Human and animal procedures in this study were approved by the Fujian University of Traditional Chinese Medicine (Fujian, China). Animal procedures were conducted in accordance with the guidelines of the Animal Committee of the University.
In a previous study, we screened DEGs on 14 pairs of CRC primary lesions and surrounding non-cancerous tissues (GEO submission: GSE113513) . We selected 10 of these genes and examined their expression in the GEPIA database (http://gepia.cancer-pku.cn/). Data were extracted from this database to examine association of NUFIP1 expression with clinicopathological stage of CRC and DFS.
The R2 application (http://r2.amc.nl) was used to explore the correlation between NUFIP1 expression and RFS of CRC patients using the log-rank method and the data in the dataset “Tumor Colon-CIT (Combat)-Marisa-566 rma-u133 p2; Tumor Colon (Core-Exon) Sveen-333 rma_sketvh-huex10p”.
Antibodies and reagents
Antibodies against HP1γ, H3k9me3 and HMGA2, and the Senescence β-Galactosidase Staining Kit were purchased from Cell Signaling Technology (CST; Beverly, MA, USA). Anti-NUFIP1 antibody, fetal bovine serum (FBS), trypsin-EDTA (0.25%), Pierce TM BCA Protein Assay kit, and FxCycleTMPI/RNase Staining Solution were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). The Annexin V-AbFluor™ 647 Apoptosis Detection kit and antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Abbkine (Wuhan, Hubei, China). UA was purchased from Millipore Sigma (Billerica, MA, USA).
Cell lines and cell culture
Human CRC cell lines (RKO, HCT-116 and HT-29 cells) were purchased from the Cell Bank of the Shanghai Institutes for Biological Science of the Chinese Academy of Sciences (Shanghai, China) and routinely cultured in RPMI1640 (Thermo Fisher Scientific; Carlsbad, CA, USA) or McCoy’s 5A medium (KeyGEN; Jiangsu, China) supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37 °C and 5% carbon dioxide. During long-term use of HCT116 and HT-29 cells, cells were checked using short tandem repeat genotyping and examined for mycoplasma contamination using real-time PCR.
Lentiviral transduction and cell growth analysis
Lentivirus encoding control shRNA or shRNAs targeting the 10 DEGs were constructed by Shanghai GeneChem (Shanghai, China). RKO cells were seeded in 12-well plates for 16 h prior to viral infection, then transduced with lentivirus at a multiplicity of infection of 10 in cell culture medium for 6-8 h, and then were changed into fresh medium and culture for total 72 h. Transduced cells were reseeded into 96-well plates at a density of 2,000 cells/well in 100 μL of completed medium. Cell growth was monitored every day for five days using the Pathway 855 high-content image analysis platform (BD Biosciences, San Jose, CA, USA), and cells were counted.
Quantitative PCR analysis
Total RNA was extracted from cell line samples using TRIZOL (Thermo Fisher Scientific; Carlsbad, CA, USA) and converted to cDNA using reverse transcription-PCR (Thermo Fisher Scientific; Carlsbad, CA, USA). The resulting cDNA, or a commercial tissue cDNA array (Shanghai Outdo Biotech; Shanghai, China), was used to measure levels of NUFIP1 mRNA using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the SYBR Premix Ex Tag (Thermo Fisher Scientific; Carlsbad, CA, USA). The following cycling conditions were used: 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Levels were quantified using the comparative Ct method and normalized to levels of GAPDH mRNA. The sequences of primers are listed in Supplementary TableS2.
TMA and survival analysis
TMA slides of CRC tissue samples and their corresponding noncancerous tissues were obtained from Shanghai Outdo Biotech (cat. no. HColA180Su15, Shanghai, China). All pathology specimens were collected, along with complete clinical and pathologic data. The TMA was incubated with antibody against NUFIP1 (1:1000) using standard techniques. Antibody binding was captured using a Nano Zoomer 2.0 HT slide scanner (Hamamatsu Photonics) and processed using Nano Zoomer Digital Pathology View 1.6 software. The intensity and extent of IHC staining were assessed independently by two experienced pathologists blinded to the clinical and pathologic data. Staining intensity was assessed using a four-point scale (0, undetectable; 1, weak; 2, moderate; 3, strong), while the percentage of positively stained cells was expressed as one of four categories (0–25%, 26–50%, 51–75%, and 76–100%). The two scores were multiplied together to yield the final overall NUFIP1 score.
Based on the overall scores from the TMA slides, patients were assigned to groups showing high NUFIP1 expression (score 9-12) or low expression (score 0–8). The relationship between NUFIP1 expression and overall survival was evaluated using Kaplan-Meier analysis and assessed for significance using the log-rank test.
Western blot analysis
Total proteins were extracted from cells using lysis buffer (Beyotime Biotechnology; Jiangsu, China) supplemented with protease inhibitor. The protein concentration was measured with BCA Protein Assay Kit (Thermo Fisher Scientific; Carlsbad, CA, USA), and equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%). Proteins were then transferred onto polyvinylidene fluoride membranes. After blocking with blocking buffer (Beyotime Biotechnology; Jiangsu, China) for 2 h, the membranes were incubated with a primary antibody overnight at 4 °C. The membranes were washed with TBST buffer, followed by incubation with the secondary antibody conjugated to horseradish peroxidase. GAPDH was used as loading control. Protein bands were detected with a chemiluminescence kit (Thermo Fisher Scientific; Carlsbad, CA, USA) and analyzed using the ImageLab software.
