Sw620

Human colorectal cancer cell lines HCT116 and SW620 were purchased from the Chinese Academy of Sciences (Shanghai) Cell Bank. Human umbilical vascular endothelial cells were purchased from Beijing Huizhiheyuan Biotechnology Co. HCT116, SW620 and HUVEC cells were cultured in DMEM medium (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (Thermo, USA). All cell lines were incubated in a 37°C incubator with 5% CO2 according to the manufacturer’s instructions. Cell line morphology was evaluated regularly to ensure cell viability and quality.

Cell lines and culture. Human colorectal cancer cell lines SW480 and SW620 were procured from BCRC (Hsinchu, Taiwan, ROC) and maintained in Dulbecco’s Modified Eagle’s medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). The cells were cultured in a humidified atmosphere at 37˚C with 5% CO₂.
Western blotting. The antibody against Bmi1 (GTX114008) was acquired from GeneTex International (Irvine, CA, USA). Antibodies against Tubulin (#2144), GAPDH (#97166), β-actin (#3700), and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology (Danvers, MA, USA), whereas antibodies against MDK (ab52637) were obtained from Abcam (Cambridge, MA, USA). Cells were washed with ice-cold phosphate-buffered saline and lysed. Equal amounts of cellular proteins were collected, loaded onto a sodium dodecyl sulfate-polyacrylamide gel, and then transferred onto nitrocellulose membranes. Immunoblotting was performed using primary and secondary antibodies, followed by visualization and comparison of protein bands on the membranes using enhanced chemiluminescence reagents.
Immunohistochemistry staining and scoring. A human colorectal cancer tissue microarray (CDA3, Biomax Inc. and Super Bio Chips Laboratories, Seoul, Republic of Korea) was utilized. Detailed instructions regarding patient and tumor demographics and clinicopathological details are available on the manufacturer’s website. Tissue sections were stained with primary antibodies against Bmi1 and midkine (MDK), appropriate secondary antibodies, and the Envision system (Dako, Glostrup, Denmark). Subsequently, the sections were counterstained with hematoxylin and examined under a microscope. Expression intensity was categorized as "low" or "high" based on the proportion of positively stained tumor cells (18 (link)). We collected 43 clinical tissue specimens from patients with varying stages of colorectal cancer, monitored from 2009 to 2019 at E-Da Hospital, Kaohsiung, Taiwan, ROC (EMRP-109-012 and BIRB-109-002). All patients underwent pathological diagnosis of CRC through endoscopic or surgical biopsy, followed by regular post-diagnosis follow-up until death or the conclusion of the study period. Clinical data, including age, sex, histological grade, comorbidities, treatment regimen (e.g., irinotecan, bevacizumab), recurrence, and survival status, were retrieved from the Cancer Database of E-Da Hospital. Expression levels of Bmi1 and its target proteins in these specimens were assessed.
Zebrafish assay. Zebrafish [strain fli1: enhanced green fluorescent protein (EGFP)] were sourced from the Zebrafish Core Facility at the Center for Laboratory Animals, Kaohsiung Medical University, Kaohsiung, Taiwan. The care and maintenance of the zebrafish were conducted in strict compliance with standard animal care regulations and protocols of the Animal Center at Kaohsiung Medical University, Taiwan, ROC. These zebrafish feature eGFP under the fli1 promoter, specifically expressed in endothelial cells, enabling visualization of both the blood and lymphatic vascular systems. To elaborate, shLuci and shBmi1 SW-620 cancer cells were labeled using the PKH26 Red Fluorescent Cell Linker Kit (Merck KGaA, Darmstadt, Germany) and subsequently microinjected into the perivitelline cavity of 2-day-old zebrafish embryos. Upon confirmation of the localized PKH26-labeled cell mass at the injection site, the zebrafish embryos were transferred to fresh water and maintained at 32.5˚C. Vascular sprouts were then observed utilizing a Nikon Eclipse Ti-S 217 microscope (Tokyo, Japan) (19 (link)).
Animal study. BALB/c-null mice were procured from the National Laboratory Animal Center in Taipei City, Taiwan, ROC. At 8 weeks of age, six mice from each group underwent anesthesia with isoflurane, followed by injection of SW620 cells (2×10⁶; 1:1 mixed with Matrigel) into the cecum. The mice were carefully returned to the peritoneal cavity and sutured (20 (link)). After two months, the mice were weighed and anesthetized using CO₂. All experimental procedures were conducted in accordance with the Animal Care and Use Guidelines of Kaohsiung Medical University, Taiwan, ROC and approved by the Animal Care and Use Committee of Kaohsiung Medical University (IACUC protocol No. 107202).
