Abstract
Objective
To investigate the role of p65 (RelA, a key subunit of canonical NF-κB pathway) in colorectal cancer (CRC) cell proliferation, migration, invasion and its regulatory effect on NF-κB signaling.
Methods
p65 expression (total and nuclear) was detected in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell line (NCM460) by Western blot and qRT-PCR. p65 was overexpressed via plasmid (pcDNA3.1-p65) or knocked down via siRNA in HCT116 cells. Cell proliferation (CCK-8), migration (scratch assay), invasion (Transwell) and canonical NF-κB-related proteins (nuclear p65, p-p65 Ser536, IκBα, MMP-9) were analyzed.
Results
p65 was upregulated in CRC cells compared with NCM460 (P<0.01), with higher nuclear p65 and p-p65 levels in metastatic SW480. p65 overexpression increased HCT116 cell proliferation (OD450 at 72h: 1.42±0.14 vs. 0.95±0.10, P<0.05), migration rate (73.2±6.1% vs. 45.5±4.6%, P<0.01) and invasive cell number (135±12 vs. 59±7, P<0.01), while enhancing nuclear p65 accumulation, IκBα phosphorylation and MMP-9 expression (P<0.05). p65 knockdown showed opposite effects.
Conclusion
p65 promotes CRC progression by activating canonical NF-κB signaling and regulating pro-oncogenic genes, serving as a potential therapeutic target.
Keywords: Colorectal Cancer; Cell Proliferation; Transwell
Introduction
Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, with ~935,000 annual fatalities1. The canonical NF-κB pathway, activated by pro-inflammatory stimuli (e.g., TNF-α, LPS), is constitutively active in over 70% of advanced CRC cases-its core transcriptional subunit p65 (RelA) forms heterodimers with p50, translocates to the nucleus and drives expression of pro-oncogenic genes (e.g., MMP-9, Bcl-2) involved in cell survival, invasion and angiogenesis2,3. Clinical studies have shown elevated nuclear p65 expression in CRC tissues, correlating with tumor grade, lymph node metastasis and poor 5-year survival4,5. However, p65’s functional role in CRC cell behaviors and its mechanism of regulating canonical NF-κB activation remain to be fully clarified. This study uses CRC cell lines to verify p65’s effect on tumor progression and its association with NF-κB signaling.
Materials and Methods
Cell culture
HCT116 (low-metastatic CRC), SW480 (high-metastatic CRC) and NCM460 (normal colonic epithelial) cells were purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C, 5% CO₂. For canonical NF-κB stimulation, cells were treated with 10 ng/mL TNF-α (R&D Systems, Minneapolis, MN, USA) for 24h.
Transfection
p65 overexpression plasmid (pcDNA3.1-p65) and empty vector were from Addgene (Cambridge, MA, USA). p65 siRNA (si-p65) and negative control siRNA (si-NC) were from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells (5×10⁵ cells/well) were transfected with plasmids/siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at 60-70% confluency. p65 expression was verified by Western blot/qRT-PCR 48h post-transfection.
qRT-PCR and western blot
qRT-PCR: Total RNA was extracted with TRIzol (Thermo Fisher Scientific). cDNA was synthesized with PrimeScript RT Kit (Takara, Kyoto, Japan). p65 primers: Forward 5'-ATGACCGAGTACGAGAAGCC-3', Reverse 5'-TCAGCTGCTTCTCGTTGCTC-3'; GAPDH as internal control. Relative expression via 2⁻ΔΔCt method.
Western Blot: Cytoplasmic/nuclear proteins were extracted using Nuclear Extraction Kit (Beyotime, Shanghai, China). Equal amounts of protein (30μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, MA, USA) and probed with antibodies against p65 (total/nuclear), p-p65 (Ser536), IκBα, MMP-9 (Cell Signaling Technology, Danvers, MA, USA), Lamin B1 (nuclear loading control) and GAPDH (cytoplasmic control, Beyotime) at 4°C overnight. Bands were visualized with ECL kit and quantified by ImageJ.
