Cell culture
The CRC cell lines and HEK293T involved in our study were from American Type Culture Collection (ATCC, Manassas, VA, USA), and were cultured in RPMI1640 or DMEM containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, Massachusetts, USA) in the incubator at 37°C in a 5% CO2, 95% humidity atmosphere. Mycoplasma contamination was detected by PCR-based method every three months, and cell line authentication was performed using STR DNA profiling.
Clinical Samples And Immunohistochemistry
Primary CRC tissues and paired adjacent normal colon epithelium tissues were collected from surgical specimen of 125 patients clinically and histologically diagnosed with CRC (from 2012 to 2017) by the Sun Yat-sen Memorial Hospital, Sun Yat-sen University, and received no chemotherapy or radiotherapy before surgery. Prior patient consents were obtained for research purposes. The study was carried out in accordance with the Declaration of Helsinki, and approved by the Institutional Research Ethics Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University (SYSKY-2022-170-01). IHC was performed as previously described [36, 37]. The antibodies used were as follows: anti-PRPF19 (#39117, Signalway Antibody Biotech, Maryland, USA), and anti-MYL9 (#15354, Proteintech, Wuhan, China).
Sirna, Plasmids, And Cell Transfection
SiRNAs were synthesized by the GenePharma Company (Shanghai, China), and the sequences were shown in Supplementary Table S1. Flag-PRPF19 (EX-V1631-M12) and HA-MYL9 plasmids (EX-A4086-M06) were purchased from the GeneCopoeia Company (Guangzhou, Guangdong, China). The His-Ub, His-Ub-K48O, His-Ub-K63O, His-Ub-K48R, and His-Ub-K63R, constructed with pcDNA3.1 were purchased from the Gene Bank (Guangzhou, Guangdong, China). Cells were transfected with the indicated plasmids or siRNA using Lipofectamine 3000 (Thermo Fisher Scientific, USA) following the manufacturer’s instructions.
Rna Extraction And Quantitative Reverse Transcription Pcr (Qrt-pcr)
Total RNA was isolated from the indicated cells or tissues using Trizol reagent (Thermo Fisher Scientific, USA) following the manufacturer's instructions. cDNA was synthetized using PrimeScript RT Master Mix (#R323-01, Vazyme, Nanjing, Jiangsu, China), and qRT-PCR was performed using a SYBR Green Master Mix (#Q711-02, Vazyme, Nanjing, Jiangsu, China). GAPDH expression was used as an endogenous control. The primer used are shown in Supplementary Table S2.
Western Blot And Co-immunoprecipitation Assays
Western blot was performed as described previously [36]. For co-IP experiments, total cell lysates were obtained using Pierce IP Lysis buffer (#87787, Thermo Fisher Scientific, USA) containing protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA), and the supernatant was collected. For immunoprecipitation (IP), cell lysates were mixed with the indicated antibody overnight and recovered with Protein A/G Magnetic Beads (HYK0202, MedChem Express, Monmouth Junction, NJ, USA). The immunoprecipitates were washed three times, and then analyzed by immunoblot, or silver staining and LC-MS/MS. The antibodies used were summarized in Supplementary Table S3. Silver staining was performed according to the protocol of the Fast Silver Stain Kit (P0017S, Beyotime, China). The LC-MS/MS tests were performed by the Shanghai luming biological technology co.Ltd.
Stable Cell Line Construction
For stable knockdown of PRPF19, short hairpin RNA (shRNA) was synthetized by the GenePharma Company (Shanghai, China), and the sequences were shown in Supplementary Table S1. Briefly, HEK293T cells were co-transfected with viral packaging plasmids PMD2.G, pSPAX2 and lentiviral constructs (pHBLV-U6-MCS-PGK-PURO) containing the indicated shRNA or control shRNA for 48h, and virus supernatant was harvested to infect the indicated cells. The stably infected cells above were selected with puromycin (2 µg/mL) for two weeks.
