The microRNA expression signature of pancreatic ductal adenocarcinoma by RNA sequencing: anti-tumour functions of the microRNA-216 cluster

Abstract

We analysed the RNA sequence-based microRNA (miRNA) signature of pancreatic ductal adenocarcinoma (PDAC). Aberrantly expressed miRNAs were successfully identified in this signature. Using the PDAC signature, we focused on 4 clustered miRNAs, miR-216a-5p, miR-216a-3p, miR-216b-5p and miR-216b-3p on human chromosome 2p16.1. All members of the miR-216 cluster were significantly reduced in PDAC specimens. Ectopic expression of these miRNAs suppressed cancer cell aggressiveness, suggesting miR-216 cluster as anti-tumour miRNAs in PDAC cells. The impact of miR-216b-3p (passenger strand of pre-miR-216b) on cancer cells is still ambiguous. Forkhead box Q1 (FOXQ1) was directly regulated by miR-216b-3p and overexpression of FOXQ1 was confirmed in clinical specimens. High expression of FOXQ1 predicted a shorter survival of patients with PDAC by Kaplan-Meier analysis. Loss-of-function assays showed that cancer cell migration and invasion activities were significantly reduced by siFOXQ1 transfectants. We investigated pathways downstream from FOXQ1 by using genome-wide gene expression analysis. Identification of the miR-216-3p/FOXQ1-mediated network in PDAC should enhance understanding of PDAC aggressiveness at the molecular level.

Keywords: FOXQ1; expression signature; miR-216b-3p; microRNA; pancreatic ductal adenocarcinoma.

Figure 1. Schematic representation of the human miR-216 family in chromosomal location

The miR-216 family members and miR-217 are located on human chromosome 2q16.1. Mature miRNAs, miR-216a-5p (guide strand) and miR-216a-3p (passenger strand) are derived from pre-miR-216a. Likewise pre-miR-216a, miR-216b-5p (guide strand) and miR-216b-3p (passenger strand) are derived from pre-miR-216b. Seed sequences of miR-216a-5p and miR-216b-5p are identical. In contrast, seed sequences of miR-216a-3p and miR-216b-3p are independent sequences.

Figure 2. Expression levels of miR-216 family members

Expression levels of miR-216 family members in clinical specimens and PDAC cell lines were determined using qRT-PCR. Data were normalized to RNU48 expression.

Figure 3. Effects of miR-216a-5p, miR-216a-3p, miR-216b-5p and miR-216b-3p transfection into PDAC cells

(A) Cell growth was determined using XTT assays 72 h after transfection with 10 nM miR-216 family members. *, P < 0.0001; **, P < 0.05. (B) Cell migration activity was determined using BD Falcon Cell Culture Inserts. *, P < 0.0001. (C) Cell invasion activity was determined using Matrigel invasion assays. *, P < 0.0001.

Figure 4. Flow chart illustrating the analytic strategy for miR-216b-3p target genes

Strategy for identification of putative candidate genes regulated by miR-216b-3p in PDAC cells. Approach to identifying miR-216b-3p target genes by in silico analysis of genome-wide gene expression, TargetScan and GEO database analysis of miR-216b-3p transfectants and PDAC clinical specimens.

Figure 5. Expression of FOXQ1 (mRNA and protein) in PDAC and association of FOXQ1 with overall survival

(A) Expression levels of FOXQ1 in clinical specimens and PDAC cell lines were determined using qRT-PCR. Data were normalized to GUSB expression. (B) Correlation between miR-216-3p and FOXQ1 expression. (C) Immunohistochemical staining of FOXQ1 in PDAC clinical specimens. Overexpression of FOXQ1 was observed in cancer lesions (original magnification ×400). In contrast, negative staining of FOXQ1 was observed in normal tissues. (IHC_13) Positively stained cancer lesion (T3N1M0). (IHC_18) Negatively stained cancer lesion (T3N0M0). (IHC_29) Negatively stained normal pancreas tissue. (D) TCGA-based large cohort study of high expression of FOXQ1 in 174 PDAC patients. (E) Kaplan-Meier patient survival curves for overall survival rates based on FOXQ1 expression in 35 patients with PDAC. P-values were calculated using the log-rank test.

Figure 6. Direct regulation of FOXQ1 by miR-216b-3p in PDAC cells

(A) FOXQ1 mRNA expression was evaluated using qRT-PCR in PANC-1 and SW 1990 cells 72 h after transfection with miR-216b-3p. GUSB was used as an internal control. *, P < 0.0001. (B) FOXQ1 protein expression was evaluated by Western blotting in PANC-1 and SW 1990 cells 72 to 96 h after transfection with miR-216b-3p. GAPDH was used as a loading control. (C) miR-216b-3p binding sites in the 3’-UTR of FOXQ1 mRNA. Dual luciferase reporter assays using vectors encoding putative miR-216b-3p target sites of the FOXQ1 3’-UTR (positions 463-468 and 720-727) for both wild-type and deleted regions. Data were normalized by expressing ratios of Renilla/firefly luciferase activities. *, P < 0.0001.

Figure 7. Effects of FOXQ1 silencing on PDAC cell lines

(A) FOXQ1 mRNA expression was evaluated using qRT-PCR analysis of PANC-1 and SW 1990 cells 72 h after transfection with si-FOXQ1-1 or si-FOXQ1-2. GUSB was used as an internal control. (B) FOXQ1 protein expression was evaluated using Western blotting analysis of PANC-1 and SW 1990 cells 72 to 96 h after transfection with miR-216b-3p. GAPDH was used as a loading control. (C) Cell proliferation was determined using XTT assays 72 h after transfection with 10 nM si-FOXQ1-1 or si-FOXQ1-2. *, P < 0.0001; **, P < 0.05. (D) Cell migration activity was determined using BD Falcon Cell Culture Inserts. *, P < 0.0001. (E) Cell invasion activity was determined using Matrigel invasion assays. *, P < 0.0001.