Differential Expression of MicroRNAs in Tissues and Plasma Co-exists as a Biomarker for Pancreatic Cancer

Abstract

Objective: Pancreatic cancer (PC) is a lethal disease with disappointing results from current treatment modalities, suggesting that novel therapeutic strategies are urgently needed. Since microRNAs (miRNAs) are important player in biology, the clinical utility of miRNAs for designing novel therapeutics is an active area of research. The objective of the present study was to examine differentially expressed miRNAs between normal and tumor tissues, and in plasma samples obtained from PC patients, chronic pancreatitis (CP) patients and healthy subjects (HC).

Material and methods: The miRNA expression profiling using formalin-fixed paraffin embedded (FFPE) tissues from normal and tumor specimens was accomplished using miRBase version 19 (LC Sciences, Houston, TX, USA). Quantitative real-time PCR (qRT-PCR) was subsequently performed in individual samples for 7 selected miRNAs. In addition, qRT-PCR was also performed for assessing the expression of 8 selected miRNAs in plasma samples.

Results: A significant difference in the expressions of miR-21, miR-205, miR-155, miR-31, miR-203, miR-214 and miR-129-2 were found in tumor tissue samples. Lower expression of miR-214 was found to be associated with better overall survival. We also observed differential expression of 8 miRNAs in plasma samples of CP and PC patients compared to HC. Interestingly, over expression of miR-21, and miR-31 was noted in both tumor tissues and in the plasma.

Conclusion: We found deregulated expression of miRNAs that could distinguish normal from PC in two different types of samples (tissues and plasma). Interestingly, lower expression of miR-214 was found to be associated with better overall survival. Although not statistically significant, we also observed higher expression of let-7a and lower expression of miR-508 to be associated with overall better survival. We conclude that our study nicely lays the foundation for detailed future investigations for assessing the role of these miRNAs in the pathology of pancreatic cancer.

Keywords: Chronic Pancreatitis; FFPE; Pancreatic Cancer; Plasma; miRNAs; qRT-PCR.

Figure 1

Comparative expression analysis of miR-21 and miR-205 in 37 pancreatic cancer patient’s tumor specimens compared to 24 pooled normal samples of FFPE tissue blocks (1A&1D), comparative expression analysis in 24 paired samples of FFPE tissue blocks of tumor and normal tissue samples from the same patient (1B&1E) quantitated individually using qRT-PCR. The Kaplan-Meier curve and Log-ranks tests for miR-21 (1C) and miR-205 (1F) expression and survival of patients are also presented. There was a significant up-regulation of miRNA-205 in almost all tumor samples when compared to Normal (1D&1E). Overall, a larger percentage of miR-21 appears to be up-regulated, in tumor samples than normal samples (1A&1B). The miRNAs expression was normalized using RNU48 miRNA. P values represent comparison between normal and tumor paired samples (1B,1E) using Wilcoxon matched pairs t-test.

Figure 2

Comparative expression analysis of miR-155 and miR-31 in 37 pancreatic cancer patient’s tumor specimens compared to 24 pooled normal samples of FFPE tissue blocks (2A&2D), comparative expression analysis in 24 paired samples of FFPE tissue blocks of tumor and normal tissue samples from the same patient (2B&2E) quantitated individually using qRT-PCR. The Kaplan-Meier curve and Log-ranks tests for miR-155 (2C) and miR-31 (2F) expression and survival of patients are also presented. There was a significant up-regulation of miRNA-155 in all except two tumor samples when compared to normal (2A). Similarly, miR-31 appears to be up-regulated in most of the tumor samples compared to normal samples (2D&2E). The miRNAs expression was normalized using RNU48 miRNA. P values represent comparison between normal and tumor paired samples (2B, 2E) using Wilcoxon matched pairs t-test.

Figure 3

Comparative expression analysis of miR-203 and miR-214 in 37 pancreatic cancer patient’s tumor specimens compared to 24 pooled normal samples of FFPE tissue blocks (3A&3D), comparative expression analysis in 24 paired samples of FFPE tissue blocks of tumor and normal tissue samples from the same patient (3B&3E) quantitated individually using qRT-PCR. The Kaplan-Meier curve and Log-ranks tests for miR-203 (3C) and miR-214 (3F) expression and survival of patients are also presented. There was a significant up-regulation of miRNA-203 and miR-214 in all except few tumor samples when compared to normal (A,B,D and E). Furthermore, Kaplan-Meier curve for miR-214 (3F) demonstrated a significant longer survival of patients with low miR-214 expression. The miRNAs expression was normalized using RNU48 miRNA. P values represent comparison between normal and tumor paired samples (3B,3E) using Wilcoxon matched pairs t-test.

Figure 4

Comparative expression analysis of miR-129-2-3p in 37 pancreatic cancer patient’s tumor specimens compared to 24 pooled normal samples of FFPE tissue blocks (4A), comparative expression analysis in 24 paired samples of FFPE tissue blocks of tumor and normal tissue samples from the same patient (4B) quantitated individually using qRT-PCR. The Kaplan-Meier curve and Log-ranks tests for miR-129-2-3p expression and survival of patients are also presented in 4C. There was a significant down-regulation of miRNA-129-2-3p in most of the tumor samples when compared to normal (4A). The miRNAs expression was normalized using RNU48 miRNA. P values represent comparison between normal and tumor paired samples (4B) using Wilcoxon matched pairs t test.

Figure 5

Comparative expression analysis of miR-221 (5A), let-7a (5B), miR-155 (5C) and miR-31 (5D) from plasma samples of HC, CP and PC quantitated individually using qRT-PCR (n=20/group). In miR-221, there appears to be an increase in the expression in CP and PC compared to HC, suggesting an oncogenic role of miR-221 (5A). In contrast, the expression of let-7a was significantly decreased in PC compared to both HC and CP patient’s plasma samples indicating a tumor suppressor role of let-7a (5B). Both miR-155 and miR-31 expression was mostly found to be up-regulated in CP patient samples followed by PC and primarily lower expression was found in HC plasma (5C&5D). The plasma miRNAs concentration was calculated using the standard miRNA concentration in 10-2 pM units using the Quantity value *3.125/6.02/1000. P values represent comparison between HC vs CP and HC vs PC using t-test.

Figure 6

Comparative expression analysis of miR-21 (6A), miR-181a (6B), miR-935 (6C) and miR-508 (6D) in plasma samples of HC, CP and PC subjects quantitated individually using qRT-PCR (n=20/group). There appears to be a gradual increase in the expression in CP and PC compared to HC in all four miRNAs as presented in Figure 6. The plasma miRNAs concentration was calculated using the standard miRNA concentration in 10-2 pM units using the Quantity value *3.125/6.02/1000. P values represent comparison between HC vs CP and HC vs PC using t-test.