Antisense inhibition of microRNA-21 and -221 targeting side population abolish tumor growth of gemcitabine resistant pancreatic cancer cells

Introduction: Our preliminary studies identified a small population, named as side population (SP) cells in pancreatic cancer cells with stem-like properties, which were able to induce fast and aggressive tumor formation in nude mice. Furthermore, gene expression analysis showed a significant difference in the expression of more than 1300 genes in SP cells, among which the difference in microRNA expression such as miR-21 and miR-221 between SP and non-SP cells were identified as most interesting candidate for further therapy studies.

Material and methods: Pancreatic SP cells from the highly metastatic cell line L3.6pl were identified by flow cytometry using Hoechst 33342 dye staining. The gene expression was assessed by Affymetrix array. The antagomir transfection was performed using miR-21 and -221 oligonucleotide antisense. Tumor cell apoptosis, cell cycle progression, chemoresistance, and invasion were quantitated. For the in vivo application, SP cells were sorted from L3.6pl gemcitabine resistant cells and implanted orthotopically in nude mice with or without miR-21 and -221 antisense mono- and combination therapy.

Results: miR-21, miR-221 were significantly upregulated in stem-like SP from L3.6pl cells. Interestingly, in these cells both miR-21 and miR-221 were involved in the modulation of expression of more than 200 genes including RASSF6, RAB2B, TP63, TP53INP1, TP53INP2, MAPK10, MAP2K6, TNFRSF11B and SMAD7. The administration of antagomir-21 and -221 significantly reduced the SP fraction, decreased SP cell differentiation, positively affected L3.6pl cell proliferation, invasion, and chemoresistance against gemcitabine and 5-Fluorouracil. Combination of antagomir-21 and -221 therapy showed a better inhibitory effect on tumor growth than single antagomir treatment, especially, in gemcitabine resistant SP induced pancreatic cancer in vivo.

Conclusion: Our results demonstrate the significance of both miR-21 and -221 regarding tumor initiatiation of SP cells in pancreatic cancer. Both microRNAs contribute to the most important biological functions of pancreatic cancer including apoptosis, metastasis, and chemoresistance. Antagomir-21 and -221 treatments may be beneficial in overcoming gemcitabine associated chemoresistance in pancreatic cancer in the future.