KCNE4 expression in colon cancer tissues was compared with that in normal tissues using the UALCAN database (http://ualcan.path.uab.edu), and the associations between KCNE4 expression and the pathological stages of colon cancer and the survival probability of patients with colon cancer were also investigated.

The LinkedOmics database (http://www.linkedomics.org/) is a web-based platform for the analysis of 32 The Cancer Genome Atlas cancer-associated multi-dimensional datasets. The KCNE4-associated genes were analyzed using Pearson's correlation coefficient and presented in a volcano plot, heat map and scatter plot.

The human colon cancer cell lines HCT-116 (cat. no. TCHu 99), SW480 (cat. no. TCHu172), SW1116 (cat. no. TCHu174), Caco2 (cat. no. TCHu146) and LoVo (cat. no. TCHu 82) were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The NCM460 normal colonic mucosa cell line (cat. no. CP-H040) was purchased from Procell Life Science & Technology Co., Ltd. All cells were cultured in Dulbecco's modified Eagle's medium (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; Hyclone; Cytiva), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere with 5% CO₂ at 37˚C.

Small interfering RNA (siRNA) targeting KCNE4 (si-KCNE4; 5'-GATTGGCTGTAAGTATCTCTA-3') and scrambled negative control (si-NC, 5'-GGATCACTAGTTACGATTGTT-3') were obtained from Shanghai GenePharma Co., Ltd. The EFEMP2 overexpression plasmid (Ov-EFEMP2) was established by inserting the EFEMP2 gene into a pcDNA3.1 vector (Shanghai GenePharma Co., Ltd.), whereas an empty vector served as the NC (Ov-NC). Transfection with si-KCNE4 (50 nM), si-NC (50 nM), Ov-EFEMP2 (0.5 µg) or Ov-NC (0.5 µg) was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 4 h strictly according to the manufacturer's guidelines. After another 48 h of incubation at 37˚C, the cells were collected for subsequent experiments.

The viability of HCT-116 cells was determined using a CCK-8 assay. Cells (5,000 cells/well) grown in 96-well plates were cultured for 48 h. Afterwards, 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added to each well and incubated at 37˚C for 2 h. The optical density at 450 nm was detected using a microplate-reader (Bio-Rad Laboratories, Inc.).

The colony-forming ability of HCT-116 cells was determined via colony formation assay. Cells plated at a density of 500 cells/well in 6-well plates were maintained in complete medium for 10 days. After that, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% crystal violet solution (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 10 min. Visible colonies were photographed under a light microscope (Olympus Corporation) and were counted using ImageJ software (Version 1.46; National Institutes of Health). Colonies consisted of ≥50 cells.

The migratory ability of HCT-116 cells was assessed using a wound healing assay. Cells (1x10⁵ cells/well) were seeded into 6-well plates and cultured up to 90% confluence. Then, the cells were scratched with a sterile 200-µl pipette tip and the detached cells were washed away with PBS. Afterwards, the cells were incubated in serum-free medium at 37˚C for 24 h and the area of the wound was photographed at 0 and 24 h under a microscope (magnification, x100). Migration was assessed as follows: Migration (%)=[(0 h average scratch distance-24 h average scratch distance)/0 h average scratch distance] x100.

The invasive ability of HCT-116 cells was assessed by performing a Transwell assay. The cells were re-suspended in serum-free medium and seeded at a density of 2x10⁴ cells/well into the upper compartment of a Transwell chamber (Corning, Inc.), which was precoated with 50 µl Matrigel (Beijing Solarbio Science & Technology Co., Ltd.) at 37˚C for 30 min. The lower chambers were filled with 600 µl FBS-containing medium. After 24 h of incubation at 37˚C, the invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution at room temperature for 20 min. Finally, the invaded cells were photographed and counted under a light microscope (magnification, x200).