Cell confluence and cell counting
Cell confluence was observed by microscope (Leica Microsystems; Wetzlar, Germany) at 200× magnification. Then the cells were stained using 0.4% trypan blue, and analyzed using a Countstar Automated Cell Counter (Shanghai, China).
In vivo experiments
Male nude mice (4-6 weeks old, 22-24 g) were purchased from Shanghai SLAC Laboratory Animal Company (Shanghai, China) and maintained in a special pathogen-free facility. Transduced HCT116 or HT-29 cells (1 × 106) in 100 μL of M5A medium containing 50% Matrigel (BD Pharmingen; Franklin Lake, NJ, USA) were injected subcutaneously into the flank of nude mice (n=6). Tumor volume was measured using a standard caliper once every other day for 19 days, starting from the third day after first injection. The tumor volume was calculated using the formula: (length × width2) /2.
Mice were anesthetized with isoflurane and analyzed using an IVIS whole-animal imaging system (PerkinElmer; Santa Clara, CA, USA). Then mice were sacrificed, and tumor tissues were collected and weighed.
HCT116 or HT-29 cells were transduced with swh-NUFIP1 or sh-Ctrl and treated (or not) with 10 μM UA. At the indicated time points, cell viability was measured by adding 10 μL Cell Counting Kit-8 (Abbkine; Wuhan, Hubei, China) into 100 μL medium per well. The cells were cultured for an additional 2 h at 37 °C, and the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific; Carlsbad, CA, USA).
Colony formation assay
Cell survival was analyzed by colony formation assay. HCT116 or HT-29 cells were reseeded into 12-well plates (500 cells/plate) and incubated for about 10-12 days at 37 °C in a humidified atmosphere containing 5% carbon dioxide. Culture medium was changed every three days. At the end of experiment, the cells were fixed with 4% paraformaldehyde for 20 min, then stained with 0.01% crystal violet for 15 min. Colonies were counted and photographed.
Cell cycle analysis
Cell cycle progression was analyzed using an FxCycleTMPI/RNase Staining Solution in accordance with the manufacturer's instructions. Briefly, cells were seeded into six-well plates, incubated for an additional 72 h, collected, and fixed with 70% cold ethanol (4 °C) for about 24 h. Washed cells were incubated with a mixture of FxCycleTMPI/RNase Staining Solution for 40 min at room temperature in the dark, then analyzed by flow cytometry (FACS Caliber; Becton Dickinson, San Jose, CA, USA).
Cell apoptosis analysis
Apoptotic status was determined using annexin-V staining using Annexin-V-AbFlourTM647 Apoptosis detection Kit (Abbkine; Wuhan, Hubei, China) with or without propidium iodide staining, followed by flow cytometric analysis. Briefly, cells were transduced with shRNA-encoding lentivirus, treated or not with UA, collected and incubated with the solution from the apoptosis detection kit. The percentage of apoptosis was analyzed by flow cytometry on a FACS Caliber (Becton Dickinson, San Jose, CA, USA).
iTRAQ-based quantitative proteomic study and pathway analysis
Proteins were identified and quantified by secondary mass spectrometry using Thermo Scientific's Q Exactive mass spectrometer (Thermo Fisher Scientific; Carlsbad, CA, USA). DEPs were identified based on the following criteria: ½fold change½ in protein expression >1.5 and P<0.05.
KEGG (http://www.genome.jp/kegg) analysis was performed to analyze pathway enrichment in DEPs, while gene ontology (GO) (http://www.geneontology.org) was used to analyze signal transduction pathways. Enrichment of KEGG pathway and GO was considered significant when P < 0.05 (Fisher’s exact test).
Senescence β-galactosidase staining
Transduced cells were washed with PBS and fixed with 1× fixing solution. After 10-15 min at room temperature, fixed cells were washed and incubated with β-galactosidase staining solution at 37 °C overnight in a dry incubator. Images of β-galactosidase staining were taken using a microscopy (Leica; Wetzlar, German) at magnification of 400×.
Immunofluorescence staining was performed following standard protocols. Cells were washed with PBS, fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton-X-100. After blocking in 1% BSA, cells were incubated with diluted primary antibody (1:200) overnight at 4 °C. After washing with PBS, cells were subjected to the secondary antibody for 1 h at room temperature, followed by Hoechst staining (Beyotime Biotechnology; Jiangsu, China). All images were captured using microscopy (Leica; Wetzlar, German) at magnification of 400×.
Statistical analysis was performed using SPSS 26.0 software (IBM, Armonk, NY, USA). Data were presented as mean ± standard deviation. Differences between two groups were assessed using the independent Student's t test, and differences among three or more groups were assessed using one-way ANOVA. Kaplan–Meier survival differences were assessed using the log-rank test. P < 0.05 was considered significant.