Immunohistochemistry (IHC)
The LOXL1 expression levels were assessed using IHC on the paraffin-preserved tissue sections of 30 pairs CRC patients and 15 pairs CRC with liver metastasis patients. Primary LOXL1 antibody were obtained from sigma (HPA042111, anti-LOXL1 diluted 1:50). Each specimen was scored according to the proportion of positive cancer cell as: 1, 0−25%; 2, 25−50%; 3, 50−75%; and 4, > 75%. Specimens were also scored according to the staining intensity of cancer cells as follows: 0, negative; 1, light yellow; 2, dark yellow; 3, brown. The IHC staining score was calculated by multiplying the proportion of positive cancer cells by the staining intensity of cancer cells. All samples were obtained with approval from the Institutional Ethics Committee of The First Affiliated Hospital of Soochow University (authorisation number ECSU-2019000212)
Cell Culture
Human colorectal cancer cell lines such as DLD1, HCT116, HCT8, HT29, LoVo, SW480, SW620, and RKO were purchased from American Type Culture Collection (ATCC). All CRC cell lines were maintained in RPMI 1640 supplemented with 10% foetal bovine serum and 1% antibiotic (penicillin and streptomycin) at 37oC in an atmosphere of 5% CO2 and 95% air.
Lentiviral Vector Construction And Packaging
Lentiviral constructs of LOXL1 encoding pLent-EF1a-FH-CMV-GFP-P2A-puromycin were prepared as described previously [17]. Lentivirus expressing LOXL1 was produced in HEK293T cells and then packaged using pMD2.G and psPAX2. The HCT8 and SW480 cell lines were infected with the viral supernatant using 8 µg/mL polybrene (Sigma) and the infected cells were incubated for 48 h. Single colonies were obtained through puromycin selection (8 µg/mL, 2 m), which were detected using western blotting.
Wound Healing Assay
First, 1 × 106 cells were cultured in six-well plates and incubated for 24 h. The cultured cells were rinsed thrice using phosphate buffered saline (PBS) and three wounds (scratches) were created in parallel using a sterile 200-µL pipette tip. The wells were washed thrice with PBS to discard any floating cells. Representative images of their migration were captured immediately using a microscope, 24 h and 48 h after scratching.
In vitro transwell migration and invasion
Cell migration and invasion experiments were performed using 24-well plates with 8 µm-polycarbonate filter inserts (CORNING). HCT8-N/HCT8-LOXL1, SW480-N/SW480-LOXL1 and HT29-N/HT29-LOXL1 (KO), and RKO-N/RKO-LOXL1 (KO) were seeded at densities of 2 × 105 cells/200 µL and 1 × 105 cells/200 µL per well, respectively, in serum free RPMI 1640. All cells were either uncoated or Matrigel-coated (Biocoat) and incubated in chambers containing 600 µL of RPMI 1640 with 10% foetal serum as a chemoattractant. The cells were imaged and their migration and invasion was captured using a microscope. The migrating and invading cells were eluted using acetic acid and quantified by measuring their absorbance at 570 nm. All experiments were performed thrice, independently.
Plate Colony Formation Assay
HCT8-N/HCT8-LOXL1, SW480-N/SW480-LOXL1, RKO-N/RKO-LOXL1 (KO), and HT29-N/HT29-LOXL1 (KO) cells were independently seeded in six-well plates at densities of 5000 cells/well at 37oC. The medium, RPMI 1640 containing 10% foetal serum, was changed every alternate day. After 10 d, the cells forming colonies were immersed in 4% paraformaldehyde for 20–30 min, stained using crystal violet for 2 h, and rinsed thrice with PBS to remove the excess crystal violet. Finally, images were captured using a microscope and the number of colony-forming units were counted.
Luciferase Reporter Assay
The promoter of 3 × GTIIC was subcloned into the XhoI/HindIII site of the pGL4.2 vector (Promega). HEK293T cells were transiently co-transfected with the pGL4.2-3 × GTIIC, pcDNA3.1-YAP, and LOXL1/LOXL1 ∆SP/LOXL1 mutants. The pRL-TK vector was co-transfected in each experimental sub-hole as an internal control. After 24 h of transfection, the cells were collected and analysed using the Dual-Luciferase Reporter Assay Kit (Promega).
