had not previously received hormone therapy, radiotherapy or chemotherapy prior to surgery. In addition, 29 age- and gender-matched healthy individuals were enrolled into the control group. The CRC patients with lymph node metastasis included 11 males and 15 females, with ages of 34–85 years (median age, 61 years). CRC patients without lymph node metastasis included 17 males and 15 females, aged between 28–76 years (median age, 58 years). In the control group, there were 13 males and 16 females, aged from 22–72 years (median age, 55 years). Prior written and informed consent was obtained from each patient and the study was approved by the Ethics Review Board of the Fourth Hospital of Hebei Medical University.

The following specimens were collected from the subjects in the present study: i) The excised tumor and adjacent tissues, which were stored in liquid nitrogen; ii) the lymph nodes in proximity to the surgical site, which were removed during surgery and stored in liquid nitrogen; and iii) the peripheral blood collected under fasting conditions in the morning, which was mixed with EDTA anticoagulant (cat. no. 367525; BD Biosciences, San Jose, CA, USA) and stored at −20°C.

The mRNA expression levels of MAP4K4 and miRNA-141 were detected with the use of RT-qPCR. Briefly, for detection of the expression levels in tumor and in lymph nodes, total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The serum RNA was extracted with the miRNeasy Serum/Plasma kit (Guangzhou Jianlun Biological Technology Co., Ltd., Guangzhou, China). RT was performed to obtain the cDNA with the RevertAid First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The 25 µl PCR system contained 2 liters template, 1 liter each primer, 12.5 liters TransStart Top Green qPCR SuperMix (Transgen Biotech, Inc., Beijing, China), and 8.5 liter double distilled H₂O.

For the detection of MAP4K4, the primer sequences were as follows: MAP4K4 forward, 5′-AAGGAGAGAGCGGGAAGCTA-3′, and reverse, 5′-TTGTTGCAACTGCCTCTGGA-3′; GAPDH forward, 5′-GTTGGAGGTCGGAGTCAACGGA-3′, and reverse, 5′-GAGGGATCTCGCTCCTGGAGGA-3′. The PCR conditions consisted of denaturation at 94°C for 5 min, followed by 94°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec, for a total of 35 cycles. For the detection of miRNA-141, the primer sequences were as follows: miRNA-141, 5′-CCGGTAACACTGTCTGGTAA-3′, and U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′. The PCR conditions were as follows: Denaturation at 95°C for 5 min, followed by 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec, for 40 cycles. The relative expression levels of target genes were calculated with the 2^−ΔΔCq method (15).

Normal, tumor and lymph node metastasis tissues were lysed on ice with lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), and the protein concentration was determined with a BCA kit (cat. no. P0009; Beyotime Institute of Biotechnology). Protein samples (20 mg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electronically transferred onto a nitrocellulose membrane. The blot was blocked with 5% non-fat milk at room temperature for 1 h, and then incubated with rabbit anti-human anti-MAP4K4 polyclonal primary antibody (1:1,000; cat. no. ab155583; Abcam, Cambridge, MA, USA) or rabbit anti-human anti-GAPDH polyclonal primary antibody (1:5,000; cat. no. ab9485; Abcam) at 4°C overnight. The membrane was then incubated with goat anti-rabbit immunoglobulin G (1:3,000; cat. no. ab6721; Abcam) at room temperature for 1 h. Subsequently, the blot was developed using an enhanced chemiluminescence system (cat. no. P0018A; Beyotime Institute of Biotechnology), and the protein bands were analyzed with the Image Lab software version 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Serum MAP4K4 contents were detected with an ELISA kit (cat. no. CSB-EL013439HU; Weijia Technology Co., Ltd., Guangzhou, China) according to the manufacturer's instructions. Blood sample was centrifuged at 4°C at 800 × g for 10 min, separating the serum from the blood cells. Sample (10 µl) was then added into the 96-well plates in the ELISA microplate, followed by the addition of 40 µl diluting solution. The experiment was performed in triplicate. Subsequently, 100 µl horseradish peroxidase-labeled antibody (contained within the ELISA kit) was added into each well, and the plate was placed in an incubator for 1 h. After washing five times with ddH₂O, 50 µl substrate A and 50 µl substrate B were added into each well, respectively, and the plate was incubated at 37°C for 15 min. The reaction was stopped by adding 50 µl stopping solution, and the optical density at 450 nm was read on a microplate reader within 15 min.