Migration and invasion assay. Transwell units were employed for conducting migration and invasion assays. Briefly, 3,000 cells in 100 μl of medium with vehicle were placed in the upper part of the Transwell unit and allowed to invade for 24 h. The lower part of the Transwell unit was filled with lymphatic endothelial cells. Following incubation, invading cells on the bottom surface of the membrane were fixed in formaldehyde, stained with Giemsa solution, and enumerated under a microscope.
RNA interference transfection. To down-regulate Bmi1 expression in SW620 cells, small hairpin RNAs (shRNAs) were employed. The sh-Bmi1 and sh-luciferase (sh-Luci) plasmids were sourced from the National RNAi Core Facility (Academia Sinica, Taiwan, ROC). The sequences for sh-Bmi1#1 and sh-Bmi1#2 were as follows: sh-Bmi1#1: 5’-CAGATTGGATCGGAAAGTAAA-3’; sh-Bmi1#2: 5’- ATTGATGCCACAACCATAATA-3’; sh-Luci: 5’-CTTCGAAAT GTCCGTTCGGTT-3’. Cells were transfected with appropriate amounts of non-targeting and specific shRNAs using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), following the manufacturer’s instructions. Transfected cells were selected using puromycin, and the efficacy of Bmi1 silencing was evaluated using real-time reverse transcription-polymerase chain reaction.
Flow cytometry analysis. SW620 cells transfected with sh-Bmi1 were harvested by trypsinization and fixed with 70% ice-cold ethanol overnight at –20˚C. The following day, the cell pellet was suspended in propidium iodide (PI) staining buffer (50 μl/ml PI, RNAse A, Beckman Coulter, Brea, CA, USA) and then incubated for 15 min at 37˚C for subsequent cell cycle analysis. The distribution of the cell cycle was assessed using FACSCalibur (BD Biosciences, San Diego, CA, USA) and analyzed with ModFit software.
Quantitative real-time polymerase chain reaction (qPCR). Total RNA was isolated using the TRIzol reagent (Invitrogen) and reverse-transcribed with SuperScript III reverse transcriptase (Invitrogen) following the manufacturer’s protocol. The resulting cDNA was utilized as a template for PCR amplification. Quantitative real-time PCR was conducted in a 20 μl reaction volume using the standard protocols provided with the Roche LightCycler 480 II system (Basel, Switzerland). The primer sequences were as follows: MDK forward: 5’-AAGGAGTTTGGAGCCGACTG-3’, reverse: 5’-CATT GTAGCGCGCCTTCTTC-3’.
Chromatin immunoprecipitation (ChIP) assay. ChIP assays were conducted utilizing a Millipore ChIP kit (Merck Millipore, Darmstadt, Germany), with modifications to the manufacturer’s protocol. During the DNA fragmentation step, SW620 cells, along with SW620 cells depleted of Bmi1 or treated with PTC209, underwent sonication for 45 min each using high and low sonication settings, respectively. Protein pull-down assays utilized specific antibodies against RNA polymerase II. Subsequently, DNA from the pull-down was purified employing the MiniElute PCR purification kit following the manufacturer’s guidelines (Qiagen, VIC, Australia). The purification process was accompanied by the following PCR protocol: 95˚C for 2 min, followed by 35 cycles of 95˚C for 45 s, 58˚C for 45 s, and 72˚C for 45 s, with a final extension at 72˚C for 7 min. Primer pairs utilized for the MDK promoter chip sequences were as follows: (forward) 5’-GGCGGCCGGAGCGGGACGGG-3’ and (reverse) 5’-GGGG CGGCCCCTCGCCGCTA-3’.
Statistical analysis. Associations among various groups of cell-based experiments were evaluated using a two-tailed Student’s t-test. Differences in immunohistochemical staining intensity among subgroups were assessed using either the chi-square test or Fisher’s exact test. Pearson analysis was employed to examine the correlation between Bmi1 and target proteins. Kaplan–Meier survival analysis was utilized to illustrate prognostic differences between the subgroups. Statistical significance was defined as p<0.05. Statistical analyses were conducted using the Statistical Package for the Social Sciences (Chicago, IL, USA) version 25.0.