Expression features of PNN in digestive tract cancers
The target data included in the gene transcripts acquired in this study were as follows: 162 ESCA and 11 adjacent normal samples, 375 STAD and 32 adjacent normal samples, 480 COAD and 41 adjacent normal samples, 167 READ and 10 adjacent normal samples. The PNN mRNA expression were observed significantly upregulated in four types of digestive tract cancers, including ESCA, STAD, COAD and READ, compared with corresponding normal tissues (Figure 1A). Correlations between PNN mRNA expression and tumor clinical stages in ESCA, STAD, COAD, READ were also analyzed respectively. In general, the expression of PNN were positively associated with tumor stages in digestive tract cancers (Figure 1B). Patients with advanced clinical stage tended to have higher expression of PNN and the highest expression was observed in stage III. However, we noticed that the expression of PNN in stage IV were lower than in stage III, it may be due to the small sample number in stage IV.
Relationships between DNA methylation and PNN expression
Studies have suggested that gene promoter region methylation is a significant cause affecting gene expression, contributing to the progression of human cancer. Current research assessed the effect of promoter DNA methylation on PNN expression in four types of digestive tract cancers using Pearson correlation. We found a significant negative correlation between PNN expression and promoter region methylation level, especially in STAD (Figure A). It indicated that in STAD, abnormal methylation of promoter region is one of the important causes of PNN gene overexpression, but in other digestive tract cancers, there may exist other more regulatory mechanisms influencing the expression of PNN.
The prognostic value of PNN in digestive tract cancers
Kaplan-Meier analyses indicated that PNN played different prognostic role in different types of digestive tract cancers (Figure 3). In ESCA, patients with higher PNN expression level associated with poorer OS (p=0.076) and PFS (p=0.044). In STAD, patients with high PNN expression had a better prognosis, but unfortunately, there was no statistically difference observed. In COAD, higher expression of PNN was significantly associated with poorer OS (p=0.003) and poorer PFS (p=0.009). And in patients with READ, the higher expression level of PNN was correlated with longer OS (p=0.02), but there was no statistical difference in PFS (p =0.082).
We further explored the genetic alterations in PNN and their effects on prognosis in patients with digestive tract cancers by utilizing cBioPortal database. The proportion of various genetic alterations of PNN in different digestive tract cancers were similar: 6% in ESCA, 8% in STAD and 6% in CRC (Figure 4). It was worth noting that the types of genetic alteration were diverse. Amplification and deep deletion were more common in ESCA, missense mutation and truncating mutation were more frequent in STAD and CRC. It was with regret that there was no significant correlation between PNN genetic alterations and OS in these cancers.
Moreover, we investigated the DNA methylation of PNN CpG sites and corresponding prognostic effects in four digestive tract cancers by the MethSurv database. The results were illustrated in Table 1. We observed that cg18648343, cg12087797, cg15592059, cg20337385 were remarkably associated with the prognosis in patients with STAD. In COAD, cg15592059, cg24034629 and cg10250651 were indicated as significant factors for prognosis. As for READ, the meaningful cg sites in prognosis included cg02969452, cg18648343 and cg12087797. However, there were no statistically significant DNA methylation CpG sites observed for predicting OS in ESCA.
Finally, considering the significantly prognostic effect of PNN expression in COAD, we performed univariate and multivariate cox regression analyses to assess the independent prognostic effect in COAD. After controlling the clinical parameters, the univariate cox regression analysis suggested that the expression of PNN had significant effect both in survival (p=0.004) and recurrence (p=0.016) (Table 2, 3). Subsequently, the expression of PNN was identified as an independent prognostic biomarker for predicting the OS in patients with COAD with multivariate cox regression analysis (p = 0.041, HR=1.7, 95% CI 1.02-2.8, Figure 5).