Functional Assays
• CCK-8 Assay: Transfected cells (2×10³ cells/well) were seeded in 96-well plates. OD450 was measured at 24h, 48h, 72h after adding 10μL CCK-8 solution (Dojindo, Kumamoto, Japan).
• Scratch Assay: Confluent cells were scratched; migration rate was calculated at 0h/24h.
• Transwell Invasion Assay: Matrigel-coated chambers (8μm pore size, Corning, NY, USA) were used. Invasive cells were counted at 24h.
Statistical analysis
Data were presented as mean ± SD (n=3). Statistical analysis was performed using SPSS 26.0 (IBM, Armonk, NY, USA) with independent samples t-test. P<0.05 was considered significant.
Results
p65 is upregulated in CRC cell lines
qRT-PCR showed p65 mRNA in HCT116/SW480 was 4.15±0.39/5.02±0.47 folds of NCM460 (P<0.01). Western blot revealed total p65 protein in HCT116 (3.05±0.28) and SW480 (3.92±0.36) was significantly higher than NCM460 (1.00±0.10, P<0.01); nuclear p65 and p-p65 (Ser536) levels were further elevated in SW480 (2.12±0.20 and 2.05±0.19 folds of HCT116, P<0.05).
p65 promotes CRC cell proliferation
p65 overexpression increased HCT116 OD450 at 48h (1.18±0.11 vs. 0.77±0.08, P<0.05) and 72h (1.42±0.14 vs. 0.95±0.10, P<0.05). p65 knockdown reduced OD450 at 48h (0.63±0.07 vs. 0.92±0.09, P<0.05) and 72h (0.76±0.08 vs. 1.38±0.13, P<0.05). TNF-α stimulation enhanced proliferation in p65-overexpressing cells (OD450 at 72h: 1.65±0.15 vs. 1.42±0.14, P<0.05).
p65 enhances CRC cell migration and invasion
p65 overexpression increased HCT116 migration rate to 73.2±6.1% (vs. 45.5±4.6% in control, P<0.01) and invasive cells to 135±12 (vs. 59±7 in control, P<0.01). p65 knockdown reduced migration rate to 36.5±4.4% (vs. 71.8±5.9% in si-NC, P<0.01) and invasive cells to 51±6 (vs. 122±10 in si-NC, P<0.01).
p65 activates canonical NF-κB signaling
p65 overexpression increased nuclear p65 (2.15±0.20 vs. 1.00±0.09, P<0.05), p-p65 (Ser536) (1.98±0.18 vs. 1.00±0.08, P<0.05) and MMP-9 (1.92±0.17 vs. 1.00±0.07, P<0.05), while decreasing cytoplasmic IκBα (0.42±0.04 vs. 1.00±0.08, P<0.05). p65 knockdown showed opposite effects: nuclear p65, p-p65 and MMP-9 decreased (P<0.05), while cytoplasmic IκBα accumulated (P<0.05).
Discussion
This study confirms p65 is upregulated in CRC cells and its overexpression promotes proliferation, migration and invasion by activating canonical NF-κB signaling-consistent with its oncogenic role in gastric and pancreatic cancer6,7. Mechanistically, p65 is phosphorylated at Ser536, promotes IκBα degradation, forms heterodimers with p50 and translocates to the nucleus to drive pro-oncogenic gene (e.g., MMP-9) expression3, which enhances CRC cell invasive capacity. Limitations include lack of in vivo validation; future studies should explore p65’s crosstalk with the Wnt/β-catenin pathway in CRC8. Targeting p65 (e.g., via phosphorylation inhibitors or nuclear translocation blockers) may be a promising strategy for CRC treatment9.
Conclusion
p65 is upregulated in colorectal cancer cell lines and promotes CRC progression by activating canonical NF-κB signaling and regulating pro-oncogenic genes, highlighting its potential as a therapeutic target for CRC.
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