Colorectal cancer and adjacent normal tissue specimens were obtained from 20 patients (10 male and 10 female) (47±5.6 years old) between October 2015 and October 2016 who were treated in the Qianfoshan Hospital (Jinan, China). Written informed consent was obtained from all patients prior to participation in the study. The experimental protocol was approved by the ethics committee of Shandong University (Jinan, China).

The human colorectal cancer cell line SW480 was purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in L-15 Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), both Thermo Fisher Scientific Inc. (Waltham, MA, USA), 100 units/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in a humidified incubator at 37°C and 5% CO₂.

Small interfering RNA (siRNA) targeting the human Lasp-1 gene and negative control (NC) siRNA were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). A total of three Lasp-1 siRNA oligonucleotides (siRNA 1, 5′-UGUAGUUCUUCAUGUUCAGUGdTdT-3′; siRNA 2, 5′-UCAAACUCCUCCUUGUAGCGCdTdT-3′; siRNA 3, 5′-AUGGUAUUUUAUGUUACUGAUdTdT-3′) and one NC siRNA (5′-CAGUCGCGUUUGCGACUGGdTdT-3′) (30 pmol per 6 wells) were used. Cells were transfected using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h, according to the manufacturer's protocol.

The coding region of the Lasp-1 primer was synthesized by Sunshine Bio-Tech Co., Ltd. (Nanjing, China) and cloned into the pIRES2 vector (pIRES2-Lasp-1; Addgene, Cambridge, MA, USA). Cells in exponential phase were seeded in 96-well plates at a density of 3×10⁵ cells/well and cultured for 24 h before transfection. Cells were divided into three groups: NC, siRNA and siRNA+Lasp-1. The siRNA + Lasp-1 group was included as a rescue group, and cells in this group were co-transfected with siRNA and the Lasp-1-expressing vector. A 1 µl volume of Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was diluted in 50 µl serum-free Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) for 5 min, prior to being mixed with NC siRNA, Lasp-1 siRNA or Lasp-1 expression vector along with Lasp-1 siRNA (30 pmol per 6 wells) to prepare the transfection solution. A total of 100 µl transfection solution was added to each well and cultured for 6 h. The medium was then changed to DMEM containing 10% FBS. After 48 h of transfection, cells were collected with scraper and washed by PBS. Cells were deposited by centrifugation at 350 × g for 8 min at room temperature for further experiments.

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA was reverse-transcribed at 42°C for 40 min into cDNA using the Takara Reverse Transcription system kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. qPCR was performed using the SYBR Green Premix kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. GAPDH was used as an internal reference gene. The following primers were used: Lasp-1, 5′-GCTTCCATTGCGAGACCTG-3′ (forward) and 5′-TGCCACTACGCTGAAACCT-3′ (reverse); ERK1/2, 5′-GTGCCGTGGAACAGGTTGT-3′ (forward) and 5′-ATGGGCTCATCACTTGGGT-3′ (reverse); and GAPDH 5′-TGTTCGTCATGGGTGTGAAC-3′ (forward) and 5′-ATGGCATGGACTGTGGTCAT-3′ (reverse). qPCR was performed on target genes under the following conditions: 95°C for 2 min, 35 cycles of 94°C for 30 sec, 58°C for 45 sec and 72°C for 35 sec. Data were collected and calculated for CT values of all samples and standards based on fluorescent quantification using GAPDH as the baseline. Standard curve was firstly plotted using CT values of standards, followed by semi-quantitative analysis by 2^−ΔΔCq method (24).

Cells were lysed using the radioimmunoprecipitation assay buffer. Total protein was quantified using the Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Protein samples (50 µg) were boiled, then separated on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Merck KGaA). Membranes were blocked by 5% BSA for 1 h at room temperature and incubated overnight at 4°C with anti-Lasp-1 (1:20,000; cat. no. ab156872), anti-ERK1/2 (1:1,000; cat. no. ab17942), anti-phospho (p-)ERK1/2 (1:500; cat. no. ab214362), anti-GAPDH (1:2,000; cat. no. ab8245) primary antibodies (Abcam, Cambridge, UK) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; cat. no., ab181658, goat anti-HRP) at room temperature for 1 h (Abcam). Immunoreactive bands were detected using enhanced chemiluminescent HRP substrate (Merck KGaA).

Cells were fixed with 4% paraformaldehyde solution for 20 min at room temperature and then washed with PBS. A solution of 0.5% TritonX-100 in PBS was used for permeabilization of cells. Blocking was performed using 10% FBS for 1 h at room temperature and cells were incubated with anti-Lasp-1, anti-ERK or anti-phospho (p-)ERK1/2 primary antibodies (1:200; cat. no. ab214362) overnight at 4°C. After washing with PBS, the cells were incubated with secondary antibody (1:500; cat. no. ab150077; goat anti-rabbit IgG H&L (Alexa Fluor^® 488); Abcam). Nuclei were counterstained with DAPI for 5 min at room temperature. Staining was observed using a fluorescence microscope (×400, magnification) (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Cells were seeded in 96-well plates at a density of 3×10⁴ cells/0.1 ml and were cultured for 24, 48 and 72 h. Subsequently, 10 µl Cell Counting Kit-8 reagent (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was added to each well. After 4 h of incubation at room temperature, the optical density at 450 nm was determined. Results are reported as the mean ± standard deviation (SD) of at least three independent experiments.