Immunoprecipitation And Western Blotting
HEK293T cells were transfected with FL LOXL1 and its mutants. After 24 h, the medium was collected and centrifuged at 1000 g for 5 min. The supernatant was subjected to immunoprecipitation using M2-conjugated magnetic beads (Sigma) by rotating for 4 h at 4oC. The immunoprecipitates were washed thrice using PBS and subjected to western blot analysis. Additionally, the cells were lysed using a lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol, 1 mM DTT, and the complete protease inhibitor cocktail) for 10 min on ice and centrifuged at 20,000 g for 30 min. The cell lysates were analysed by subjecting them to SDS-PAGE and immunoblotting with antibodies as indicated in the figures.
Total RNA isolation and Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Expressions of the genes CYR61, CDC20, CDX2A, and CTGF were detected using q-PCR and normalized to that of GAPDH. Total RNAs were extracted using TRIzol (TIANGEN), according to the manufacturer’s protocol. Total RNA (1 µg) was reverse transcribed using the PrimeScript RT reagent Kit (TaKaRa). SYBR green (Bimake) and an ABI Step One Plus real-time PCR system (Applied Biosystems) were used to conduct q-PCR. The primers used in this study are mentioned in Table 1.
Table 1
Primers used for real-time quantitative PCR (QPCR)
| | | | | | |
Target mRNA | | Sequences 5′—3′ |
GAPDH | F | 5’- GGAGCGAGATCCCTCCAAAAT-3’ |
| R | 5’- GGCTGTTGTCATACTTCTCATGG-3 |
CDX2 | F | 5’-CCAATGACAACGCCTCCTG-3’ |
| R | 5’-TGGTGCAGCCAGAAAGCTC-3’ |
CTGF | F | 5’-AAAAGTGCATCCGTACTCCCA-3’ |
| R | 5’-CCGTCGGTACATACTCCACAG- 3’ |
CYR61 | F | 5’- AGCCTCGCATCCTATACAACC- 3’ |
| R | 5’- TTCTTTCACAAGGCGGCACTC-3’ |
CDC20 | F | 5’-GACCACTCCTAGCAAACCTGG-3’ |
| R | 5’- GGGCGTCTGGCTGTTTTCA-3’ |
Animal Experiments
To carry out the xenograft tumorigenesis assays, 1 × 107 cells of HCT8-N/HCT8-LOXL1 were subcutaneously injected into 4-week-old male nude BALB/c mice. The tumour sizes were monitored every 3 d and their volumes were determined using the following formula: volume (mm3) = (length × width2)/2. Subsequently, they were subjected to H-E staining and immunohistochemistry (IHC).
To carry out the tail vein metastasis assay, cells were injected into the lateral tail veins of 4 w old male nude BALB/c mice. Eight weeks later, the mice were anesthetised using nembutal (pentobarbital, TRC). The mice were sacrificed and examined at necropsy for the presence of metastases. Their lungs, livers, and bones were fixed in formalin. Subsequently, the samples were subjected to H-E staining and IHC.
To conduct the liver metastasis assay, cells were harvested using 0.25% trypsin, washed thrice with PBS, and suspended in PBS at a final concentration of 1.5 × 107 cells/mL. The 6-week-old BALB/c nude mice were anesthetised through an intraperitoneal injection of nembutal at a dose of 75 mg/kg. Then, a small incision, approximately 10 mm in length, was made through the skin over the spleen. Using a 27 gauge needle, 100 µL of the tumour cell suspension was slowly injected into the spleen, after which it was placed back in the abdominal cavity. The incision was closed through simple continuous suturing. The mice were sacrificed after 20 d and liver metastasis was confirmed pathologically [18].
All animal experiments were approved by the Animal Care and Use Committee as well as the Ethical Committee of Soochow University (SYXK2017–0043). All surgery was performed under sodium pentobarbital anesthesia with minimum fear, anxiety and pain.
Statistical analysis
The data obtained was statistically analysed using SPSS (version 20.0; IBM, New York) and represented as the mean ± SD. A t-test (for two groups) was used to determine differences between the groups, which were considered statistically significant at P < 0.05.