Bioinformatics analysis
Limma package (version: 3.40.2) of R software was applied to screen differentially expressed mRNAs between CRC and normal intestinal tissues in The Cancer Genome Atlas (TCGA) or Genotype-Tissue Expression (GTEx). The false positive results were corrected by analyzing the adjusted P-value. Adjusted P<0.05 and Fold Change >2 were defined as the thresholds for screening. TCGA database was considered to analyze the gene expression profile of DMBX1 in different tissues by Gene Expression Profiling Interactive Analysis 2 (GEPIA2) (http://gepia2.cancer-pku.cn/#analysis).
Cell culture
CRC cell lines RKO, SW480 and HCT116 were acquired from the Chinese Academy of Sciences (Shanghai, China), and SW620 CRC cell line was purchased from Genechem (Shanghai, China). HCT116 and SW620 cells were cultivated in RPMI-1640 medium, and RKO and SW480 cells were respectively cultivated in MEM medium and L-15 medium, including 10% FBS under humidified condition with 5% CO2 at 37 ° C.
Real-time quantitative PCR (RT-qPCR)
RNA extraction from the cultured cells by using TRIzol reagent (Pufei Biotechnology, Shanghai, China) and further reversely transcribed to cDNA with M-MLV kit (Promega, Madison, Wisconsin). RT-qPCR was executed by SYBR Master Mixture (cat.no. DRR041B; Takara). GAPDH was regarded as an internal control. The specificity was confirmed via melting curve analysis, and the relative expression amount was computed by the 2−△△Ct method. Primers are shown in Additional file 1: Table S1.
Western blotting
Total protein of culture cells was isolated by lysis buffer, and further determined by the BCA Protein Assay Kit (cat. no. P0010S; Beyotime). The cell lysates were isolated on SDS-PAGE and transferred to polyvinylidene fluoride membranes (cat.no. IPVH00010; Millipore) for blotting. The membranes were blocked by TBST involves 5% non-fat milk for 1 h, and later incubated with the primary antibody at 4 ° C overnight. The primary antibodies were as follows: anti-DMBX1 (Sigma-Aldrich, St. Louis, MO), anti-GAPDH (Santa Cruz Biotechnology), anti-c-Myc (Abcam, Cambridge, MA), anti-twist (Abcam), anti-MMP2 (Abcam), anti-MMP9 (Cell Signaling Technology, Danvers, MA). After washing with TBST, the secondary antibodies were further supplemented for 1.5 h. Proteins were quantified using the Pierce™ ECL Western Blotting Substrate (Thermo).
Lentivirus transfection
The shRNA sequence targeting DMBX1 (5'-CTGGCTCCTGCATCAGCTA-3') was designed by Genechem. The T4 DNA Ligase was used to ligate the double-stranded DNA and the double digested linearized GV115 vector containing enhanced green fluorescent protein (EGFP). The recombinant plasmids were transferred into TOP10 E. coli competent cells. The 293T cells were transfect to obtain Lentivirus particles using GV115, pHelper 1.0 and pHelper 2.0 vector (Genechem). Consistently, Lentivirus vector of c-Myc overexpression containing mCherry were constructed by Genechem. Subsequently, RKO and HCT116 cells were inoculated into 6-well plates at an MOI of 10. The expression of EGFP or mCherry was observed by fluorescent microscope using a model IX71 microscope (Olympus, Tokyo, Japan) to assess positive infection efficiency after 72 h.
Celigo cell counting
The lentivirus-infected cells were seeded in 96-well plates (1500 cells/well), and further cultivated in incubators at 37 ° C containing 5% CO2. The Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, MA) was considered to analyze the number of green fluorescence emitted cells for 5 consecutive days after 24 h incubation.
MTT assay
After lentivirus infection, cells were inoculated into 96-well plates (2000 cells/well). Following 24 h, 20μl of 5 mg/ml MTT was supplemented and maintained for 4 h. The supernatants were disposed, and 100 µl of dimethyl sulfoxide was further supplemented. After oscillation for 3 min, a M2009PR microplate reader (Tecan Infinite) was used to determine the optical density (OD) at 490 nm.
Colony formation assay
After shRNA lentivirus infection, cells were inoculated into 6-well plates (600 cells/well) and cultivated for 8 days. These cells were fixed in 4% paraformaldehyde for 30 minutes, and later stained by 1000 µl crystal violet (Sangon Biotech, Shanghai, China) for 10 minutes. After rinsing with double distilled water, cell colonies were further counted below a microscope.
Cell apoptosis assay
The shRNA lentivirus-infected cells were inoculated into 6-well plates. After 2 days, these cells were harvested and washed using PBS, and stained with 10 µl Annexin V-APC (eBioscience, MA) and kept away from light for 15 min. Finally, flow cytometry was used to determine cell apoptosis.
Wound-healing assay
The shRNA lentivirus-infected cells were inoculated into the 96-well plates. A small wound area was created using a 96 Wounding Replicator after cells exceeded about 90% confluence. Then, cells were carefully rinsed using serum free medium. Celigo Image Cytometer (Nexcelom Bioscience) was used to obtain photograph and analyze the area of migrated cells at 0, 8 and 24 h.
Transwell assay
Cell migration was measured by transwell chambers (cat. no. 3422; Corning, NY). Briefly, 100 µl of cell suspension with a concentration of 1×105 cells per well was inoculated into the upper chamber, and 600 µl of 30% FBS was replenished to the lower chamber. After 24 h, cells were fixed with 4% paraformaldehyde for 30 min and later stained with crystal violet for 3 min. Photomicroscope was used to obtain images at ×200 magnification.
Statistical analysis
Data were presented as mean ± standard deviation. Student’s t-test (two-tailed) was used to analyze statistical significance between experimental group and normal control group. P<0.05 was regarded as a statistical difference.