Patients and serum samples

This project was carried out with the permissions from patients and was approved by the ethics committee of the People’s Hospital of Xinjiang Uyghur Autonomous Region University. All procedures performed in the studies involving human participants were in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Clinical cancer tissue samples from 60 CRC patients at the People’s Hospital of Xinjiang Uyghur Autonomous Region Hospital from 2016 to 2017 were collected for ZIC5 examination. Paired normal tissues were also harvested for negative control. Samples were collected with liquid nitrogen and stored at –80°C. All these patients received no radiotherapy or chemotherapy before surgery.

Cell culture

Human colon carcinoma cells including SW620 and HCT116 were purchased from the Bena culture collection Co., Ltd (Jiangsu, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, USA). A cell incubator with a humidified atmosphere containing 5% CO₂ was applied for all cell cultures. In addition, flavopiridol (4.5 mmol/l) was prepared in water and then added into the colon cancers for functional assay.

RNA extraction and real-time PCR analysis

We used the mirVana miRNA kit (Takara, China) to extract total RNAs from CRC samples or cultured cells following the manufacturer’s instructions. U6 small RNA was used as an internal control. For detection of ZIC5 mRNA expression, PrimeScript RT reagent kit (Takara, Dalian, China) was applied to synthesize the first-strand cDNA. The expression of ZIC5 was quantified by real-time PCR mixture assays (Takara). GAPDH was used as the internal control. Primers were designed as follows:

ZIC5_Forward: 5′-GCGCUGAGAUUAGCGGAUUTT-3′.

ZIC5_Reverse: 5′-AAUCCGCUAAUCUCAGCGCTT-3′

GAPDH_Forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′

GAPDH_Reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′

Cell transfection

Cells transfection was used for ZIC5 overexpression or inhibition. In brief, we amplified the human ZIC5 cDNA through PCR and then subcloned into the pcNDA3.1 vector (Invitrogen). siZIC5 were designed as follow:

siZIC5_Forward: 5′-GCGCUGAGAUUAGCGGAUUTT -3′

siZIC5_Reverse: 5′-AAUCCGCUAAUCUCAGCGCTT -3′

Lipofectamine 2000 (Invitrogen, USA) was applied to efficiently transfect ZIC5 or siZIC5 in CRC cells according to the manufacturer’s instructions.

MTT assay

The MTT assay kit (Thermo Fisher, USA) was used to assess the viability of SW620 and HCT116 cells transfected with ZIC5 or siZIC5. In brief, 20 μl of MTT reagent was added into each well and then these cells were incubated at 37°C for 4 h. We aspirated the medium and added 100 μl of DMSO to dissolve the formazan crystals. A microplate reader was used to examine the absorbance at 450 nm.

Western blot assays

Expression levels of proliferative cell cycle proteins, such as CDC25C, CDK1, and cyclin B1, were determined by western blot. Briefly, CRC (SW620 and HCT116) cells were lysed using RIPA buffer. Primary antibodies, such as rat anti-cyclin B1 (Santa Cruz, 1 : 500), rat-anti CDC25C (Sigma, 1 : 1000) and mouse anti-CDK1 (Santa Cruz, 1 : 1000) were incubated with PVDF membrane at room temperature for 3-4 h. Secondary antibodies conjugated with HRP were used to detect the protein expression at room temperature for 1 h through chemiluminescence reagent. β-actin was used as an internal reference.

Cell cycle analysis

Trypsin was applied to digest the transfected CRC cells, which were subsequently fixed in 70% ethanol. Cells were washed with 1 × PBS and treated with 10 μg/ml RNase for 1 h at 37°C, and propidium iodide (100 μg/ml) was used for staining at room temperature for 30 min in dark. A flow cytometer (BD Bioscience, San Jose, CA, USA) was used to analyze cell cycle distribution and the data were analyzed by FlowJo.

Wound healing assay

Wound healing assay was used to detect cell migration. Briefly, transfected cells (1 × 10⁴ cells/well) were seeded in 6-well plates, and then an artificial homogeneous wound was created via a sterile plastic micropipette tip. Next, cells were cultured under standard conditions for 24 h. Cells were imaged from 0 to 24 h after wounding. Cells that migrated from the wound edge in five random fields were counted through a microscope (Olympus, Japan)

Cell invasion assay

Capacity of CRC cell invasion was evaluated by transwell chambers (BD Biosciences). In brief, 5 × 10⁴ CRC cells were placed into the upper chamber with Matrigel (BD, USA) for 24 h. Then 10% FBS was added as a chemoattractant in the bottom chamber. 48 h after seeding, we removed the upper chamber cells via a cotton swab and fixed the bottom chamber cells with 70% ethanol for 30 min. Finally, these cells were stained with 0.2% crystal violet for 10 min. A microscope (Olympus, Japan) was applied to count the fixed cells of five randomly selected fields.