Table 1
The prognostic effect of CpGs in PNN
Tumor
|
Gene-CpG
|
HR
|
p-value
|
Esophageal carcinoma
|
PNN-5′UTR;1stExon-Island-cg02969452
|
0.641
|
0.06
|
|
PNN-5′UTR;1stExon-Island-cg18648343
|
0.769
|
0.3
|
|
PNN-TSS200-Island-cg10035432
|
1.205
|
0.41
|
|
PNN-Body-Island-cg12087797
|
0.664
|
0.083
|
|
PNN-Body-Island-cg15592059
|
1.204
|
0.42
|
|
PNN-Body-Island-cg20337385
|
1.046
|
0.84
|
|
PNN-Body-Island-cg24034629
|
0.646
|
0.076
|
|
PNN-Body-S_Shore-cg03045079
|
1.417
|
0.2
|
|
PNN-Body-S_Shelf-cg06918918
|
0.792
|
0.31
|
|
PNN-TSS1500-N_Shore-cg10250651
|
0.839
|
0.5
|
|
PNN-TSS1500-N_Shore-cg16408528
|
0.837
|
0.53
|
|
PNN-TSS1500-N_Shore-cg19599972
|
0.863
|
0.52
|
|
PNN-TSS1500-N_Shore-cg24138021
|
0.869
|
0.55
|
Stomach Adenocarcinoma
|
PNN-5′UTR;1stExon-Island-cg02969452
|
0.816
|
0.22
|
|
PNN-5′UTR;1stExon-Island-cg18648343
|
1.494
|
0.014*
|
|
PNN-TSS200-Island-cg10035432
|
0.815
|
0.22
|
|
PNN-Body-Island-cg12087797
|
0.639
|
0.0075*
|
|
PNN-Body-Island-cg15592059
|
1.558
|
0.013*
|
|
PNN-Body-Island-cg20337385
|
1.402
|
0.043*
|
|
PNN-Body-Island-cg24034629
|
1.284
|
0.13
|
|
PNN-Body-S_Shore-cg03045079
|
1.295
|
0.18
|
|
PNN-Body-S_Shelf-cg06918918
|
0.743
|
0.13
|
|
PNN-TSS1500-N_Shore-cg10250651
|
1.299
|
0.15
|
|
PNN-TSS1500-N_Shore-cg16408528
|
1.066
|
0.7
|
|
PNN-TSS1500-N_Shore-cg19599972
|
0.778
|
0.19
|
|
PNN-TSS1500-N_Shore-cg24138021
|
1.214
|
0.29
|
Colon Adenocarcinoma
|
PNN-5′UTR;1stExon-Island-cg02969452
|
1.589
|
0.079
|
|
PNN-5′UTR;1stExon-Island-cg18648343
|
1.098
|
0.7
|
|
PNN-TSS200-Island-cg10035432
|
1.214
|
0.42
|
|
PNN-Body-Island- cg15592059
|
0.583
|
0.025*
|
|
PNN-Body-Island-cg20337385
|
1.068
|
0.81
|
|
PNN-Body-Island-cg24034629
|
0.423
|
0.0078*
|
|
PNN-Body-S_Shore-cg03045079
|
0.793
|
0.35
|
|
PNN-Body-S_Shelf-cg06918918
|
0.832
|
0.51
|
|
PNN-TSS1500-N_Shore-cg10250651
|
0.595
|
0.041*
|
|
PNN-TSS1500-N_Shore-cg16408528
|
0.796
|
0.38
|
|
PNN-TSS1500-N_Shore-cg19599972
|
1.36
|
0.21
|
|
PNN-TSS1500-N_Shore-cg24138021
|
1.245
|
0.37
|
Rectum Adenocarcinoma
|
PNN-5′UTR;1stExon-Island-cg02969452
|
0.154
|
0.016*
|
|
PNN-5′UTR;1stExon-Island-cg18648343
|
3.34
|
0.034*
|
|
PNN-TSS200-Island-cg10035432
|
0.412
|
0.082
|
|
PNN-Body-Island-cg12087797
|
0.318
|
0.048*
|
|
PNN-Body-Island-cg15592059
|
1.515
|
0.4
|
|
PNN-Body-Island-cg20337385
|
0.674
|
0.44
|
|
PNN-Body-Island-cg24034629
|
0.756
|
0.58
|
|
PNN-Body-S_Shore-cg03045079
|
0.716
|
0.5
|
|
PNN-Body-S_Shelf-cg06918918
|
1.677
|
0.3
|
|
PNN-TSS1500-N_Shore-cg10250651
|
0.385
|
0.055
|
|
PNN-TSS1500-N_Shore-cg16408528
|
0.42
|
0.14
|
|
PNN-TSS1500-N_Shore-cg19599972
|
0.304
|
0.071
|
|
PNN-TSS1500-N_Shore-cg24138021
|
0.424
|
0.087
|
* p<0.05. |
Table 2
Univariate cox regression analysis of PNN expression as survival predictors in COAD
Parameter
|
Univariate analysis
|
Hazard Ratio
|
95% CI
|
P value
|
Age
|
1.023
|
1.005-1.042
|
0.012*
|
Gender
|
1.162
|
0.769-1.757
|
0.476
|
T stage
|
2.777
|
1.842-4.187
|
<0.001*
|
N stage
|
2.550
|
1.673-3.886
|
<0.001*
|
M stage
|
3.519
|
2.312-5.356
|
<0.001*
|
PNN expression
|
2.064
|
1.256-3.392
|
0.004*
|
