Sampling

Clinical samples were obtained from 100 patients with colorectal cancer who had not received any anti-tumour therapies prior to sample collection. Resected colorectal cancer tissue in wax blocks was collected from our hospital for study purposes after diagnosis by the pathologist. The study protocol was reviewed and approved by the medical ethics committee and informed consent was obtained from the patients.

Follow-up registration, clinicopathological features and prognosis of colorectal cancer

Clinicopathological data of enrolled patients with colorectal cancer were collected, including gender, age, tumour location, gross type, tumour size, neurovascular involvement, differentiation, primary tumour invasion, lymph node metastasis and distant metastasis. Pathological patterns and differentiation rankings of tumours were defined according to the 2010 World Health Organization diagnostic criteria for gastrointestinal tumours [8]. The tumour, nodes, metastasis (TNM) staging was done as per the 2009 American Joint Committee on Cancer and the International Union Against Cancer TNM staging system for colorectal cancer (7th edition) [20]. The survival status of patients included in the study was assessed by follow-up telephonic consultations and household surveys by the public security systems. To record the current living status and to calculate the three-year survival time in months, we used the following codes: death = 0, survival = 1, and lost to follow-up = 2.

Immunohistochemical staining to detect in situ NudCD1 expression in tissue samples

The paraffin tissue was sectioned, baked at 65 °C overnight, dewaxed in xylene, washed with alcohol and water and then soaked in 3% H2O2 solution for 15 min in the dark. The tissue was then immersed in 0.01 mol/L citrate buffer (pH 6.0) for three minutes in a pressure cooker for antigen retrieval. Next, it was cooled with tap water, washed with phosphate buffer saline (PBS) and incubated with the NudCD1 primary antibody (1:200, ab126902, abcam) at 4 °C overnight. Langerhans cells of human skin were used as a positive control, and a sample stained with PBS in place of the primary antibody was used as a negative control.

The following day, the sectioned piece was washed with PBS and incubated with goat anti-rabbit HRP IgG secondary antibody at room temperature for 15 min. Next, it was washed with PBS and incubated with 3,3’-diaminobenzidine (DAB) solution for a length of time appropriate for colouration under a microscope. The sample was then stained with Harris haematoxylin for two minutes, differentiated with hydrochloric acid in ethanol and dehydrated with gradient alcohol. Next, it was made transparent with xylene, mounted on slides with soluble resin and examined under a microscope.

Positive staining was defined by the presence of brown or tan particles in the cytoplasm of parenchymal cells. Five fields (200X) in each section were examined. Scoring based on the percentage of positive cells was as follows: 0 ~ 5% positive cells – 0 points, 5% ~ 25% – 1 point, 26% ~ 75% – 2 points, and 76% ~ 100% – 3 points. According to the IHC staining intensity, the scoring was as follows: colourless – 0 points, yellowish – 1 point, brownish – 2 points, and tan – 3 points. Stained tissues were read and scored by two pathologists who were blinded to each other’s results, take the average value of the results read by the two doctors as the final score. Tissue staining was scored according to staining intensity and the percentage of positive cells, with 0–4 representing low expression and 5–9 representing high expression. A chi-square test was used to compare differences between the groups.

Cell culture

Colon cancer cell lines (LoVo, SW620, HCT116, HT-29) were cultured in an L-Glutamine Dulbecco’s Modified Eagle Medium (L-DMEM) and incubated at 37 °C with 5% CO2 and saturated humidity in a cell culture incubator (Forma, USA). All cell lines were purchased from Nanjing Cobioer Bioscience Co. Ltd. The cells were digested with 0.25% trypsin for passage, and logarithmic growth-phase cells were collected for the assays described below.

Real time quantitative polymerase chain reaction to detect NudCD1 expression in different cancer cell lines

The colon cancer cell lines (LoVo, SW620, HCT116, HT-29) were lysed withTRIzol Reagent (Invitrogen Corp., CA, USA) at room temperature for five minutes. Then one-fifth the volume of chloroform (Sinopharm, Beijing, China) was added with oscillation for 15 s. When the solution emulsified completely, it was allowed to sit at room temperature for five minutes and then centrifuged at 12,000xg for 15 min at 4 °C. The supernatant was transferred into another RNase-free Eppendorf (EP) PCR tube with an equal volume of isopropanol (Sinopharm, Beijing, China) and mixed thoroughly. MonScript™ RTIII All-In-One Mix (Monad Biotech Co. Ltd., Wuhan, China) was used for synthesis of cDNA. This was then mixed with ds DNase (Monad Biotech Co. Ltd., Wuhan, China) according to the manufacturer’s protocol. MonAmp™ ChemoHS qPCR Mix (Monad Biotech Co. Ltd., Wuhan, China) was used for performing real time quantitative polymerase chain reaction (qPCR). Samples were incubated at 50 °C for 15 min, followed by 95 °C for 5 min, followed by 32 PCR cycles with the following temperature profile: 95 °C for 15 s, 60 °C for 30 s and 72 °C for 1 min. The primers were as follows: NudCD1-forward, 5’-CCTGCTTCTGTTTGCGCCATG-3; NudCD1 -reverse, 5’-GAAGGCACTCACAAAGGGCTG -3’; GAPDH-forward, 5’-ACAACTTTGGTATCGTGGAAGG-3; and GAPDH-reverse and 5’-GCCATCACGCCACAGTTTC-3’. The relative expression of the corresponding gene mRNA was analysed and expressed as 2 −ΔΔCt (△ threshold cycle [CT] is the CT value of the target gene in the same sample minus the internal reference CT value). The relative mRNA expression was standardised to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.

