Effect of miR-21 on resistance to 5-fluorouracil and regulation of MSH2.

Abstract

431 Background: MicroRNAs are small non coding RNAs controlling cell homeostasis. Defects in mismatch repair (MMR) genes cause resistance to 5-fluorouracil (5FU). miR-21 is up-regulated in colorectal cancer (CRC) and is associated with poor benefit from adjuvant 5FU. We aimed at studying if miR-21 may induce 5FU resistance by down-regulating MSH2. Methods: Fresh frozen (32) and paraffin-embedded (50) cases of CRC and matched normal tissues were studied for miR-21 expression (Northern Blotting and in situ Hybridization) and MSH2 expression (Western Blotting and Immunohistochemistry). CRC Colo-320DM, SW620 and isogenic Lovo cells with [Lovo(MSH2+)] and without MSH2 [Lovo(MSH2-)] were used. Pre-miR-21 was used for over-expression experiments. Luciferase vectors with MSH2 (Luc-MSH2) and MSH6 (Luc-MSH6) 3'UTRs downstream of the Luciferase gene were used. Cell cycle modifications after 5FU (10uM) were assessed by FACS analysis. Lentiviral vectors encoding for miR-21 or siRNA to MSH2 or empty vectors were used for stable infection. Stable clones were injected in the flank of nude mice. Mice were treated with 5FU i.p. for 2 weeks. Tumor volume was measured once a week and calculated according to the formula Volume=LxW2/2. Results: A statistically significant inverse correlation between miR-21 and MSH2 expression was observed by Parson's test in the two CRC cohorts. miR-21 over-expression caused reduction in MSH2 and MSH6 protein expression and in Luciferase activity after transfection with Luc-MSH2 or Luc-MSH6 vectors confirming that miR-21 directly regulates MSH2 and MSH6. miR-21 up-regulation reduced 5FU induced apoptosis and G2/M arrest at the same extent of siRNA to MSH2 in all MMR proficient cells while no significant effect was observed in Lovo(MSH2-). Complementation experiments with plasmid encoding for MSH2 promoted 5FU induced apoptosis that was inhibited by co-transfection with miR-21. Xenograft tumors over-expressing miR-21 or siRNA anti MSH2 achieved the same response to 5FU and both showed to be less responsive to 5FU than controls. Conclusions: miR-21 causes resistance to 5FU in a MSH2 dependent manner and might be a useful marker in predicting therapeutic outcome in CRC patients. No significant financial relationships to disclose.