PPFIA mRNA and protein expression levels were associated with the development of CRC
The Oncomine and UALCAN databases were used to compare the expression of the mRNA level expression of PPFIA in human CRC samples and normal colon epithelium samples. The results from the two databases demonstrated that the mRNA expression levels of PPFIA1, PPFIA3, and PPFIA4 were significantly higher in CRC samples than in normal colon epithelium. PPFIA2 mRNA expression was not significantly different between normal colon epithelial samples and CRC tissues (Figs. 1A–a to d, 1B, 2A–a, 2B–a, 2C–a, 2D–a ). In addition, the mRNA expression levels of PPFIA1, PPFIA3, and PPFIA4 were associated with tumor stage, lymph node metastasis, and TP53-mutation status (Figs. 2A–b to d, 2B–b to d, 2C–b to d, and 2D–b to d). Results of CCLE database analysis also confirmed that the mRNA expression levels of PPFIA1, PPFIA3, and PPFIA4 in CRC cell lines were higher than those in normal colon cell lines (Fig. 3A – a to c). Subsequently, RT-PCR and western blotting were performed to detect the mRNA and protein levels of PPFIA1, PPFIA3, and PPFIA4 in normal colon (NCM460) and CRC (LoVo and Hct116) cell lines. The results confirmed that both mRNA (Fig. 3B – a to c) and protein (Figs. 3C and 3D– a to c) expression of PPFIA1, PPFIA3, and PPFIA4 were increased in CRC cell lines compared to the normal colon cell line, and the differences were statistically significant (**P < 0.01, ***P < 0.001).
Prognostic value of PPFIA mRNA expression levels in CRCs
To evaluate the prognostic value of PPFIA1, PPFIA2, PPFIA3, and PPFIA4 in CRC patients, the GEPIA server and TCGAportal database were used to analyze the survival of CRC patients. The results indicated that the mRNA expression levels of PPFIA1, PPFIA2, and PPFIA3 were not significantly correlated with overall survival (OS) or disease-free survival (DFS) in CRC patients (Figs. 4A–a to c, 4B–a to c). However, patients with high PPFIA4 mRNA expression levels of CRCs had shorter DFS and OS than those with low PPFIA4 mRNA expression levels, and the difference was statistically significant (Fig. 4A–d, P = 0.00028; Fig. 4C, P = 0.00039). These results are consistent with the TCGA portal database (Fig. 4C). The results from the two databases demonstrated that high PPFIA4 mRNA expression levels were associated with poor prognosis in patients with CRC.
PPFIA4 IHC staining in human CRC tissues
PPFIA4 was selected for further study based on the results of the bioinformatics analysis described above. According to the Human Protein Atlas website, IHC analysis showed that the expression of PPFIA4 protein was higher in CRC tissues than in normal tissues (Fig. 5A). In this study, IHC analysis was performed to detect the protein expression of PPFIA4 in five normal colon tissue samples and 219 cases. The results showed that the protein expression of PPFIA4 was higher in CRC tissues than in normal colon tissues (Figs. 5B–a to e and Table 1). The expression of PPFIA4 was significantly lower in Group I than in Groups II (P = 0.009), III (P = 0.029), IV (P = 0.000), and V (P = 0.000). PPFIA4 expression was higher in groups IV (P = 0.000) and V (P = 0.000) than in group III. In addition, PPFIA4 expression was also higher in groups V (P = 0.004) and IV (P = 0.000) than in group II. The differences were not statistically significant between groups II and III (P = 0.299) or between groups IV and V (P = 0.128).
