Identification and validation of the role of matrix metalloproteinase-1 in cervical cancer

Abstract

Lymph node (LN) metastasis at an early stage of cervical cancer is often an indicator of poor prognosis and is critical for subsequent adjuvant therapy. The current study aimed to identify aberrant gene signatures and biomarkers of metastasis for patients with cervical cancer. RNA-sequencing data of 132 LN negative (N0) and 60 LN positive (N1) cervical cancer samples obtained from The Cancer Genome Atlas database were analyzed. Differentially expressed genes were identified using R packages 'edgeR' and 'limma'. Kyoto Encyclopedia of Genes and Genomes pathway enrichment and Gene Set Enrichment Analysis (GSEA) were conducted. The GSE9750 dataset obtained from Gene Expression Omnibus was analyzed to identify genes that are persistently aberrantly expressed during the development of cervical cancer. The peroxisome proliferator-activated receptor (PPAR) signaling pathway was screened out to be significant during LN metastasis. In the two analyzed datasets, 11 genes were aberrantly expressed, while matrix metalloproteinase 1 (MMP1) was the only gene that was persistently overexpressed. Cell viability, wound healing and Transwell assays were performed to evaluate the effects of MMP1 knockdown in cervical cancer cell lines, and the expression of epithelial mesenchymal transition (EMT) markers was detected. Finally, the clinical significance of MMP1 was investigated. The current study identified that MMP1 was overexpressed and the PPAR signaling pathway was associated LN metastasis in patients with cervical cancer. Following knockdown of MMP1, the proliferation, migration and invasion of cervical cancer cell lines were weakened, the expression of epithelial marker E-cadherin was increased, and the expression of metastasis-associated gene vimentin was decreased. MMP1 was an independent prognostic factor for cervical cancer. The current study indicated that MMP1 has a key role in the regulation of cervical tumor growth and LN metastasis via EMT to a certain extent. The results suggest that MMP1 may be a biomarker for LN metastasis of cervical cancer, and further validation should be performed.

Figure 1

Work flow and heatmap of the top 25 DEGs. (A) Work-flow of the current study. (B) Top 25 up- and downregulated DEGs in a heatmap. The upper section represents the hierarchical clustering results, with yellow and grey bars indicating N0 and N1 samples respectively. Genes are listed on the right and red grids represented high expression while blue grids represented low expression in N1 vs. N0. TCGA, The Cancer Genome Atlas; DEG, differentially expressed gene; GSEA, Gene Set Enrichment Analysis; DAVID, Database for Annotation, Visualization and Integrated Discovery; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPAR, peroxisome proliferator-activated receptor; MMP1, matrix metalloproteinase 1; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats.

Figure 2

Identification of upregulated pathways based on ClueGO. (A) Significantly upregulated pathways of upregulated DEGs between N0 and N1 based on ClueGO. (B) Significantly downregulated pathways of downregulated DEGs between N0 and N1 based on ClueGO. Large circles are pathways and small circles indicate genes in the pathways. If the genes were part of more than two pathways, it is indicated as a fusion of the colors from the two pathways. P<0.05 was considered to indicate a statistically significant difference, pathways including four or more DEGs are presented in ClueGo-Kyoto Encyclopedia of Genes and Genomes figures. DEGs, differentially expressed genes; PPAR, peroxisome proliferator-activated receptor; cAMP, cyclic adenosine monophosphate.

Figure 3

Identification of key gene signatures and key genes in cervical cancer. (A) Venn diagram illustrating the overlapped pathways among KEGG and GSEA (left) and the representative GSEA curve of mutual upregulated pathway PPAR signaling (right) with NES=1.413 and P=0.030 when comparing N1 and N0. (B) Venn diagram illustrating the overlapped pathways between KEGG and GSEA for downregulated pathways. (C) Venn-diagram illustrating the overlapped DEGs between another GEO cohort (normal vs. cancer, GSE 9750; N0 vs. N1, TCGA; left) and a heatmap illustrating the log fold change of the 11 overlapped genes in the two groups (right). MMP1 is marked as red for its elevation in both groups. KEGG, Kyoto Encyclopedia of Genes and Genomes; PPAR, peroxisome proliferator-activated receptor; GSEA, Gene Set Enrichment Analysis; NES, normalized enrichment score; DEGs, differentially expressed genes; ABCA8, ATP binding cassette subfamily A member 8; APOD, apolipoprotein D; CHGB, chromogranin B; CXCL13, C-X-C motif chemokine ligand 13; DSC1, desmocollin 1; IL1R2, interleukin 1 receptor type 2; KRT2, keratin 2; MMP1, matrix metalloproteinase 1; PTPRN, protein tyrosine phosphatase, receptor type N; UPK1A, uroplakin 1A; WISP3, WNT1 inducible signaling pathway protein 3.

Figure 4

Knockdown of MMP1 alters malignant behavior in vitro. (A) Efficacy of MMP1 CRISPR was demonstrated by western blotting. (B) Cell viability of MMP1 knockdown and control cells was detected by PrestoBlue, relative growth was represented by fluorescence at a wavelength of 570 nm. MMP1 knockdown cells grew more slowly on day 5 and 6 compared with control cells. (C) Cell motility was analyzed by wound healing assay, bar chart displayed the 36 h relative wound closure of SiHa and HeLa MMP1 knockdown and control cells. (D) Cell invasion was analyzed Transwell assay, images at 36 h are presented and bar chart displays the cell count per field from random three fields. Data are presented as the mean ± standard error (n=3; ^*P≤0.05 vs. dox-). MMP1, matrix metalloproteinase 1; sgRNA, single guide RNA; dox, doxycycline hyclate.

Figure 5

Effect of MMP1 knockdown on epithelial-mesenchymal transition markers. (A) Protein expression of E-cadherin and vimentin as detected by western blotting in each group. The expression of E-cadherin was increased and vimentin was decreased in MMP1 knockdown cervical cancer cell lines. (B) Relative mRNA expression of E-cadherin and vimentin were detected by reverse transcription-quantitative polymerase chain reaction. ^*P<0.05 vs. dox-. MMP1, matrix metalloproteinase 1; sgRNA, single guide RNA; dox, doxycycline hyclate.

Figure 6

Clinical significance of MMP1 in cervical cancer. (A) The survival curve of patients with N0 and N1 cervical cancer based on The Cancer Genome Atlas demonstrates that N1 patients had poor prognosis (P=0.03789). (B) Expression level of MMP1 are significantly associated with the overall survival of patients with cervical cancer and highly expressed MMP1 patients had a poor prognosis (P=0.0036). (C) MMP1 expression was significantly increased in samples from N1 compared with that in N0 (P=0.0434). (D) A receiver operating characteristic curve of MMP1 was shown for discriminating between N0 and N1. The sensitivity and specificity of MMP1 were >60% (60.00% and 68.90%, respectively). N0, lymph node-negative; N1, lymph node-positive; MMP1, matrix metalloproteinase 1; PPV, positive predictive value; NPV, negative predictive value; AUC, area under curve.