Histology and immunohistochemistry

A microtome was used to harvest CRC tissue sections (5 μm thickness). Sections were first put into xylene and graded alcohol for deparaffinization. Citrate buffer was used to expose the antigen by heating at 95°C for 15 min. After the sections cooled down, we washed the sections with 1 × PBS and then added the blocking solution to neutralize the peroxidase activity for 1 h. Primary antibodies (ZIC5, R&D, 1 : 200) were incubated with sections for 12 h at 4°C and then secondary antibodies were added for 1 h incubation at 37°C. Finally, DAB solution was added for target antigen detection. Mayer’s hematoxylin was used as a counter stain. In negative controls, the primary antibodies were omitted.

Tumor xenograft study

Four-week-old male immune deficiency nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. Animal experiments were approved by the People’s Hospital of Xinjiang Uyghur Autonomous Region University. 2 × 10⁶ SW620 cells were transfected with ZIC5. These cells were then suspended in sterile saline (0.2 ml) and subcutaneously injected into the axillary fossae of each nude mouse. After implantation of cancer cells, mice were killed at different time points (1w, 2w, 3w, 4w, 5w, 6w) and consequently xenograft tumors were harvested intact. Fresh tissues were stored in liquid nitrogen for next-step RNA and/or protein extraction.

Data analysis

Data are presented as mean ± standard deviation. Statistical analyses between two groups were performed using Student’s t-test via SPSS 16.0. Statistical analyses between multiple groups were performed using one-way analysis of variance (ANOVA) followed by the least significant difference post-hoc test. Differences with values of p < 0.05 were regarded as statistically significant.

Ethical approval

The study was approved by the Animal Ethics Committee of the People’s Hospital of Xinjiang Uyghur Autonomous Region, and the experiments with rats were in full compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) or with the Guidelines laid down by the NIH in the US.

Zinc finger of the cerebellum 5 was up-regulated in CRC patients

To examine the role of ZIC5 in CRC, samples derived from 60 patients were collected to examine the expression levels of ZIC5. Consistently, we found that ZIC5 was significantly increased compared to paired normal tissues (Figures 1 A, B). We further assessed the correlation between ZIC5 and clinicopathological parameters; 60 CRC patients were chosen to examine the effect on ZIC5 in CRC prognosis. As shown in Table I, higher expression of ZIC5 was observed in highly malignant primary tumors (T2/3) (p = 0.026). As for lymph node metastasis, higher expression of ZIC5 in CRC patients was prior to occur (p = 0.004). In addition, CRC patients with higher ZIC5 expression showed a significant increase in CRC distal metastasis (p = 0.002). However, there were no significant relations observed in ZIC5 and other clinicopathological parameters.

Figure 1

Zinc finger of the cerebellum 5 expression was significantly increased in CRC tissue samples. A – qRT-PCR results revealed that expression of ZIC5 in CRC patients was significantly higher than that in paired normal tissues. B – ZIC5 expression was elevated in CRC patients compared with paired normal tissues (n = 3 patients; ***p < 0.001; scale bar = 100 μm)

CDK1/CDC25A signaling cascade-mediated ZIC5 induced malignant CRC cell proliferation

To assess the functional role of ZIC5 in CRC, two CRC cell lines (SW620 and HCT116) were used to elevate or knock down the expression of ZIC5. The efficiency of ZIC5 expression was examined by qRT-PCR and Western blot (Figures 2 A, B). The capacity of cell proliferation showed an increased tendency in a time-dependent manner (Figure 2 C) upon ZIC5 overexpression in SW620 cells, whereas the proliferation of HCT116 cells was down-regulated (Figure 2 D) under ZIC5 knockdown. Next, cell cycle analysis showed a higher proportion of SW620 cells maintained in the S phase upon ZIC5 overexpression, illustrating a higher proliferation rate (Figure 2 E). In contrast, a large amount of HCT116 cells stayed at G0/G1 phase in the condition of ZIC5 deficiency, demonstrating that inhibition of ZIC5 could induce CRC cell cycle arrest at G0/G1 phase. As we know, CDC25/CDK/cyclin signaling was essential for cell cycle regulation, so we further investigated whether the CDC25/CDK/cyclin pathway was activated. As expected, the expression levels of CDC25c, CDK1 and cyclin B1 were all elevated. Considering the major role of CDC25c in the nucleus during mitosis, nuclear proteins from CRC cells were further extracted to verify the CDC25c function (Figures 2 F, G) upon ZIC5 overexpression of knockdown. CDC25c expression was significantly increased in the nucleus but not in the cytoplasm. Oppositely, ZIC5 deficiency caused lower expression of nuclear CDC25c. Taken together, the results obtained from this section showed that ZIC5 played a pivotal role in CRC proliferation.

