Engulfment and Cell Motility 1 (ELMO1) Regulates Tumor Cell Behavior and Predicts Prognosis in Colorectal Cancer

Materials and Methods

Cell culture and siRNA transfection. DKO1, COLO205, HCT116, SW480 and DLD1 cells (human CRC cell lines) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml gentamycin (Gibco). Cultures were kept at 37°C with 5% CO₂ in a humidified atmosphere. Small interfering (si)RNA (5′-GACAUGAUGAG CGACCUGA-dTdT-3′) and scrambled control used as negative control siRNA (cat. No. #SN-1002) were purchased from Bioneer (Daejeon, Republic of Korea). Transfection of the siRNA was performed using Lipofectamin™ RNAiMAX (Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol. Briefly, SW480 and DLD1 cells were seeded into 6-well plates such that they would be 40-60% confluent at the time of transfection. A total of 100 μM of ELMO1 siRNA and negative control siRNA was transfected with 5 μl Lipofectamine™ RNAiMAX reagent (Thermo Fisher Scientific, Inc.), respectively. After incubation for 48 h at 37°C, identification of ELMO1 expression was performed by western blotting and transfected cells and siRNA-transfected cells were applied to each experiment.

Cell proliferation assay. The effects of ELMO1 siRNA on cell proliferation in SW480 and DLD1 cells were determined by the water-soluble tetrazolium salts (WST)-1 assay (Daeil lab Inc., Seoul, Republic of Korea) assay. In summary, cells were dispersed within culture plates. A total of 10% WST-1 reagent was added to each well 1 h before the end of incubation. The optical density (OD) values at 450 nm in each well were determined by a microplate reader (Infinite M200; Tecan Austria GmbH, Grödig, Austria). Each experiment was performed in triplicate wells and was repeated at least thrice.

Western blotting. Total protein extracts were prepared using the RIPA^® reagent (Thermo Fisher Scientific, Inc.) containing protease and phosphatase inhibitors. Protein quantification of each sample was performed using the BCA™ protein assay (Thermo Fisher Scientific, Inc.). Subsequently, total protein samples were subjected to 8~12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Billerica, MA, USA). The blots were blocked with 5% BSA at room temperature and incubated overnight at 4°C with primary antibodies at 1:1,000 dilution. The blots were then washed with Tris-buffered saline/0.1% Tween-20 and incubated with horseradish peroxidase (HRP) conjugated secondary antibody (Cell Signaling, Danvers, MA, USA) at a dilution of 1:2,000. Protein bands were visualized using a chemiluminescent HRP substrate (Millipore) and the LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan). Immunoblots were quantified using Multi-Gauge gel analysis software (ver 3.0) (Fujifilm). Antibody against ELMO1 and GAPDH was purchased from Abcam (Cambridge, UK) (cat. No. ab2239) and Santa Cruz Biotechnology (Santa Cruz, CA, USA) (cat. No. FL-335), respectively. Antibodies against cleaved caspase-3 (cat. No. #9664), cleaved caspase-7 (cat. No. #8438), cleaved poly (ADP-ribose) polymerase (PARP, cat. No. #5625), Mcl-1 (cat. No. #2453), cyclin-dependent kinase (CDK) 2 (cat. No. #2546), CDK4 (cat. No. #2906), CDK6 (cat. No. #3136), cyclin D1 (cat. No. #2926), cell division cycle (CDC) 25C (cat. No. #4688), phospho-Akt (cat. No. #4060), phospho-glycogen synthase kinase-3β (phospho-GSK3β, cat. No. #5558), phospho-phosphoinositide-dependent protein kinase 1 (phospho-PDK1, cat. No. #3438), E-cadherin (cat. No. #3195), Vimentin (cat. No. #5741), Claudin1 (cat. No. #13255) were purchased from Cell Signaling. All experiments were repeated at least 3 times and bands of immunoblot were quantified using the Multi-Gauge software (Fujifilm).