Table 1
Comparison of the PPFIA4 staining index among different groups of human CRCs
| Group | n | Staining index | Value of statistics | P* |
Normal colon tissue | I | 5 | 2.2 ± 0.83666 | χ2 = 53.837 | 0.000 |
Well-differentiated CRCs | II | 51 | 5.5882 ± 3.11882 |
Moderately differentiated CRCs | III | 54 | 4.8519 ± 2.17540 |
Poorly differentiated CRCs | IV | 51 | 7.6471 ± 3.56552 |
Metastatic lymph node foci | V | 63 | 8.6349 ± 3.27897 |
*P < 0.05: statistically significant. (P: difference between the three groups; P1 (difference between groups I and II) = 0.009; P2 (difference between groups I and III) = 0.029; P3(difference between groups I and IV) = 0.000; P4(difference between groups I and V) = 0.000. P5 (difference between groups II and III) = 0.299. P6 (difference between Groups III and IV) = 0.000. P7 (difference between Groups III and V) = 0.000. P8 (difference between Groups II and IV) = 0.004. P9 (difference between groups II and V) = 0.000. P10 (difference between Groups IV and V) = 0.128. |
CRC, colorectal cancer. |
PPFIA4 gene co-expression and functional enrichment analysis
To better understand the biological processes and signaling pathways that are involved in PPFIA4, co-expressed genes with PPFIA4 were analyzed. The UALCAN database was used to identify the top 100 genes that were positively correlated with PPFIA4 expression in CRC (Figs. 5C–a to d). Consequently, the PPI network was generated in the STRING protein interaction database to show the interaction among the 100 genes (Supplementary Fig. 1A), and then the 100 genes were imported into the Cytoscape platform (Version 3.7.1) for further visualization (Supplementary Fig. 1A). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the co-expressed genes of PPFIA4 were significantly enriched in G-protein coupled peptide receptor activity and leukotrience B4 receptor activity (Fig. 6A). The most enriched KEGG pathways were the PPAR and HIF-1a signaling pathways (Fig. 6B).
miRNAs and the expression of PPFIA4
miRNAs are a class of small non-coding RNAs that play a critical role in the initiation and progression of diverse tumors [20, 21]. To determine the correlation between miRNAs and PPFIA4, the ENCORI platform was used to predict the miRNAs involved in the regulation of PPFIA4. Eventually, it was demonstrated that a total of 17 miRNAs could regulate the expression of PPFIA4 (Table 2). Among these, 14 miRNAs were negatively correlated with PPFIA4 expression. Four miRNAs among the 14 miRNAs showed decreased expression in CRCs, and only miR-485-5P was significantly associated with the prognosis of CRC patients (Fig. 7A, P = 0.021). The results showed that miR-485-5P expression was decreased in CRC samples compared to that in normal colon tissue samples and negatively regulated the expression of PPFIA4, as determined by the ENCORI database (Figs. 7B to 7D).
Table 2
Micro-RNAs targeted PPFIA4 in ENCORI.
序号 | miRNA | Coefficient-R | p-Value |
1 | hsa-miR-101-3p | -0.051 | 2.76E-01 |
2 | hsa-miR-144-3p | -0.087 | 6.41E-02 |
3 | hsa-miR-329-3p | -0.019 | 6.86E-01 |
4 | hsa-miR-485-5p | -0.043 | 3.64E-01 |
5 | hsa-miR-580-3p | -0.022 | 6.42E-01 |
6 | hsa-miR-6884-5p | -0.097 | 4.06E-02 |
7 | hsa-miR-650 | -0.046 | 3.28E-01 |
8 | hsa-miR-660-5p | -0.032 | 4.94E-01 |
9 | hsa-miR-502-3p | -0.001 | 9.85E-01 |
10 | hsa-miR-362-3p | -0.131 | 5.41E-03 |
11 | hsa-miR-501-3p | -0.004 | 9.25E-01 |
12 | hsa-miR-138-5p | 0.021 | 6.59E-01 |
13 | hsa-miR-668-3p | 0.058 | 2.19E-01 |
14 | hsa-miR-134-5p | 0.076 | 1.06E-01 |
15 | hsa-miR-342-3p | 0.114 | 1.52E-02 |
16 | hsa-miR-423-3p | 0.09 | 5.54E-02 |
17 | hsa-miR-582-5p | 0.012 | 7.92E-01 |
To further verify whether miR-485-5P can regulate the expression of PPFIA4, the expression of PPFIA4 was detected in LoVo and Hct116 cells after transfection with miR-485-5P inhibitor and miR-485-5P mimics. Intriguingly, the results indicated that PPFIA4 expression was decreased in LoVo and Hct116 cells after treatment with miR-485-5P mimics and increased after treatment with miR-485-5P inhibitors. These differences were statistically significant (Fig. 7E and 7F, **P < 0.01). The results showed that miR-485-5P negatively modulated the expression of PPFIA4.