MicroRNA-100 regulates IGF1-receptor expression in metastatic pancreatic cancer cells

Abstract

Patients with pancreatic adenocarcinoma have the lowest 5 year survival rate and yearly rates of incidence are nearly equal to the mortality rates. Long term cure rates by standard therapies are disappointing owing to disseminated disease at diagnosis and chemotherapeutic resistance. New therapeutic targets are necessary to decrease the progression of pancreatic cancer and the ability to identify targets specific to metastasis would improve patient care. We evaluated the levels of microRNA of metastatic and non-metastatic cell lines. The expression levels of microRNAs and mRNAs were determined using microarray analysis to examine and compare five pancreatic cancer cell lines, two that can metastasize in vivo (S2VP10 and S2CP9) and three that do not metastasize (MiaPaCa2, Panc-1 and ASPC-1). MicroRNA analysis indicated an increase in miR-100 and a decrease in miR-138 expression in metastatic cancer cells. Microarray analysis of different expressions of mRNAs in metastatic and non-metastatic pancreatic cell lines also indicated significantly increased insulin growth factor-1 receptor (IGF1-R) expression in metastatic pancreatic cancer cell lines compared to non-metastatic pancreatic cancer cell lines. To confirm microarray analysis results, western blot and immunocytochemistry were performed. Western blot revealed that IGF1-R expression exhibited in metastatic cancer cell lines a seven-fold increase compared to non-metastatic cell lines. In addition, downstream expressions of the proteins, GRB2 and phosphorylated PI3K, also were increased in aggressive cancer cell lines. Immunocytochemistry confirmed the linkage of IGF1-R to miR-100, because cells transfected with miR-100 inhibitor showed a decrease in IGF1-R. Cells transfected with a miR-138 mimic, however, did not affect IGF1-R expression.

Fig. 1

MiRNA array analysis of pancreatic cancer cell lines that can metastasize in vivo. Graph shows miRNA differences between potentially metastatic and non-metastatic pancreatic cancer cell lines. MiRNA-100, -23b, -31, and -18b show greater expression in metastatic pancreatic cancer cell lines than in non-metastatic cancer cell lines (p = 0.0001, 0.001, 0.001, 0.003, respectively). MiRNA-138, -299, -183, and -126 have lower expressions in metastatic pancreatic cancer cells compared to non-metastatic cancer cells (p = 0.04, 0.07, 0.002, 0.0007, respectively).

Fig. 2

Western blot analysis of S2VP10, S2CP9, Panc-1, and Miapaca-2. Metastatic pancreatic cancer cell lines (S2VP10, S2CP9) in vitro demonstrated up-regulation of IGF1-R compared to non-metastatic pancreatic cancer cell lines (Panc-1, Miapaca-2). The downstream signals of IGF1-R, including GRB2 and PI3K, showed increased protein expression in metastatic pancreatic cancer cell lines.

Fig. 3

Immunocytochemistry of S2VP10 cancer cells. Blue indicates DAPI, red indicates IGF1-R, and green indicates miRNA transfected cells. A–C show controls, i.e., S2VP10 cells stained with DAPI and secondary antibody. D–F show additional controls, i.e., S2VP10 cells stained with IGF1-R. E, F) S2VP10 cells show red fluorescence, which indicates that IGF1-R is present in these cells. G–I) S2VP10 cells transfected with miRNA-100. Transfected cells (GFP positive) show less intense red fluorescence, which indicates that IGF1-R is down-regulated in S2VP10 cells transfected with miR-100. J–L) S2VP10 cells transfected with miR-138. Red fluorescence in transfected S2VP10 cells (GFP positive) is similar to S2VP10 control cells, which indicates that IGF1-R expression was not down-regulated in S2VP10 cells transfected with miR-138.