Table 3
Univariate cox regression analysis of PNN expression as recurrence predictors in COAD
Parameter
|
Univariate analysis
|
Hazard Ratio
|
95% CI
|
P value
|
Age
|
1.000
|
0.986-1.015
|
1.000
|
Gender
|
1.192
|
0.831-1.711
|
0.339
|
T stage
|
2.750
|
1.947-3.883
|
<0.001*
|
N stage
|
2.551
|
1.772-3.672
|
<0.001*
|
M stage
|
3.088
|
2.145-4.445
|
<0.001*
|
PNN expression
|
1.767
|
1.112-2.806
|
0.016*
|
Functional enrichment analysis of PNN expression in COAD
Owing to the independent prognostic value of PNN expression in recurrence and survival, we explored the biological functions of PNN in COAD by GO analysis based on Metascape. In this research, GO pathway and process enrichment analysis included: molecular functions (MFs, functional set), biological processes (BPs, pathway) and cellular components (CCs, structural complex). Top 15 clusters were displayed in Figure 6A, CCs, including GO: 0016607 (nuclear speck) and GO: 0000226 (microtubule cytoskeleton organization); MFs such as GO:0006397 (mRNA processing), GO:1903313 (positive regulation of mRNA metabolic process), GO: 0031124 (mRNA 3‘-end processing), GO: 0018023 (peptidyl-lysine trimethylation), and GO: 0006354 (DNA-templated transcription, elongation); BPs, such as GO 0033044: (regulation of chromosome organization), GO 0009314 (response to radiation), GO 0051056 (regulation of small GTPase mediated signal transduction) and GO 0061136 (regulation of proteasomal protein catabolic process).
The GSEA was performed for evaluating the underlying signaling pathways, which were involved in carcinogenesis of PNN in COAD. The study indicated that PNN high expression was positively associated with “spliceosome”, “basal transcription factors”, “WNT signaling pathway”, “ERBB signaling pathway”, “mTOR signaling pathway” and “adherens junction” (Figure 6B).
Associations between TIICs and PNN in COAD
In the last few years, increasing researches have revealed the crucial relationships between the immune microenvironment and cancer progression. In the current study, we investigated the effects of different PNN expression levels on tumor-infiltrating immune cells in COAD by using CIBERSORT algorithm. The results illustrated that the distribution of 22 immune cell types in each COAD sample varied markedly (Figure 7A). Furthermore, we observed that CD8+ T cells, regulatory T cells (Tregs) and resting dendritic cells were significantly enriched in low PNN expression group, nevertheless, resting NK cells and naive CD4+ T cells were markedly enriched in high PNN expression group (Figure 7B). Moreover, TIMER database analysis demonstrated that high expression of PNN was positively associated with several types of TIICs, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells (Figure 7C).
A prognostic nomogram for patients with COAD
A prognostic nomogram incorporating the PNN expression and common clinicopathological characteristics was successfully established for predicting the 3, 5, 10-year overall survival probability, which might be promisingly applied in clinical evaluation of patients with COAD (Figure 8).