A total of 30 CRC specimens were obtained for use in this study from the First Hospital of Jiaxing, China, between January 2013 and May 2014. The study was approved by the ethics committee of the same hospital, and written informed consent was obtained from all participants in accordance with the provisions of the Helsinki Declaration of 1975. One tissue microarray including 90 pairs of CRC and corresponding normal tissues was purchased from BioChip (Shanghai, China).

Anti-ZFX antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA) for immunohistochemical analysis and western blot analysis, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Total RNA was extracted from 30 pairs of matched CRC tissues using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). All tissues were examined histologically and all matched non-tumor tissues were confirmed to be normal. First-strand cDNA was synthesized using the PrimeScript™ RT reagent kit (Takara, Shiga, Japan) according to the manufacturer's instructions. qPCR was performed using an ABI Prism 7900HT sequence detection system (Applied Biosystems Life Technologies, Foster City, CA, USA) with SYBR® Premix Ex Taq™ II (Takara). 18s RNA was used as an internal control. The primer sequences were as follows: ZFX, 5′-GGCAGTCCACAGCAAGAAC-3′ (forward) and 5′-TTGGTATCCGAGAAAGTCAGAAG-3′ (reverse); 18s, 5′-CGGACAGGATTGACAGATTGATAGC-3′ (forward) and 5′-TGCCAGAGTCTCGTTCGTTATCG-3′ (reverse).

Total proteins were harvested from tissues or cells, and standard western blot analysis was performed using anti-ZFX and anti-GAPDH antibodies. All primary antibodies were used at a 1:1000 dilution. Peroxidase-conjugated anti-rabbit IgG secondary antibody was purchased from Kangchen Biotechnology (Shanghai, China) and used at a 1:5000 dilution.

IHC was conducted using a standard streptavidin-biotin-peroxidase complex method. Briefly, paraffin-embedded slices were deparaffinized and rehydrated. Endogenous peroxidase was blocked with 0.3% H₂O₂ for 15 min at room temperature, then the slides were microwaved for antigen retrieval. Following antigen retrieval, the sections were blocked with sheep serum for 30 min at room temperature and then treated with rabbit anti-ZFX (1:300) at 4°C overnight. Samples incubated with phosphate-buffered saline (PBS) were used as negative controls. Immunolabeling was detected using a diaminobenzidine detection kit (Maixin biotechnology, Fuzhou, China).

Human CRC cell lines HCT116 and LoVo were maintained in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and cultured in a humidified incubator at 37°C under 5% CO₂.

All siRNAs specifically targeting ZFX were synthesized by GenePharma (Shanghai, China) using the following sequence: 5′-GACGGGUGAUUCUAUACAUTT-3′, 5′-GUCGGAAAUUGAUCCUUGUTT-3′ and 5′-GCAACAGAGUGAGCUUAAATT-3′. The three siRNAs were used as a pool for siRNA transfection. Non-specific siRNAs (GenePharma) were used as a negative control.

HCT116 or LoVo Cells were seeded in 96-well plates at 5,000 cells/well following transfection. Cell proliferation was evaluated after 24, 48 and 72 h using cell counting kit-8 (Dojindo, Tokyo, Japan) according to the manufacturer's instructions.

Transwell cell invasion assays were performed using Boyden chambers with filter inserts (pore size, 8 µm) coated with 40 µg Matrigel in 24-well plates. Twenty-four hours after transfection with or without siRNA, cells were harvested, and 100 µl serum-free media containing 2×10⁵ cells was added into the upper chamber, while 600 µl medium with 20% fetal bovine serum was placed in the lower chamber. Following incubation for 24 h, the cells were fixed in 4% formaldehyde and stained with 0.1% crystal violet in PBS for 30 min at room temperature. Cells on the lower side of the filters were counted under light microscopy in ten randomly selected fields of each filter.

Data from at least three independent experiments are presented as the means ± standard deviation. Differences between two groups were compared using Student's paired t-test. The expression of ZFX in CRC tissues was analyzed using the χ² test. P<0.05 was considered to indicate a statistically significant difference.

First, the ZFX expression level was investigated in normal colorectal tissues and CRC tissues by qPCR and western blot analysis. The results suggested that ZFX mRNA was highly expressed in 66.7% of CRC tissues (P=0.0005, Fig. 1A and B). Western blot analysis of 20 paired CRC tissues further confirmed this finding (Fig. 1C and D).

Next, ZFX expression in 90 paired CRC tissues was analyzed by IHC, which revealed that ZFX was significantly upregulated in CRC tissues compared with the corresponding non-tumor tissues (P<0.001, Fig. 2A and B). As shown in Fig. 2, ZFX was readily detected in the plasma and part of the nuclei of CRC tissues, whereas little or no staining was observed in the normal colorectal tissues.

To test whether ZFX expression was correlated with various clinicopathological features of CRC, 90 CRC cases were stratified by ZFX expression as determined by IHC. ZFX upregulation was observed to be significantly associated with larger tumor size (P=0.01), higher pathological stage (P=0.02), depth of invasion (P=0.047), lymph node invasion (P=0.02) and higher American Joint Committee on Cancer (AJCC) stage (P=0.04), as detailed in Table I.

Given that ZFX is highly expressed in CRC tissues and is associated with various clinicopathological features of CRC, we further tested whether ZFX expression was correlated with poor prognosis in CRC patients. As shown in Fig. 3, CRC patients with higher ZFX expression exhibited a significantly shorter survival time (P=0.019). These results strongly suggest that ZFX expression is correlated with poor prognosis in CRC.

As ZFX was significantly upregulated in CRC tissues and associated with the severity of CRC, we next explored its potential function in CRC cells. To test this, specific siRNAs were employed to knock down ZFX expression in the human CRC cell lines HCT116 and LoVo. qPCR and western blot analysis revealed efficient knockdown of ZFX in HCT116 and LoVo cell lines (Fig. 4C and D). The cell counting kit-8 assay was adopted to explore the influence of proliferation by depletion of ZFX and revealed that suppression of ZFX significantly decreased the proliferation rate of HCT116 and LoVo cell lines (Fig. 4A and B). Transwell assay was used to further study the association of ZFX with cell invasion. Knockdown of ZFX significantly decreased the number of penetrated HCT116 and LoVo cells, which suggested a substantial loss of cell invasion ability by depletion of ZFX (Fig. 5A and B).