The microRNA-218 and ROBO-1 signaling axis correlates with the lymphatic metastasis of pancreatic cancer

Abstract

Pancreatic cancer is known for its poor prognosis and early lymphatic metastasis is a notable characteristic. microRNAs (miRNAs) have been shown to be involved in the initiation and progression of pancreatic cancer. We, therefore, established a screening strategy to find miRNAs related to the lymphatic metastasis of pancreatic cancer and explored the target genes of miRNAs. miRNA array profiles were analyzed in tissue samples [pancreatic ductal adenocarcinoma (PDAC) and matched adjacent benign tissues (MAT)] and cell lines (BxPC-3-LN and BxPC-3). Combined analysis of profiling data from tissue samples and cell lines was used to identify miRNAs related to the lymphatic metastasis of pancreatic cancer. The expression levels of miRNAs were confirmed by real-time reverse transcription PCR (RT-PCR) in tissue samples and cell lines. The correlation between miRNAs and clinicopathological characteristics was investigated. The expression features of miRNAs in pancreatic cancer, precursor lesions and metastatic lymph nodes were characterized by in situ hybridization (ISH). Predicted target genes of miRNAs were validated by RT-PCR and the protein levels of target genes were revealed by western blotting. Seventy and 63 miRNAs were differentially expressed in pancreatic cancer and BxPC- 3-LN, compared to MAT and BxPC-3, respectively. Combined microarray analysis found 4 co-differentially expressed miRNAs (miRNA-663, miRNA-145, miRNA-218 and let-7) related to the lymphatic metastasis of pancreatic cancer. miRNA-218 was significantly downregulated in BxPC-3-LN (fold-change >10) and the expression levels of miRNA-218 were confirmed by RT-PCR. The group with lymph node metastasis and the elder group (age >64) showed lower expression of miRNA-218 (P=0.003 and 0.002), compared to patients without lymph nodes metastasis and patients in the younger group (age ≤64), respectively. The expression of miRNA-218 showed a decreasing trend from normal acinar/ductal epithelium, intraductal papillary mucinous neoplasm (IPMN), pancreatic cancer to metastatic lymph nodes by ISH. Among 8 predicted target genes of miRNA-218, rodent bone (ROBO-1) was confirmed to be upregulated in both mRNA and protein levels in pancreatic cancer. In conclusion, we established a screening strategy based on microarray results and found miRNA-218 to be a notable gene related to lymphatic metastasis of pancreatic cancer. Downregulation of miRNA-218 and upregulation of ROBO-1 were first demonstrated in pancreatic cancer. The miRNA-218 and ROBO-1 signaling axis may contribute to the lymphatic metastasis of pancreatic cancer.