For stable overexpression of PRPF19 or MYL9, the lentiviral constructs (pHBLV-CMV-MCS-EF1-NEO) containing the indicated cDNA were generated, and were transfected into HEK293T cells along with the viral packaging plasmids PMD2.G and pSPAX2 for 48h. Cell supernatant was harvested to infect the indicated cells. G418 (0.5 mg/mL) was used to select the stably infected cells for two weeks.
Cell Migration And Invasion Assays
Transwell assays were performed using inserts (BD Biosciences, San Jose, CA, USA) coated with or without Matrigel for invasion and migration assays, respectively. 100 µL medium without FBS was added to the upper well with 2×105 cells, followed by corresponding medium containing 10% FBS added to the bottom chambers. Cells migrated across the membrane were washed, fixed and stained at the indicated time, and were counted under microscope at 100 ×magnification.
Wound Healing Assays
Cells were seeded in 6-well plates (JET BIOFIL, Guangzhou, China) and incubated for 24 h. Subsequently, artificial wounds were scratched using sterile tips, and the cells were cultured in FBS-free medium. Cell migration was assessed by measuring the movement of the cells to the scratched area. The wound healing images were captured at the indicated time point at a magnification of 100× using an inverted microscope.
Protein Half-life Assays
The indicated cells were seeded in 6-well plates for 24h before being treated with the protein synthesis inhibitor CHX (100µg/ml, R750107, Sigma-Aldrich). Cells were then collected at the indicated time point and analyzed by Western blot.
Ubiquitination Assays
For in vivo ubiquitination assays, the indicated plasmids were transfected into cells for 48 h. Cells were harvested and lysed after incubation with proteasome inhibitor MG132 (20 µM, HY13259, MedChem Express, Monmouth Junction, NJ, USA) for 5h. Cell lysates were prepared and incubated with anti-MYL9 antibody overnight, and subsequently protein A/G beads were added and incubated for 2 h. The beads were washed with IP wash buffer three times, and the immunoprecipitates were detected by immunoblot.
For in vitro ubiquitination assays, MgATP Solution (R&D Systems, B-20), UBE1 (E1) (R&D Systems, E-305-025), UbcH5c/UBE2D3 (E2) (R&D Systems, E2-627-100), PRPF19 protein (Self-laboratory purification), MYL9 protein (Proteintech, Ag7636), 10X E3 Ligase Reaction Buffer (R&D Systems, B-71), Ubiquitin Recombinant Human HA-Ubiquitin Protein (R&D Systems, U-110-01M) and ddH20 were compounded in a 25ul reaction system following the manufacturer's instructions. The reaction mixture was incubated at 37°C for 60 min. Reactions were stopped by SDS buffer and resolved by SDS–PAGE for Western blot.
Animal Experiments
All animal experiments were performed in accordance with the ARRIVE guidelines. The study was approved by the Animal Care and Use Committee of of Guangzhou Medical University (S2022-100). Male BALB/c nude mice were purchased from Guangdong Medical Laboratory Animal Center. Mice were randomly assigned to four groups for indicated cells treatment. 2 × 10 6 HCT116 cells suspended in 100µL PBS were injected into the distal tip of the spleen. After 8 weeks of injection, mice were euthanized and livers were collected, and the metastatic nodules were evaluated using a microscope. Transverse sections were stained with H&E for histology analysis.
Bioinformatics Analysis
The clinical data of CRC patients and the gene copy number variations were obtained from The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. The clinical implication of PRPF19 was determined in UALCAN (http://ualcan.path.uab.edu/index.html), an interactive web portal [38, 39], to perform indepth analyses of TCGA transcriptome data to compare the expression of PRPF19 in CRC tissues with that in normal colon epithelium tissues.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Data shown in figures was represented as mean ± SD. Comparisons between two independent groups were assessed using two-tailed t-test, and two-tailed χ 2 test was applied to analyze the correlations between PRPF19 expression and clinicopathological features. Survival curves were plotted by the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate analyses were conducted using the Cox proportional hazard regression model. Pearson’s test was applied for correlation analysis. p ≤ 0.05 was considered to indicate statistical significance.