Figure 2

Overexpression of ZIC5 promoted CRC cell proliferation through activating CDC25/CDK1/cyclin B1 signaling. SW620 cells and HCT116 cells were transfected with ZIC5 plasmid and siZIC5 respectively. A – Transfection efficiency was examined by qRT-PCR and western blot (B). C – MTT assay showed that SW620 cells transfected with ZIC5 plasmid had higher proliferation capacity, D – whereas inhibition of ZIC5 decreased HCT116 cell proliferation E – Cell cycle analysis revealed that in SW620 cells with overexpression of ZIC5, a larger proportion of cells remained in the S phase, whereas in HCT116 cells with ZIC5 knockdown, G0/G1 phase cells were increased F, G – Western blot results showed that expression of cell cycle related proteins was up-regulated and CDC25C was translocated into the nucleus and was responsible for activation of downstream cascades (***p < 0.001)

Zinc finger of the cerebellum 5 promoted CRC cell migration and invasion

The CRC patients, treated with surgery and several adjuvant therapies, usually suffered from relapse and metastasis. As shown in Table I, ZIC5 was closely associated with the metastasis of CRC. We subsequently investigate the role of ZIC5 in CRC cell migration and invasion. We found that ZIC5 overexpression accelerated the CRC cell migration and invasion. In HCT116, we knocked down the expression of ZIC5 and found that ZIC5 deficiency restricted cells’ capacity for invasion and migration (Figures 3 A, B), suggesting that ZIC5 had a versatile role in CRC.

Figure 3

Zinc finger of the cerebellum 5 facilitated CRC cell migration and invasion. A – Wound healing test showed that ZIC5 promoted SW620 cell migration and inhibition of ZIC5 ameliorated HCT116 cell migration. B – ZIC5 promoted SW620 cell invasion and inhibition of ZIC5 prevented the invasion of HCT116 cells

CDK/CDC25c signaling in CRC was positively regulated by ZIC5

To further confirm the causal relationship between ZIC5 and CDK/CDC25c signaling, we next performed a rescue experiment in CRC cells. ZIC5 was initially overexpressed in HCT116 cells, and then malignancy was examined. We found that CRC cell proliferation, migration and invasion abilities were all elevated (Figures 4 A, C, D). Accordingly, the expression of cyclin B1, CDK1 and CDC25c was also increased (Figure 4 B). Next, the effect of flavopiridol (an inhibitor of CDK) on CRC cell proliferation, migration and invasion was examined. As shown in Figure 4, flavopiridol restricted CRC cell proliferation, migration and invasion abilities. Intriguingly, ZIC5-mediated effects of pro-tumor were all reversed (Figures 4 A–D) under flavopiridol treatment, suggesting that CDK/CDC25c signaling may be regulated by ZIC5.

Figure 4

Flavopiridol mitigated the CRC malignancy induced by ZIC5. A – MTT assay indicated that flavopiridol restricted CRC cell proliferation induced by ZIC5 overexpression. B – Western blot results also showed the inhibitory effect of ZIC5 on the CDC25C/CDK1/cyclin B1 signaling pathway. C – Scratch assay revealed that flavopiridol inhibited CRC cell migration D – Flavopiridol inhibited ZIC5-overexpressing CRC cell invasion (***p < 0.001)

Zinc finger of the cerebellum 5 promoted tumor formation in vivo

Given the important role of ZIC5 in CRC, we next investigated whether ZIC5 facilitated tumor formation in vivo. SW620 cells that transiently overexpressed ZIC5 were subcutaneously injected into the axillary fossae of nude mice. The results showed that nude mice injected with ZIC5 overexpressing cells showed larger tumor formation compared with the control group, i.e., tumors volume was significantly increased (Figures 5 A, B).

Figure 5

Zinc finger of the cerebellum 5 promoted tumor growth in vivo. A – Following the subcutaneous injection of SW620 cells with ZIC5 overexpression, tumor volume was progressively increased in a time-dependent manner. B – Morphologic phenotype of tumor from nude mice with/without ZIC-overexpressing CRC cell injection (***p < 0.001)