Archived CRC tissues were obtained from 100 patients in 2005 and another 10 samples were obtained in 2018. Written informed consent was obtained from all patients for use of the specimens. The present study was approved (approval no. 2018-07-061) by the Institutional Review Board of Soonchunhyang University Cheonan Hospital (Cheonan, Korea). Immunohistochemistry (IHC) for LY6E was performed on paraffin-embedded tissues using 4-µm tissue sections mounted on slides. For immunohistochemistry staining of LY6E, all slides were blocked with serum and incubated with the LY6E primary antibody at 4°C, overnight (1:200; cat. no. GTX101567; GeneTex, Inc.). The slides were washed and then incubated with EnVision+ System- HRP-labelled polymer anti-rabbit (cat. no. K4002; Dako; Agilent Technologies, Inc.) for 30 min at room temperature. To evaluate the immunohistochemistry results, the antibodies were stained with 70 µl 3′-3-diaminobenzidine (DAB; cat. no. K3468; Dako; Agilent Technologies, Inc.) and visualized under a light microscope. The expression of LY6E in CRC tissues was scored in a blinded manner by independent investigators, and a consensus score was determined for each specimen. The expression scoring of LY6E was graded on a four-point scale based on the intensity and percentage of LY6E staining: The percentage of staining was scored as follows: 0, 0–5; 1, 5–25; 2, 25–50; 3, 50–75; and 4, 75–100%, whereas intensity of staining was scored in the following manner: 0, negative; 1, weak; 2, moderate; 3, strong. The final scores were calculated by multiplying of two scores: 0 points for negative, 1–3 points for weak, 4–6 points for moderate and 7–12 points for strong. The LY6E low expression group included negative and weak categories, and the LY6E high expression group included cases that scored above 4 points.

Human CRC cell lines (SW480, SW620, HCT116 and HT29) were provided by the Korean Cell Line Bank (Seoul, Korea) and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (cat. no. SH30027.01; Hyclone; Cytiva) containing 10% fetal bovine serum (FBS; cat. no. 35-015-CV; Corning, Inc.) and 1% penicillin/streptomycin (cat. no. 30-022-CI; Corning, Inc.) at 37°C in a 5% CO₂ incubator. The identities of CRC cells were confirmed by short tandem repeat (STR) profiling by KCLB and all cell lines were each purchased within the last 5 years.

CRC cell lines were transfected with 100 nM of the appropriate small interfering RNA (siRNA) with 12 µl of HiPerFect Transfection Reagent (cat. no. 301705; Qiagen GmbH) in 12-well plates. After 72 h, the cells were used for experiments. The targeting sequences for LY6E-specific siRNA were siRNA1: 5′-GCAUUGGGAAUCUCGUGACTT-3′, siRNA2: 5′-GCUUCUCCUGCUUGAACCATT-3′, and siRNA3: 5′-GCCAGAGCUUUCUGUGCAATT-3′. Non-targeting control siRNAs of AccuTarget™ Negative Control siRNA (cat. no. SN-1002; Bioneer Corporation) was used as control. The LY6E-specific siRNAs were used in combination.

Total RNA from CRC cell lines was extracted from the control group and LY6E-siRNA treated group using RiboEx Total RNA Solution (cat. no. 301-001; GeneAll Biotechnology). Using 1 µg of the extracted RNA, cDNA was synthesized using the ReverTra Ace™ qPCR RT kit (cat. no.FSQ-101; Toyobo Life Science) according to the manufacturer's instructions. The PCR reaction temperature conditions were followed by 35 cycles of pre-denaturation (95°C, 10 min), denaturation (95°C, 30 sec), annealing (55–65, 15 sec) and extension (72°C, 15 sec). RT-qPCR for LY6E was performed using primers purchased from Bioneer Corporation. To confirm the expression of genes for cancer metastasis-related cell growth and proliferation-related phenotypes, primers were designed for CDKN2A, IGF1, CXCR4 and MET and determined by RT-qPCR, and GAPDH was used as an endogenous control (Table I). The target gene mRNA level was quantified as GAPDH mRNA, and the gene expression level was analyzed using the 2^−ΔΔCq method (18). The amplicon was mixed with 10X loading buffer (TaKaRa Bio, Inc.) and then agarose gel electrophoresis was performed using 2% agarose gel containing NEOgreen (NeoScience) DNA staining reagent. then FluoroBox (Neoscience) nucleic acid gel imaging system was used to visualize the amplicon, and the mRNA expression level was analyzed using image J 1.53t software (National Institutes of Health).