Cells transfected with the Lasp-1 or NC siRNA were harvested with trypsin-EDTA and resuspended in DMEM supplemented with 10% FBS. The cells (100 µl cell suspension, 1×10⁶ cells/µl) were subsequently stained with fluorescein isothiocyanate-conjugated annexin V and propidium iodide (Beyotime Institute of Biotechnology, Haimen, China). Samples were incubated for 20 min in the dark at 4°C and a Cell Analyzer instrument (Merck KGaA) along with BD FACSDiva^™ software (version 8.0.1; BD, Franklin Lakes, NJ, USA) was used to evaluate cellular apoptosis.

Cells transfected with the Lasp-1 or NC siRNA were harvested with trypsin-EDTA after 72 h. Cells (1×10⁶) were resuspended in DMEM medium containing 10% FBS. The propidium iodide/RNase staining kit (MultiSciences Biotech Co., Ltd., Hangzhou, China) was used according to the manufacturer's protocol. The proportion of cells in each phase of the cell cycle (G₀/G₁, S and G₂/M) was determined using a flow cytometer and BD FACS Diva^™ software (BD Biosciences, Franklin Lakes, NJ, USA).

Migration and invasion assays were performed using Transwell inserts (BD Biosciences). For invasion, chambers were coated with Matrigel. Cells were seeded at a density of 2×10⁵ cells/250 µl DMEM supplemented with 0.1% FBS. Medium supplemented with 750 µl 10% FBS was added to the lower chamber and served as a chemotactic agent. Following incubation at 37°C for 12 h, non-migrating cells were discarded from the upper side of the membrane and cells on the lower side were fixed using ice-cold methanol (−20°C) and then air-dried. Cells were stained using crystal violet at 37°C for 30 min and imaged with Nikon Eclipse te200 Inverted Phase Contrast Microscopeat (×100, magnification; Nikon Corporation, Tokyo, Japan).

Results are presented as the mean ± SD. Statistical analysis was performed using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). Differences between groups were determined using analysis of variance or the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

A total of 20 pairs of tumor samples (tumor, T) and adjacent normal colorectal mucosa (normal, NT) tissues were used in the present study. Lasp-1 and EKR1/2 expression levels were investigated using RT-qPCR and western blot analysis (Fig. 1). It was demonstrated that Lasp-1 mRNA expression was upregulated in tumor tissues compared with the normal controls (Fig. 1A). ERK1/2 mRNA expression was also increased in tumor tissues compared with the normal controls (Fig. 1B). Regarding the protein level, Lasp-1, EKR1/2 and p-EKR1/2 were over expressed in tumor samples (Fig. 1C and D). These results suggest that the upregulation of Lasp-1 and ERK1/2 may be associated with the progression of colorectal carcinoma.

To investigate the role of Lasp-1 in the progression of colorectal carcinoma and the involved underlying molecular mechanism, Lasp-1 was knocked down by siRNA. Three siRNA oligonucleotides were designed. Among them, siRNA 1 was the most efficient (data not shown). It was then demonstrated that siRNA targeting Lasp-1 significantly downregulated Lasp-1 and ERK1/2 mRNA expression (Fig. 2A). Furthermore, the protein levels of Lasp-1, ERK1/2 and p-ERK1/2 were also suppressed by Lasp-1 knockdown (Fig. 2B and C). By contrast, Lasp-1 and p-ERK1/2 mRNA and protein levels were rescued in the siRNA group combined with Lasp-1 overexpression (Fig. 2A and C). Using immunofluorescence staining, it was further confirmed that Lasp-1 and p-ERK1/2 were decreased following Lasp-1 knockdown (Fig. 3A and B). Notably, it was observed that Lasp-1 inducedp-ERK1/2 expression, indicating that Lasp-1 regulates ERK1/2 expression in the progression of colorectal carcinoma.

The effects of Lasp-1 knockdown on cellular proliferation, induction of apoptosis and cell cycle arrest were investigated (Fig. 4). Cellular proliferation was impaired at 48 and 72 h after Lasp-1 knockdown (Fig. 4A). Furthermore, increased early-(19.0 vs. 8.4%) as well as late-(9.3 vs. 5.7%) stage (Fig. 4B) apoptotic cell death was detected. Furthermore, Lasp-1 silencing increased the proportion of cells in the G₀/G₁ phase of the cell cycle (79.88 vs. 51.65%) and decreased the proportion of cells in the S phase (12.12 vs. 37.74%). By contrast, overexpression of Lasp-1induced cellular proliferation and inhibited the induction of apoptotic cell death, compared with cells treated with siRNA (Fig. 4B and C). These results indicate that cell-cycle arrest in the G₀/G₁ phase of the cell cycle was induced following Lasp-1 knockdown.

Cell migration and invasion following Lasp-1 knockdown were investigated using the Transwell assay (Fig. 5). It was demonstrated that cellular migration and invasion were significantly inhibited in cells treated with Lasp-1 siRNA compared with the NC group (Fig. 5B and C). Notably, migration and invasion were increased following Lasp-1 overexpression. These results indicated that Lasp-1 promotes metastasis in colorectal carcinoma cells.