Total RNA was isolated from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the RNA was reverse transcribed into cDNA using a PrimeScript RT Kit (Takara Bio, Inc.). The RT conditions were performed at 30˚C as the priming stage for 10 min, 42˚C as the RT step for 30 min and 95˚C as the RT inactivation stage for 5 min. Then, qPCR reactions were performed on an ABI PRISM 7900 Real-Time PCR system (Thermo Fisher Scientific, Inc.) using SYBR Premix Ex Taq kit (Takara Bio, Inc.). The thermocycling conditions were as follows: 95˚C for 10 min, followed by 35 cycles of 95˚C for 15 sec and 65˚C for 60 sec. The sequences of the primers were as follows: KCNE4 forward: 5'-CACCGCTACCTGAAAACCCT-3' and reverse: 5'-TTGATCGTGGCAGAGTGAGC-3'; EFEMP2 forward: 5'-GAGTGTCTGACCATCCCTGAG-3' and reverse: 5'-GCCGTGTAGGTCGTTGATGAC-3'; GAPDH forward: 5'-CAGGAGGCATTGCTGATGAT-3' and reverse: 5'-GAAGGCTGGGGCTCATTT-3'. GAPDH served as the internal control. Relative gene expression levels of KCNE4 and EFEMP2 were normalized to GAPDH and calculated via the 2^-ΔΔCq method (18).

Total protein was isolated from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) and protein concentrations were quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein samples (30 µg/lane) were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (MilliporeSigma). After blocking in 5% skimmed milk for 1 h at room temperature, the membranes were incubated with specific antibodies against KCNE4 (ab254642; 1:1,000), proliferating cell nuclear antigen (PCNA; ab92552; 1:1,000), Ki-67 (ab92742; 1:5,000), MMP2 (ab181286; 1:1,000), MMP9 (ab137867; 1:1,000), EFEMP2 (ab125073; 1:1,000) and GAPDH (ab181602; 1:10,000), all from Abcam, overnight at 4˚C. The next day, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (ab205718; 1:50,000; Abcam) for 1.5 h at room temperature. GAPDH served as the internal control. Signals of the immunoblots were developed using a BeyoECL plus kit (Beyotime Institute of Biotechnology) and analyzed with ImageJ software v1.8.0 (National Institutes of Health).

The interaction between KCNE4 and EFEMP2 was verified via Co-IP. Cells were lysed using IP lysis buffer (Beyotime Institute of Biotechnology). The supernatant was collected after centrifugation at 13,000 x g for 10 min at 4˚C. Next, anti-KCNE4 (ab254642; 1:200; Abcam) or anti-IgG (ab205718; 1:1,000; Abcam) was added to 250 µl cell lysate and incubated overnight at 4˚C. On the following day, the cell lysates were cultured with 25 µl protein A/G agarose beads (Santa Cruz Biotechnology, Inc.) for 4 h at 4˚C to bind the antibody complexes. Following centrifugation at 1,000 x g for 2 min at 4˚C, the agarose beads were washed in lysis buffer 3-4 times. Finally, a total of 15 µl 2X SDS sample buffer was added and the samples were boiled at 100˚C for 5 min, followed by western blot analysis to detect EFEMP2 and KCNE4, as aforementioned.

Data from three independent experiments are displayed as the mean ± standard deviation (SD). One-way analysis of variance followed by Tukey's post hoc test was employed for analyzing the statistical significance of differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

KCNE4 expression in colon cancer tissues and normal tissues, as well as the relationships of KCNE4 expression with the pathological stage of colon cancer and the survival probability of patients with colon cancer were explored using the UALCAN database. The UALCAN data indicated that KCNE4 was upregulated in colon cancer tissues compared with normal tissues (Fig. 1A). In addition, the expression level of KCNE4 in colon cancer tissues was positively associated with the cancer stage; KCNE4 expression in late-stage colon cancer was higher than that in early-stage colon cancer (Fig. 1B). Furthermore, patients with colon cancer whose tissues had high KCNE4 expression had a shorter duration of survival than those with low KCNE4 expression (Fig. 1C). In the present study, differences in the expression levels of KCNE4 in the HCT-116, SW480, SW1116, Caco2 and LoVo human colon cancer cell lines and the NCM460 normal colonic mucosa cell line were assessed via RT-qPCR and western blot assays. The KCNE4 mRNA (Fig. 1D) and protein (Fig. 1E) expression levels in the colon cancer cells were markedly increased compared with those in NCM460 cells, and the expression levels in HCT-116 cells were particularly high. Therefore, HCT-116 cells were selected for subsequent experiments.