Total cellular protein was extracted using PRO-PREP (Intron Biotechnology, Inc.) and quantified using a BCA kit (cat. no. 23227; Thermo Fisher Scientific, Inc.) and an xMark Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Inc.). The 30 µg protein was analyzed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and β-actin was used as a loading control. Then proteins were transferred to a polyvinylidene fluoride membrane (cat. no. ISEQ00010; MilliporeSigma). The polyvinylidene fluoride membrane was incubated with anti-LY6E antibody (1:100; cat. no. ABIN517581; Antibodies-online GmbH) overnight at 4°C, and the appropriate horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. 32260; Thermo Fisher Scientific, Inc.) was reaction for 2 h at room temperature. Protein was visualized with the addition of enhanced chemiluminescence (cat. no. 34577; Thermo Fisher Scientific, Inc.). Western blot results were visualized using a Molecular Imager ChemiDoc XRS + system (Bio-Rad Laboratories, Inc.) and quantified using the ImageJ software (National Institutes of Health).

The control group and LY6E-siRNA treated group of cells were collected, and 2×10⁵ cells were resuspended in 200 µl of medium and inserted into the upper of 6.5 mm Transwell^® with 8.0 µm Pore Polycarbonate Membrane Insert with serum-free RPMI-1640 medium. In the lower chamber, 600 µl of supplemented medium was added, and the cells were cultured at 37°C in a humidified incubator for 48 h. The remaining cells in the upper chamber that could not pass through the 8 µm pore-size filter were removed using a cotton swab. The cells were fixed with 3.7% neutral-buffered formalin at room temperature for 3 min and stained with 0.005% crystal violet at room temperature for 20 min. Cell images were obtained, and cells were counted using an inverted microscope. Invasion assays were performed as aforementioned for the migration assay, except that the Transwell chamber was coated with 50 µl Matrigel 3 h before the experiment and stored at 37°C.

Post 48-h transfection with LY6E-inhibited and scramble control siRNA, nine replicates (1×10⁴ cells/well) were uniformly dispensed in a 96-well plate and were incubated at 37°C, 5% CO₂ for 24, 48 and 72 h. Next, WST-1 assay reagent (EZ-CYTOX; cat. no. EZ-1000; DogenBio; http://www.dogenbio.com/) was added 10 µl (5 mg/ml) at every set time and reacted for 1 h. The absorbance was measured at 562 nm using a microplate reader.

As basement agar, 0.5% agarose was prepared in RPMI-1640 medium supplemented with 10% FBS. As top agar, the control and LY6E-siRNA treated groups were plated in six-well dishes (5×10³ cells/well) in 0.35% agarose in RPMI-1640 supplemented with 10% FBS. After 10–14 days, the colonies were stained for 1 h at room temperature with 0.005% crystal violet and images were captured after removing the growth medium. Numbers of colonies with a diameter greater than 30 µm were quantified after 2 weeks and analyzed using Image J 1.53t software (National Institutes of Health).

The data are presented as the average of the values of three replicates. Analysis was conducted using SPSS v19 software (IBM Corp.) was used for statistical analysis using unpaired Student's t-test. Mann Whitney U test and Kruskal-Wallis followed by Dunn's or Steel-Dwass post hoc tests were used to compare for analyzing the IHC scores. Cox regression and Kaplan-Meier curves were used to analyze the associations between LY6E expression and patient outcomes. P≤0.05 was considered to indicate a statistically significant difference.