NK and T cells in the peripheral blood were detected by flow cytometric analysis with CD3/CD4/CD8 and CD3/CD16+56 agents (cat. nos. IM1650 and IM2076, respectively; Immunotech; Beckman Coulter, Inc., Marseille, France), according to the manufacturer's instructions. Briefly, 20 ml staining agent was added into the anticoagulated whole blood at room temperature for 25 min, and 2 ml hemolytic agent was then added at room temperature for 10 min. Following centrifugation at 450 × g at 4°C for 5 min, the supernatant was discarded. After washing with phosphate-buffered saline twice, 500 µl 1% paraformaldehyde was added for fixation. The sample was detected with a FACScan flow cytometer (BD Biosciences), and the data were analyzed using Cellquest software version 3.1 (BD Biosciences).

To determine the target gene of miRNA-141, bioinformatics analysis was performed with the following online software and/or websites: miRanda (http://www.microrna.org/microrna/getExprForm.do), TargetScan (www.targetscan.org), PiTa (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) and PICTA (http://pictar.mdc-berlin.de/).

Data are expressed as the mean ± standard deviation. SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. One-way analysis of variance was performed for the comparison between the groups, along with the least-significant difference and Student-Newman-Keuls tests (for equal variance), or the Tamhane's T2 and Dunnett's T3 tests (when equal variance was not assumed). P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the mRNA expression levels of MAP4K4 in the tumor tissues, lymph nodes and serum in CRC patients with or without lymph node metastasis, RT-qPCR was performed. The present results indicated that, compared with the control group, the mRNA expression levels of MAP4K4 were significantly elevated in the tumor tissues, lymph nodes and serum in patients with CRC (all P<0.05; Fig. 1). Furthermore, within the CRC patients, the mRNA expression levels of MAP4K4 in the tumor tissues, lymph nodes and serum in cases with lymph node metastasis were all significantly higher compared with those in cases without lymph node metastasis (P<0.05; Fig. 1). Thus, the results suggest that MAP4K4 may be upregulated in CRC, particularly in cases with lymph node metastasis, and this may contribute to the disease pathogenesis.

The protein expression levels of MAP4K4 in the tumor tissues and lymph nodes were detected by western blot analysis, while the serum expression was detected by ELISA. The results of western blot analysis indicated that, compared with the control group, the protein expression levels of MAP4K4 were significantly increased in CRC tissues (P<0.05) and more evidently in the lymph nodes (P<0.01; Fig. 2A and B). In addition, the protein expression levels of MAP4K4 in the tumor and lymph nodes in CRC patients with lymph node metastasis were all significantly higher compared with those without lymph node metastasis (P<0.05; Fig. 2A and B).

Similar results were obtained for the protein expression of MAP4K4 in the serum. ELISA analysis revealed that, compared with the control group, the serum MAP4K4 expression was significantly increased in CRC patients with and without lymph node metastasis (P<0.01). However, the expression was elevated to a greater extent in the cases with lymph node metastasis compared with cases without metastasis (P<0.01; Fig. 2C). In accordance with the alteration of the mRNA expression levels of MAP4K4, the aforementioned results indicated that MAP4K4 may be involved in the pathogenesis of CRC, particularly in the development of metastasis via the lymphatic and blood circulation.

It has been reported that MAP4K4 may be regulated by miRNA-141 in cases of pancreatic cancer (16). To further investigate the underlying mechanisms through which MAP4K4 is modulated in CRC, bioinformatics analysis was performed. Results from the miRanda, TargetSean, PiTa, RNAhybrid, and PICTA analyses revealed that MAP4K4 may be the target gene of miRNA-141 (Fig. 3), and may be involved in the pathogenesis of CRC. RT-qPCR was then performed to detect the expression levels of miRNA-141 in the tumor tissues, lymph nodes and serum from CRC patients. The current results demonstrated that, compared with the control group, the expression levels of miRNA-141 in the tumor tissues, lymph nodes and serum were significantly decreased in CRC patients (P<0.05; Fig. 4). Furthermore, of the CRC patients, the expression levels of miRNA-141 in the tumor tissues, lymph nodes and serum in cases with lymph node metastasis decreased to a significant extent when compared with those in patients without lymph node metastasis (P<0.05; Fig. 4). The results suggest that miRNA-141 may participate in the pathogenesis and development of CRC, and contribute to lymph node metastasis.

To investigate the anti-tumor response in CRC, the NK cells and T-lymphocyte subsets were detected with flow cytometry. The results revealed that, compared with the control group, the percentage of NK, CD3⁺ T and CD4⁺ T cells in the peripheral blood of CRC patients were significantly decreased, while the CD8⁺ T cells were significantly increased (P<0.05 and P<0.01; Fig. 5A), resulting in significantly decreased CD4⁺/CD8⁺ ratios (P<0.01; Fig. 5B). Furthermore, the alterations were significantly more marked in the CRC patients with lymph node metastasis compared with those without lymph node metastasis (P<0.05). Thus, the results reveal declined anti-tumor responses in CRC patients, which is worse in cases with lymph node metastasis compared with those without metastasis.