Analysis of apoptosis by flow cytometry. Apoptosis of siRNA-transfected cells was analyzed by staining cells with Annexin V-APC/7-amino-actinomycin D (7-AAD) (BD Biosciences, San Diego, CA, USA). Cell apoptotic rate (%) was determined as the sum of the 7AAA- annexin V+ population (% early apoptotic cells) and 7AAD+/annexin V+ population (% late apoptotic cells). Cells were washed with PBS once prior to trypsinization. The trypsinized cell pellet was gently resuspended in 1x binding buffer (BD Biosciences). Subsequently, cells were stained by the addition of Annexin V-APC and 7-AAD. Cells were incubated in the dark for 10 min and subjected to detection by BD FACSCalibur flow cytometry (Becton Dickinson, San José, CA, USA). The apoptotic cell population was analyzed on the BD Cell Quest^® version 3.3 (Becton Dickinson) and WinMDI version 2.9 (The Scripps Research Institute, San Diego, CA, USA). All experiments were repeated at least 3 times.

Detection of cell-cycle distribution using flow cytometry. Cell-cycle distribution of siRNA-transfected cells was determined by flow cytometry DNA analysis. Cells were removed from plates by trypsin and fixed in 70% ethanol at −20°C. Fixed cells were washed with PBS and resuspended with 10 μg/ml ribonuclease A (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Resuspended cells were stained with 50 mg/ml propidium iodide (Sigma-Aldrich) for 15 min in the dark. DNA content was evaluated in BD Cell Quest^® version 3.3 (Becton Dickinson) and WinMDI version 2.9 (The Scripps Research Institute). All experiments were repeated at least 3 times.

Gelatin invasion assay. The invasion ability of siRNA-transfected cells was observed using Transwell chambers with 8-μm pores size (Corning Inc., NY, USA). A quantity of 1% gelatin with RPMI1640 was pipetted into the upper chamber (culture insert), which was placed in a lower chamber and dried on a clean bench. Cells transfected (1×10⁵ cells/well) with siRNA with 0.2% bovine serum albumin were seeded in the upper chamber and then transferred to the lower chamber consisting of 400 μl 0.2% BSA medium containing human plasma fibronectin (Calbiochem, La Jolla, CA, USA). After overnight incubation at 37°C, the upper chamber was fixed with 70% ethanol and stained with Diff-Quik solution (Sysmex, Kobe, Japan). Non-migrated cells on the inner surface of the upper chamber were wiped off using a cotton swab. Under an inverted microscope, invaded cells of the lower surface were then observed and counted in 5 selected fields under a light microscope. Results are expressed as mean±SD of the number of cells/field on three individual experiments.

Wound healing assay. The wound-healing assay was subjected to Culture Inserts (Ibidi, Regensburg, Germany). Cells transfected with siRNA were seeded in Culture Inserts. After becoming confluent, the inserts were gently removed using sterile forceps. Next, cells were cultured with a new culture medium, and wound-healing was photographed at 3 random sites at 0, 24, and 48 h of culture. The gap distance for each hour was converted to 1 cm and statistically analyzed. All experiments were repeated at least 3 times.

Patients and tissue samples. Fresh CRC tissues and adjacent paired normal tissues were collected from 20 CRC patients who underwent colonoscopic biopsy at the Chonnam National University Hwasun Hospital (Jeonnam, Republic of Korea). The biopsy of CRC tissues and adjacent normal tissues was quickly frozen in liquid nitrogen and stored at −80°C until RNA was extracted. Sera from 126 CRC patients who were histopathologically diagnosed in our Hospital were obtained. CRC patients did not receive any therapy before serum and tissue sample collection and had no other severe systemic diseases. Sera from a healthy individual group were obtained from 126 healthy subjects who visited for routine health screening. Sera was stored at −80°C until use. For immunohistochemistry, formalin-fixed and paraffin-embedded tissue samples were obtained from 425 patients who underwent surgery for pathologically confirmed colon cancers at the Chonnam National University Hwasun Hospital (Jeonnam, Republic of Korea) between July 2009 and June 2011. None of the patients had received preoperative radiotherapy or chemotherapy. The tissue blocks examined the original pathological slide, and selected blocks showed the junction between the normal colonic epithelium and the tumor region. Tumor staging was performed according to the American Joint Committee on Cancer (AJCC) staging system (29). Overall survival was analyzed from the date of initiation of the operation until 31 December 2017. This study was approved by the Institutional Review Board (IRB No. CNUHH-2019-046) of the Chonnam National University Hwasun Hospital. Also, ethical approval was obtained from the Institutional Review Board of the Chonnam National University Hwasun Hospital. The biospecimens and data used for this study were provided by the Biobank of Chonnam National University Hwasun Hospital, a member of the Korea Biobank Network with the approval of IRB. All participants gave their written consent for storage information in the hospital database and later use for research.