To investigate the genes affecting among the LY6 family in CRC, the expression of LY6 family (LY6D, LY6E, LY6H, LY6K) was confirmed in colon adenocarcinoma and rectal adenocarcinoma based on the The Cancer Genome Atlas database (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). As a result, it was confirmed that only LY6E was specifically overexpressed among LY6 family in CRC and genes other than LY6E were rarely expressed in CRC (Fig. 1A). The mRNA expression was analyzed from 275 colon cancer tissues, 349 relevant normal tissues, 92 rectal cancer tissues, and 318 normal rectum tissues from the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/index.html) to investigate the role of LY6E in CRC. LY6E was found to show higher mRNA expression in tumor tissues than in normal tissues (Fig. 1B and C). Immunohistochemical staining was performed using tissues from 110 patients obtained from Soonchunhyang University Cheonan Hospital. Low LY6E expression was detected in 78 (70.9%), while high LY6E expression was found in 32 (29.1%) CRC tissues (Fig. 1D). However, lymphostromal cells did not express LY6E as revealed by immunohistochemistry. High LY6E expression was significantly associated with pN (P<0.0001) and clinical stage (P<0.0001, Fig. 1E and F, Table II). Univariate and multivariate cox proportional hazard regression analysis were performed to further evaluate according to clinical pathological factors whether expression of LY6E can be used as independent predictive biomarkers to predict treatment response. Univariate and multivariate analysis were expressed using hazard ratio (HR) according to clinicopathological factors (Table III). The univariate HR values for vascular invasion were 2.622 (1.151-5.974, P=0.022), and the univariate and multivariate HR values for lymphatic invasion were 3.216 (1.683-6.144, P<0.001), 2.272 (1.019-5.066, P<0.001), and metastasis were 6.860 (2.800-16.804, P<0.001), 6.472 (2.252-18.594, P<0.001). Also, in a Kaplan-Meier analysis of 110 patients, high LY6E expression was significantly associated with overall survival. High expression of LY6E significantly reduced cancer-specific survival in patients compared with those with low LY6E expression (log-rank test; P=0.048, Fig. 1G).

The expression of LY6E was confirmed using RT-qPCR and immunoblotting in four CRC cell lines (SW480, SW620, HCT116 and HT29). Then, four CRC cell lines were transfected by LY6E-targeted siRNA to inhibit LY6E expression. The mRNA level of LY6E decreased significantly in SW480 (57.8±4.6%), SW620 (72.7±2.5%), HCT116 (58.9±5.3%), and HT29 (50.9±4.6%) cells compared with that in the control group (Fig. 2A and B, P<0.001). Additionally, the protein level of LY6E decreased in SW480 (63.9±3.9%), SW620 (78.5±3.3%), HCT116 (55.3±3.2%) and HT29 (69.3±7.2%) cells compared with that in the control group (Fig. 2C and D, P<0.001). These results indicated that LY6E expression was successfully reduced at both the mRNA and protein levels in CRC cells.

The effect of LY6E downregulation on cell migration and invasion was examined. LY6E downregulation notably reduced cell migration into the lower chamber compared with that of control cells (SW480, 64.3±8.2%; SW620, 73.5±5.3%; HCT116, 58.3±7.9%; and HT29, 52.1±8.0%) (Fig. 3A and B, P<0.001). siRNA_LY6E cells had significantly reduced invasive properties (SW480, 57.3±7.3%; SW620, 69.3±8.4%; HCT116, 76.7±7.7%; and HT29, 55.4±7.0%) (Fig. 3C and D, P<0.001). To confirm whether LY6E plays an important role in CRC cell colonization, a colony formation assay was performed. Cells in which LY6E was downregulated showed decreased colony formation compared with the control group (SW480, 65.3±14.7%; SW620, 79.2±10.6%; HCT116, 69.5±9.7%; and HT29, 79.8±8.0%) (Fig. 3E and F, P<0.001).

Proliferation assay (WST-1 assay) was performed to determine whether the decreased LY6E gene is involved of cell proliferation in CRC. Cells in which LY6E was downregulated showed significant absorbance values compared with the control group (SW480, 1.81±0.09; SW620, 1.72±0.11; HCT116, 1.86±0.07; and HT29, 1.50±0.12) (Fig. 4A-D, P<0.001). The expression of genes for phenotypes related to cell growth and cell proliferation was compared between CRC cells in which LY6E expression was suppressed and a control. MET, IGF1, CDKN2A and CXCR4 are cell growth and cell proliferation-related genes that affect cancer metastasis (19) and were verified using RT-qPCR. Expression of MET, IGF1, CDKN2A, and CXCR4 was confirmed in four CRC control cell lines. By contrast, LY6E-suppressed CRC cells were confirmed that the expression of MET, IGF1, CDKN2A and CXCR4 was reduced (Fig. 4E and F).