Total 15 patients that have never received therapy before surgery were recruited from Affiliates Hospital of Beihua University in present study. Tissues were collected after acquirement of informed consent from patients. CRC tissues and paired adjacent normal tissues were collected and rapid frozen in liquid nitrogen for subsequent real time PCR analysis. All protocols have been approved by Ethics Committee of Affiliates Hospital of Beihua University.
Colorectal carcinoma cell lines
Colorectal cancer cell lines HCT116 and SW480 were obtained from the ATCC and cultured in RPMI 1640 (Hyclone) with supplement of 10 % FBS (Gibco-BRL; Invitrogen) at a humidity of 5 % CO2 at 37 °C.
Quantitative real time PCR
Total RNA was extracted from colorectal tissues of CRC patients or CRC cells with Trizol Reagent (Qiagen, Hilden). Complementary DNA (cDNA) was synthesized with equal 1 μg of total RNA by using superscript III reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). One microliter of the cDNA was amplified by real time PCR for AFAP1-AS1 using Bio-Rad iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA) with SYBR Green qPCR Super Mix (Invitrogen, Carlsbad, CA). Relative expression was calculated with the comparative Ct method and normalized against the Ct of β-Actin. The primers for qRT-PCR are as follows:AFAP1-AS1, Forward 5′-TCGCTCAATGGAGTGACGGCA-3′; Reverse 5′-CGGCTGAGACCGCTGAGAACTT-3′; AFAP1, Forward 5′-AGAGTGTCCTCCTCCACCAA-3′ Reverse 5′-CTTGGCCTCTGATTTGGAAC-3′ GAPDH, Forward 5′- CACCCACTCCTCCACCTTTG-3′; Reverse 5′-CCACCACCCTGTTGCTGTAG-3′.
To knockdown AFAP1-AS1, the siRNA oligonucleotides targeting AFAP1-AS1 and control (as mock indicated in manuscript) transfected into SW480 cells using Lipofectamine RNAiMAX (Invitrogen, CA) according to the manufacturer’s protocols. The sequences of the AFAP1-AS1 targeting siRNAs were 5′-GGGCTTCAATTTACAAGCATT-3′ (Si-RNA1) and 5′-CCTATCTGGTCAACACGTATT-3′ (Si-RNA 2).
CRC cell proliferation was evaluated by MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) assay. In brief, 1 × 104 cell/well cells were seeded in triplicate in 48-well plates and cultured in normal condition for 24 h under. Cells were transfected with si-AFAP1-AS1 for 24 h for subsequent MTT assay with MTT cell proliferation assay kit (Abnova, Walnut, CA), according to manufacturer’s protocols. Absorbance of each well was measured at 570 nm with a spectrophotometer (ELISA reader, Dynatech Laboratories, Chantilly, VA).
Cell in logarithmic phase was digested with 0.25 % trypsin and suspended in RPMI 1640 medium. A total of 400 cells were uniformly inoculated into 10 ml petri dish and cultured in normal condition for 2 weeks till visible colon. Colons were then fixed with 4 % paraformaldehyde for 15 min for subsequent staining with GIMSA. Colony number was calculated under optical microscope.
Wound scratch assay
A total of 5 × 105 cells were seeded in six-well culture plates and grown to 85–90 % confluence. Scratch was then vertically created by a p200 pipet tip in the cell monolayer. After a period of 24 h incubation in normal condition, images for cell growth around wounding were captured. Number of cell number around wounding calculated and was normalized to Mock.
Transwell invasion assay
SW480 cell invasion capacity was assessed using Transwell Chamber Cell Culture (10-μm pore membrane, BD Biosciences). A total of 1 × 105 cells in 100 μl of serum-free medium were added to the top chamber of 24-well plates that has been pre-added with Matrigel. The bottom well contained growth medium with 20 % FBS. Transwell chambers were placed at 37 °C for 36 h. Cells in chamber were fixed with methanol for 30 min and then staining with Giemsa for 15–30 min. Invaded cells were finally observed under a microscope and the number was counted with randomly nine field for each experiment.
Protein expression of N-cadherin, vimentin, Fibronectin E-cadherin and MMP-9 was performed with immunoblot analysis in cell lysates. After total protein quantification by BCA kit, an equal quantity 30–40 μg protein of all samples was separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and then electrophoretic transferred onto PVDF membrane. Membranes were experienced blockage with 5 % skim milk in TBS buffer, incubation with primary antibodies for target gene, and subsequent incubation with horseradish peroxidase-conjugated secondary antibody. Between each step, membranes was washed with TBS suppled with 20 % Tween20. In the present experiment, anti-E-cadherin antibody, anti-N-cadherin antibody, anti-Vimentin antibody, anti-Fibronectin antibody and anti-MMP9 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-GAPDH antibody and anti-AFAP1 antibody were purchased from Proteintech (Wuhan, China). The signal was finally visualized using an ECL detection reagent on Bio-Rad ChemiDoc MP. Visualization of GAPDH expression was used as a loading control.
Male C57BL/6 nude mice with 7-week-age were obtained from SLAC Laboratory Animals Co Ltd (Shanghai, China). Animals protocols have been approved by animal Ethics Committee of Affiliates Hospital of Beihua University and performed according to the institutional guidelines.
Subcutaneous tumor transplantation was performed by 106 of SW480 cells injection in the right flank of mice. Tumor was removed after 4 weeks transplantation and tumor weight was measured.
Hepatic metastasis of colonic carcinoma was performed by 106 of SW480 cells injection into spleen of nude mice. Mice was euthanatized after 4 weeks injection. Hepatic metastasis nodules were then counted.
Statistical analysis was performed using SPSS software (version 16.0; Chicago, IL). Student’s t tests were used to evaluate significant differences between any two groups of data, or one-way analysis of variance with the Bonferroni post-test was performed for three or more comparisons. All data are represented as mean ± standard deviation. A p values <0.05 was considered significant.