Differential expression profiles of microRNAs in highly and weakly invasive/metastatic pancreatic cancer cells

Abstract

Pancreatic cancer is the eighth-leading cause of cancer-associated mortality worldwide. To date, the cellular and molecular mechanisms associated with the invasion and metastasis of pancreatic cancer remain unclear. To examine these mechanisms, a microRNA (miRNA/miR) microarray with 1,965 genes was hybridized with labeled miRNA probes from invasive PC-1.0 and non-invasive PC-1 cells for molecular profiling analysis. In addition, reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was utilized to validate the microarray results. Online miRNA target prediction algorithms online were used to predict the target genes of the differentially expressed miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) term enrichment analysis were performed for the potential targets of the differentially expressed miRNAs. The results demonstrated that 54 miRNAs were differentially expressed, of which 33 were upregulated and 21 were downregulated in the PC-1.0 cell line compared with the PC-1 cell line. A total of 6 upregulated miRNAs (miR-31, -34a, -181a, -181b, -193a-3p, and -193b) and 4 downregulated miRNAs (miR-221, -222, -484, and -502-3p) were selected from these 54 miRNAs and validated by RT-qPCR. The differentially expressed miRNAs were further validated by RT-qPCR in the human pancreatic cancer cell lines AsPC-1 (highly invasive) and CAPAN-2 (less invasive). The results revealed that 2 upregulated miRNAs (miR-34a and -193a-3p) and 4 downregulated miRNAs (miR-221, -222, -484, and -502-3p) exhibited a consistent expression pattern between the PC-1.0/PC-1 and AsPC-1/CAPAN-2 pancreatic cancer cells. The GO and KEGG enrichment analysis indicated that the mRNAs potentially targeted by miRNAs were involved in a range of biological functions. These results suggest that different miRNA expression profiles occur between highly and weakly invasive and metastatic pancreatic cancer cell lines, and may affect a variety of biological functions in pancreatic cancer.

Keywords: invasion; metastasis; microRNA; microarray; pancreatic cancer.

Figure 1.

miRNA chip overlay images. (A) Original image of two-channel miRNA microarray; PC-1.0 was labeled with red fluorescent Cy5 dye and PC-1 was labeled with green fluorescent Cy3 dye. (B) Original image of two-channel miRNA microarray; PC-1.0 was labeled with green fluorescent Cy5 dye and PC-1 was labeled with red fluorescent Cy3 dye. (C) Scatter plot of PC-1.0 and PC-1 data. miRNA, microRNA.

Figure 2.

Reverse transcription-quantitative polymerase chain reaction analysis of highly (PC-1.0) and weakly (PC-1) invasive and metastatic hamster pancreatic cancer cells. A total of 10 miRNAs were selected to verify the reliability of the miRNA microarray data. Of the 10 miRNAs, 6 were upregulated and 4 were downregulated. These results were very similar to the miRNA microarray data, supporting its reliability. Black bar, PC-1.0 cell line. White bar, PC-1 cell line. *P<0.05. miRNA, microRNA.

Figure 3.

Reverse transcription-quantitative polymerase chain reaction analysis of highly (AsPC-1) and weakly (CAPAN-2) invasive and metastatic human pancreatic cancer cells. To further investigate the association of miRNAs with invasion and metastasis in pancreatic cancer, two human pancreatic cancer cell lines were analyzed. The results were partially different from those in the PC-1.0/PC-1 cells; of the 10 miRNAs, 2 were upregulated and 8 were downregulated. Black bar, AsPC-1 cell line. White bar, CAPAN-2 cell line. *P<0.05. miRNA, microRNA.

Figure 4.

Downstream target genes of the upregulated microRNAs.

Figure 5.

Downstream target genes of the downregulated microRNAs.

Figure 6.

Cellular components for the differentially expressed microRNAs identified with Gene Ontology term enrichment analysis.

Figure 7.

Molecular functions for the differentially expressed microRNAs identified with Gene Ontology term enrichment analysis.

Figure 8.

Biological processes for the differentially expressed microRNAs identified with Gene Ontology term enrichment analysis.

Figure 9.

Kyoto Encyclopedia of Genes and Genomes term enrichment analysis.

Figure 10.

Kyoto Encyclopedia of Genes and Genomes term pathways common or unique to the up- and downregulated microRNAs.