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. 2024 Feb 18;16(4):824.
doi: 10.3390/cancers16040824.

Identification of Tissue miRNA Signatures for Pancreatic Ductal Adenocarcinoma

Carlo Caputo  1 , Michela Falco  1   2 , Anna Grimaldi  3 , Angela Lombardi  3 , Chiara Carmen Miceli  4 , Mariateresa Cocule  4 , Marco Montella  5 , Luca Pompella  4 , Giuseppe Tirino  4 , Severo Campione  6 , Chiara Tammaro  1 , Antonio Cossu  7 , Grazia Fenu Pintori  8 , Margherita Maioli  8   9 , Donatella Coradduzza  8 , Giovanni Savarese  10 , Antonio Fico  10 , Alessandro Ottaiano  11 , Giovanni Conzo  12 , Madhura S Tathode  1 , Fortunato Ciardiello  1 , Michele Caraglia  1   2 , Ferdinando De Vita  4 , Gabriella Misso  1
Affiliations

Identification of Tissue miRNA Signatures for Pancreatic Ductal Adenocarcinoma

Carlo Caputo et al. Cancers (Basel). .

Abstract

Pancreatic ductal adenocarcinoma (PDAC), a neoplasm of the gastrointestinal tract, is the most common pancreatic malignancy (90%) and the fourth highest cause of cancer mortality worldwide. Surgery intervention is currently the only strategy able to offer an advantage in terms of overall survival, but prognosis remains poor even for operated patients. Therefore, the development of robust biomarkers for early diagnosis and prognostic stratification in clinical practice is urgently needed. In this work, we investigated deregulated microRNAs (miRNAs) in tissues from PDAC patients with high (G3) or low (G2) histological grade and with (N+) or without (N-) lymph node metastases. miRNA expression profiling was performed by a comprehensive PCR array and subsequent validation by RT-qPCR. The results showed a significant increase in miR-1-3p, miR-31-5p, and miR-205-5p expression in G3 compared to G2 patients (** p < 0.01; *** p < 0.001; *** p < 0.001). miR-518d-3p upregulation and miR-215-5p downregulation were observed in N+ compared to N- patients. A statistical analysis performed using OncomiR program showed the significant involvement (p < 0.05) of two miRNAs (miR-31 and miR-205) in the histological grade of PDAC patients. Also, an expression analysis in PDAC patients showed that miR-31 and miR-205 had the highest expression at grade 3 compared with normal and other tumor grades. Overall, survival plots confirmed that the overexpression of miR-31 and miR-205 was significantly correlated with decreased survival in TCGA PDAC clinical samples. A KEGG pathway analysis showed that all three miRNAs are involved in the regulation of multiple pathways, including the Hippo signaling, adherens junction and microRNAs in cancer, along with several _target genes. Based on in silico analysis and experimental validation, our study suggests the potential role of miR-1-3p, miR-31-5p, and miR-205-5p as useful clinical biomarkers and putative therapeutic _targets in PDAC, which should be further investigated to determine the specific molecular processes affected by their aberrant expression.

Keywords: biomarkers; diagnosis; microRNAs; pancreatic ductal adenocarcinoma; prognosis.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Research design.
Figure 2
Figure 2
The expression level of the most significantly dysregulated miRNAs in N+ vs. N− comparison. ** p < 0.05.
Figure 3
Figure 3
The expression level of the most significantly dysregulated miRNAs in G3 vs. G2 comparison. The p-value was calculated by t-test ** p < 0.05; *** p < 0.005.
Figure 4
Figure 4
Validation of candidate miRNAs expression in PDAC Tissues. The expression levels of (A) miR-1-3p, (B) miR-31-5p, (C) miR-205-5p, (D) miR-215-5p, (E) miR-518d-3p, (F) miR-519a-3p, and (G) miR-576-5p were validated in PDAC tissues and the paired control ones using RT-qPCR. For the normalization, U6 snRNA was used as the endogenous control. Each sample was run in triplicate. Error bars show mean ± SD. A t test was used for the calculation of p values. *** p < 0.005, ** p < 0.05, * p < 0.05.
Figure 5
Figure 5
ROC curve analysis of each validated miRNA for an evaluation of biomarker potential. (A) miR-1-3p in G3 tissues compared to the G2 tissues (sensibility = 0.63; specificity = 0.61; AUC = 0.62, p-value: 0.2242); (B) miR-31-5p in G3 tissues compared to the G2 tissues (sensibility = 0.88; specificity = 0.47; AUC = 0.85; p-value: 0.0003); (C) miR-205-5p in G3 tissues compared to the G2 tissues (sensibility = 0.77; specificity = 0.61; AUC = 0.72; p-value: 0.0194). (D) miR-215-5p in N+ tissues compared to the N− tissues (sensibility = 0.79; specificity = 0.42; AUC = 0.65, p-value: 0.1439); (E) miR-576-5p in N+ tissues compared to the N− tissues (sensibility = 0.57; specificity = 0.64; AUC = 0.64, p-value: 0.1306); (F) miR-519a-3p in N+ tissues compared to the N− tissues (sensibility = 0.63; specificity = 0.54; AUC = 0.54, p-value: 0.6916); (G) miR-518d-3p in N+ tissues compared to the N− tissues (sensibility = 0.57; specificity = 0.67; AUC = 0.58; p-value: 0.4036). The red dashed line represents a classifier with the random performance level.
Figure 6
Figure 6
Kaplan–Meier survival analysis. Kaplan–Meier survival analysis of PDAC patients expressing low or high levels of miR-1-3p (A), miR-31-5p (B), and miR-205-5p (C). *: hsa-miR-31-5p and has-miR-205-5p were significantly associated with PDAC.
Figure 7
Figure 7
UALCAN analysis of DEMs expression in different PDAC grades. Grade-wise expression of miR-31 (A) and miR-205 (B), derived using UALCAN program. Analysis showed the highest expression of these two miRNAs in grade 3, in comparison to normal and grades 1, 2, and 4.
Figure 8
Figure 8
Venn diagram of cross-regulated DEMs’ _targets. The count of _target genes predicted for miR-1-3p, miR-31-5p, and miR-205-5p dysregulated in G3 versus G2 in pancreatic adenocarcinoma using three different gene prediction tools.
Figure 9
Figure 9
Heatmap of significant KEGG pathways predicted by DIANA-mirPath v3.0, regulated by three differentially expressed miRNAs. The color code represents log (p-value), where the most pathway is colored in red.
Figure 10
Figure 10
Heatmap of significant gene ontologies: cellular components predicted by DIANA-mirPath v3.0, regulated by three differentially expressed miRNAs. The color code represents log (p-value), where the most significant gene ontology term is colored in red.
Figure 11
Figure 11
Heatmap of significant gene ontologies: molecular functions predicted by DIANA-mirPath v3.0, regulated by three differentially expressed miRNAs. The color code represents log (p-value), where the most significant gene ontology term is colored in red.
Figure 12
Figure 12
Heatmap of significant gene ontologies: biological processes predicted by DIANA-mirPath v3.0, regulated by three differentially expressed miRNAs. The color code represents log (p-value), where the most significant gene ontology term is colored in red.
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