Matrix metalloproteinase 3 facilitates colorectal cancer progression by enhancing cell proliferation and invasion

Figure 1 Matrix metalloproteinase 3 is upregulated in colorectal cancer and associated with poor clinical outcomes. A: Analysis of matrix metalloproteinase 3 (MMP3) mRNA expression in colon adenocarcinoma tissues compared with normal adjacent tissues using The Cancer Genome Atlas (TCGA) data; B: MMP3 mRNA expression in rectal adenocarcinoma vs matched normal tissues based on the TCGA dataset; C: Reverse transcription-PCR analysis of MMP3 mRNA levels in 40 pairs of colorectal cancer (CRC) and matched adjacent normal tissues; D: Representative immunohistochemical (IHC) images displaying MMP3 expression in CRC and corresponding normal tissues; E: Quantitative analysis of IHC staining scores for MMP3 in 86 CRC samples from a commercial tissue microarray; F: Kaplan-Meier survival analysis based on IHC data indicates that elevated MMP3 protein expression correlates with reduced overall survival in CRC patients. MMP3: Matrix metalloproteinase 3; COAD: Colon adenocarcinoma; TCGA: The Cancer Genome Atlas; READ: Rectal adenocarcinoma; IHC: Immunohistochemical; HR: Hazard ratio.

Figure 2 Matrix metalloproteinase 3 regulates colorectal cancer cell proliferation, migration, invasion, and apoptosis. A: Matrix metalloproteinase 3 (MMP3) expression levels analyzed by quantitative real-time-PCR (qRT-PCR) in NCM460 and colorectal cancer (CRC) cell lines (SW480, SW620, HT-29, HCT116, LOVO); B: MMP3 expression levels analyzed by western blot in NCM460 and CRC cell lines (SW480, SW620, HT-29, HCT116, LOVO); C: Validation of MMP3 knockdown in SW480 and SW620 cells by qRT-PCR; D: Validation of MMP3 knockdown and overexpression in CRC cells by western blot; E: Wound healing assay to evaluate the impact of MMP3 on cell migration; F: Transwell assays to assess cell migration and invasion in response to MMP3 modulation; G: Colony formation assay showing the effect of MMP3 on CRC cell proliferation; H: Flow cytometric analysis of apoptosis in cells with MMP3 silencing or overexpression. MMP3: Matrix metalloproteinase 3.

Figure 3 Proteomic analysis identifies endothelial PAS domain protein 1 as a downstream target of matrix metalloproteinase 3 in colorectal cancer. A: SDS-PAGE and Coomassie Brilliant Blue staining to evaluate protein integrity in colorectal cancer (CRC) cell lines; B: Bar graph depicting the distribution of differentially expressed proteins (DEPs) across various comparison groups; C: Hierarchical clustering of DEPs, revealing distinct expression patterns in CRC cells; D: Volcano plot showing DEPs between MMP3 siRNA and control groups, highlighting key proteins such as endothelial PAS domain protein 1 (EPAS1); E: Bar chart of Cluster of Orthologous Groups analysis for DEPs; F: Pie chart illustrating the subcellular localization of DEPs; G: Gene ontology enrichment analysis of DEPs, identifying key biological processes; H: Bubble plot displaying Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of DEPs; I: PPI network for DEPs, identifying key protein interactions; J: Quantitative real-time-PCR validation of EPAS1 expression in MMP3-knockdown CRC cells; K: Protein interaction network within the P53 signaling pathway involving EPAS1.

Figure 4 Matrix metalloproteinase 3 knockdown impairs tumor growth in a colorectal cancer xenograft model. A: Representative images of subcutaneous tumors derived from control and matrix metalloproteinase 3 (MMP3)-knockdown SW620 cells in nude mice (n = 5); B and C: Tumor volume measurements and growth curves recorded over time; D: Immunohistochemical staining of MMP3, EPAS1, and Ki-67 in xenograft tumor sections; E: TUNEL staining indicating apoptotic cell death in tumor tissues; F: Western blot analysis of MMP3 and EPAS1 protein levels in excised tumors; G: Tumor weight measurements in the xenograft model. MMP3: Matrix metalloproteinase 3.