Enzyme-linked immunosorbent assay (ELISA). Blood samples were precipitated at room temperature for 30 min and centrifuged to separate serum. All serum samples were kept at −80°C before use. Serum levels of ELMO1 protein were measured using the ELMO1 ELISA kit (MyBiosource, San Diego, CA, USA), according to the manufacturer’s instructions. Briefly, serum samples and standards were placed on a Microelisa plate. Next, HRP conjugate reagent was added to each well and incubated for 1 h at 37°C. After washing the plate 4 times, chromogen solution was added to the microplate, and the OD was determined using a microplate reader (Infinite M200; Tecan). Finally, OD values were converted to concentration using the standard curve derived from serially diluted ELMO1 standards. Each experiment was performed in triplicate.

Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from siRNA-transfected cells was isolated using TRIzol reagent (Thermo Fisher Scientific, Inc.). After quantifying the amount of total RNA, 1 μg total RNA from each sample was converted to cDNA using 50 ng/μl oligo-dT and MMLV transcription reagents (Promega, Madison, WI, USA). Next, PCR amplification was performed using gene-specific primers and Go Taq^® DNA polymerase (Promega). The sequences of primers for ELMO1 were forward 5′-GCACTGAGCGATACCAGAAA-3′ and reverse 5′-CCTGTCTTCCAGGAGGTTAAAG-3′. The sequences of the primers for GAPDH were forward 5′-ACCACAGTCCATGCCATCAC-3′/5′-TCCACCACCCTGTTGCTGTA-3′. Finally, the PCR products were separated by electrophoresis on agarose gel and quantified on using Multi-Gauge software (Fujifilm).

Immunohistochemistry. Paraffin-embedded sections (4 μm) were dewaxed and rehydrated with graded alcohols. Antigen recovery was carried out in a citrate buffer (pH 6.0, Dako, Carpentaria, CA, USA) using a pressure cooker. The 10% goat serum and Dako REAL™ peroxidase blocking solution (Dako) were then used to block endogenous antigen and endogenous peroxide activity, respectively. Sections were incubated with primary antibodies against ELMO1 (Thermo Fisher Scientific, Inc.) at 1:600 dilution overnight at 4°C. TBST stained sections were stained using the Dako REAL™ Envision HRP/DAB detection system (Dako) and counterstained with hematoxylin for 30 s. Stained sections were observed and photographed using a light microscope.

Evaluation of ELMO1 expression. ELMO1 stained sections were divided into 2 groups (negative and positive) based on the percentage and intensity of stained cells. The percentage of stained cells was classified as follows: 0 (none), 1 (<10%), 2 (10-50%) and 3 (>50%). The degree of intensity of staining was classified as follows: 0 (no staining), 1 (weakly staining), 2 (moderately staining) and 3 (strongly staining). The overall score was defined as the value of the percent staining rate and intensity scores. The mean overall score of ≤3 was defined as negative, and of >3 was defined as positive. All samples were evaluated by two independent observers without the knowledge of the patient clinical outcome data.

Statistical analysis. Statistical analysis was performed using statistical software the Statistical Package for Social Sciences (SPSS) software version 20.0 (IBM Corporation, Armonk, NY, USA). The relationship between ELMO1 expression and clinicopathological parameters was investigated using the Pearson’s Chi-square test. The diagnostic value of serum expression of ELMO1 was evaluated using receiver operating characteristic (ROC) analysis. Survival results were reported using the Kaplan-Merrier method, and the significance of the difference was confirmed using the logarithmic rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. The results of the intergroup comparison were expressed as mean values±standard deviation (SD) of at least 3 independent experiments. The student’s t-test was performed for the comparison of 2 groups. Significance was set at p<0.05.