HCT-116 cells were transfected with si-KCNE4 or si-NC and transfection efficiency was validated via RT-qPCR. Transfection with si-KCNE4 significantly downregulated KCNE4 expression compared with transfection with si-NC (Fig. 2A). CCK-8 assay results indicated that KCNE4 knockdown significantly reduced the viability of HCT-116 cells (Fig. 2B). Furthermore, reduced expression levels of PCNA and Ki-67 in the HCT-116 cells transfected with si-KCNE4 were observed, which also suggested that the viability of the colon cancer cells was repressed by the downregulation of KCNE4 (Fig. 2C). In addition, the colony formation assay revealed that the downregulation of KCNE4 suppressed the colony formation ability of the HCT-116 cells (Fig. 2D).

Wound healing and Transwell assays demonstrated that the migratory and invasive abilities of HCT-116 cells were significantly suppressed by KCNE4 knockdown (Fig. 3A and B). MMP2 and MMP9 are gelatinases that are able to degrade and remodel the extracellular matrix (19). Therefore, MMP2 and MMP9 play important roles in promoting the metastasis of cells. Decreased expression levels of MMP2 and MMP9 in the HCT-116 cells transfected with si-KCNE4 compared with those transfected with si-NC also indicated that the downregulation of KCNE4 restrained the migration and invasion of the colon cancer cells (Fig. 3C).

Genes associated with the key gene KCNE4 were identified using the LinkedOmics database. The genes indicated to be highly associated with KCNE4 in colon cancer are displayed in a volcano plot (Fig. 4A). The top 50 genes significantly positively correlated with KCNE4 in colon cancer are shown in a heat map (Fig. 4B). Using Pearson's correlation analysis, it was revealed that KCNE4 exhibited a strongly positive correlation with EFEMP2 (Fig. 4C). Moreover, the interaction between KCNE4 and EFEMP2 was validated via Co-IP analysis. EFEMP2 protein was detected in the anti-KCNE4 group, indicating that KCNE4 interacted with and bound to EFEMP2 (Fig. 4D).

Expression differences of EFEMP2 in the HCT-116 and SW1116 human colon cancer cell lines and the NCM460 normal colonic mucosa cell line were assessed via RT-qPCR and western blot assay. In comparison with those in NCM460 cells, EFEMP2 mRNA (Fig. 5A) and protein (Fig. 5B) expression levels in both colon cancer cell lines were significantly upregulated.

Rescue experiments were carried out to explore whether KCNE4 mediates colon cancer progression via the regulation of EFEMP2 expression. The transfection efficiency of Ov-EFEMP2 was validated via RT-qPCR, and this vector markedly upregulated EFEMP2 expression (Fig. 6A). The downregulation of KCNE4 reduced the viability of the HCT-116 cells, and this reduction was attenuated by EFEMP2 overexpression (Fig. 6B). Increased PCNA and Ki-67 expression levels in the HCT-116 cells co-transfected with si-KCNE4 and Ov-EFEMP2 compared with those transfected with si-KCNE4 and Ov-NC also indicated that the upregulation of EFEMP2 abolished the suppressive effects of KCNE4 knockdown on the viability of colon cancer cells (Fig. 6C). In addition, the decreased colony formation of HCT-116 cells transfected with si-KCNE4 was attenuated by EFEMP2 overexpression (Fig. 6D). In summary, these results indicate that KCNE4 knockdown suppressed the proliferative and colony formation abilities of colon cancer cells via the downregulation of EFEMP2 expression.

The downregulation of KCNE4 repressed the migratory and invasive abilities of colon cancer cells, and these changes were reversed by EFEMP2 overexpression (Fig. 7A-D). Furthermore, increased MMP2 and MMP9 expression levels in the HCT-116 cells transfected with si-KCNE4 and Ov-EFEMP2 compared with those transfected with si-KCNE4 and Ov-NC also indicated that upregulation of EFEMP2 abolished the suppressive effects of KCNE4 knockdown on the migration and invasion of colon cancer cells (Fig. 7E). In conclusion, these results suggest that KCNE4 knockdown weakens the migratory and invasive capacities of colon cancer cells via the downregulation of EFEMP2 expression.