Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer

Abstract

Pancreatic cancer (PC) has the poorest overall survival rate among all human cancers because of late diagnosis and absence of screening tools. We compared the expression profile of microRNAs (miRNAs) in the plasma of patients diagnosed with PC (n=50) with healthy volunteers (n=10). Data was further validated by quantitative realtime PCR and cell-based assays. Thirty-seven miRNAs were down-regulated and 54 were up-regulated in plasma from patients with PC. The expression of miR-21 was significantly higher, and the expression of let-7 family (especially let-7d) and miR-146a was significantly lower in PC. Most interestingly, the expression of miR-21 was correlated with worse survival, and the expression of let-7 was inversely correlated with survival in this pilot study with mixed patient population. Moreover, we found that miR-21 family was markedly over-expressed in chemo-resistant PC cell lines, which was consistent with the plasma data from PC patients. Our previous studies have shown increased expression of miR-21 with concomitant loss of PTEN expression in PC cells, which is consistent with our current findings showing the loss of three additional targets of miR-21 (PDCD4, Maspin and TPM1). These results suggest that identifying and validating the expression of miRNAs in newly diagnosed patients could serve as potential biomarker for tumor aggressiveness, and such miRNAs could be useful for the screening of high-risk patients, and may also serve as targets for future drug development.

Keywords: Drug Resistance; PTEN; Pancreatic cancer; let-7d; miR-21; miR-221.

Figure 1

A schematic presentation of microRNAs in pancreatic cancer (PC). Various families of microRNAs are shown as either up-regulated or down-regulated miRNAs with their corresponding targeted genes.

Figure 2

Comparative expression analysis of differentially expressed miRNAs by microarray profiling in plasma from PC patients and healthy controls. (A) The expression of miR-21 and miR-214 (left panel) and the miR-21 family (right panel), (B) The expression of let-7a, let-7b and let-7c (left panel) and the let-7 family (right panel), (C) The expression of let-7d, let-7i, miR-146a (left panel) and combined let-7 family and miR-146a (right panel). There was a significant up-regulation of miR-21 family in PC patients compared to normal subjects. Conversely, let-7 family and miR-146a expression showed a significant down-regulation compared to normal subjects.

Figure 3

Comparative expression analysis of miRNAs (miR-21, miR-221 and miR-146a) in the plasma of 32 PC patients analyzed individually compared to plasma samples obtained from 10 normal subjects by using qRT-PCR. The line drawn at 1.0 represents average of normal subjects (n=10). The results showed a significant increase in the expression of miR-21 and miR-221 (oncogene) over the cutoff of 1.0 by almost all PC patients. In contrast, a significant down-regulation of miR-146a (tumor suppressor gene) was observed in PC patients compared to normal subjects.

Figure 4

Comparative expression analysis of miRNAs (miR-200b, miR-200c, let-7b and let-7d) in the plasma of 32 PC patients analyzed individually compared to plasma obtained from 10 normal subjects by using qRT-PCR. The line drawn at 1.0 represents average of normal subjects (n=10). The results showed a significant decrease in the expression of miR-200b, miR-200c, let-7b, let-7d (tumor suppressor genes) in almost all PC patients compared to normal subjects.

Figure 5

Comparative expression analysis of (A) The expression of miR-21 family and miR-221 in MIAPaCa-2, MIAPaCa-GR, MIAPaCa-GTR, AsPc-1, AsPc-1OR, AsPc-1GTR as determined by miRNA microarray profiling; (B) Comparative expression analysis of miR-21 and miR-221 in BxPC-3, MIAPaCa-2, MIAPaCa-GR, MIAPaCa-GTR, Aspc-1, AsPc-1OR, AsPc-1GTR cells by qRT-PCR. There was a significant up-regulation of both miR-21 and miR-221 by both the methods of microarray profiling and qRT-PCR methods. P values were calculated by the paired t-test (C) Western blots analysis showing the basal level expression of several proteins some of which are targets of miR-21 (PTEN, PDCD4, Maspin and TPM1).

Figure 6

(A) Box plot representing the expression of seven miRNAs as assessed by qRT-PCR. (B). The Kaplan-Meier curves and log-rank tests for miR-21 expression and survival; (C) The Kaplan-Meier curves and log-rank tests for let-7d expression and survival.