NudCD1 overexpression in colon cancer cell lines

Colon cancer cell lines were screened by qPCR for NudCD1 expression and divided into two groups for either empty vector control or NudCD1 overexpression (NudCD1 +). A vector for the overexpression of NudCD1 in the colon cancer cell lines was established as follows. The human source vitronectin precursor (VTN) sequence was obtained from the National Center for Biotechnology Information database (Accession No. BC000967) and primers were designed in accordance with the coding DNA sequence (CDS): VTN-F 5’-TGCTCTAGAGCCACCATGGAGGTGGCGGCTAATTG-3’ and VTN-R 5’-CCGGAATTCTTAATTCTCTGTATTTACTTTTATTAAA-3’. Total RNA was extracted by using the TRIzol method, and a reverse transcription kit was used to reverse-transcribe total RNA into cDNA. The plasmid profile of the NudCD1 overexpressing lentiviral vector system is shown in Fig. 1.

Fig. 1

Plasmid profile of NudCD1 overexpressing lentiviral vector system. Plasmid profile of NudCD1 overexpressing lentiviral vector system was structured, including pCDH-GFP vector map (A), psPAX2 vector map (B) and pMD2.G vector map (C)

Plasmid was extracted for sequencing with the following sequencing primer: CACGCTGTTTTGACCTCCATAGAA. Transfection appeared when the cell fusion degree was up to 90%–95%. Fluorescence expression was observed after four days. Polybrene was then added to a final concentration of 6 µg/mL. If the infection rate was over 90%, amplification was performed.

The determination of the cycle of colon cancer cells

Cells in the logarithmic phase in each group were collected, resuspended in PBS and counted, after which 1 × 105 cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for five minutes. The supernatant was discarded, and the cell sediment was washed with PBS. Next, 0.5 ml PBS was left in the centrifuge tube, and 5 ml of pre-cooled 75% ethanol was added, fixed and placed for 24 h at -20 °C. The ethanol was discarded after centrifugation and the cell sediment was again washed with PBS. One mL PBS was left in the centrifuge tube and the cells resuspended. This was followed by the addition of 5 µL RNase (10 mg/mL) and incubation at 37 °C for one hour. Next, the sample was stained with 5 μl propidium iodide (PI) (10 mg/mL, Sigma, USA) for 30 min at room temperature with the aim of avoiding light staining. The sample was then examined for cell cycle assay by flow cytometry (BD FACSCalibur, BD Biosciences, CA, USA).

Observation of apoptosis of the colon cancer cells

The cells in the logarithmic phase in each group were collected, resuspended in PBS and counted, after which 1 × 105 cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for five minutes. The supernatant was discarded, and the cells were gently resuspended with 195 μL Annexin V-FITC binding solution and gently mixed with 5 μl Annexin V-FITC (Annexin V-FITC/PI Apoptosis Detection Kit, Gibco, USA). After incubation in the dark at room temperature for ten minutes, the mixture was centrifuged at 1000 rpm for five minutes, the supernatant was discarded, and the cells gently resuspended with 190 μL Annexin V-FITC binding buffer. Then, 10 μl PI staining solution was added, mixed gently and placed in an ice bath in the dark. Apoptosis was measured by flow cytometry, in which Annexin V-FITC presented green fluorescence, while PI presented red fluorescence.

Detection of mRNA expression of SAC-related genes in colon cancer cell lines by qPCR

Real-time quantitative PCR was used to detect the mRNA levels of SAC-related genes, including BUB1, BUBR1, MAD1, CDC20 and MPS1/TTK, as well as downstream molecules in the LIS 1/dynein pathway, namely, LIS 1, DYNC1H1 and DYNLL1. The qPCR conditions were the same as the detection method for NudCD1 expression described earlier. Primer sequences and synthesis conditions (following the same detection procedures as in subsection 2.5) are shown in Table 1. The relative mRNA expression was standardised to the expression level of GAPDH mRNA.

Table 1 Primer sequence and synthesis conditionStatistical analysis

The statistical package IBM SPSS Statistics for Windows, Version 19.0. (Armonk, NY) was used for data analysis. All data were expressed as mean ± standard deviation. Measurement data were compared using t-tests, and the count data were compared using the chi-square test. The Kaplan–Meier survival analysis was used for univariate survival analysis of colorectal cancer patients. The log-rank test was used to draw the survival curve. A P value < 0.05 was considered statistically significant.