miR-192 Regulates dihydrofolate reductase and cellular proliferation through the p53-microRNA circuit

Purpose: The purpose of this study is to investigate the molecular mechanism of miR-192 in colon cancer. Experimental Design: Human colon cancer cell lines with different p53 status were used as our model system to study the effect of miR-192 on cell proliferation, cell cycle control, and mechanism of regulation. Results: Our results show that one of the key miR-192 target genes is dihydrofolate reductase (DHFR). miR-192 affects cellular proliferation through the p53-miRNA circuit. Western immuno-blot analyses indicated that the expression of DHFR was significantly decreased by miR-192. Further investigation revealed that such suppression was due to translational arrest rather than mRNA degradation. More profound inhibition of cellular proliferation was observed by ectopic expression of miR-192 in colon cancer cell lines containing wild-type p53 than cells containing mutant p53. Thus, the effect of miR-192 on cellular proliferation is mainly p53 dependent. Over-expression of miR-192 triggered both G ₁ and G ₂ arrestin HCT-116 (wt-p53) cells but not in HCT-116 (null-p53) cells. The cell cycle checkpointcontrol genes p53 and p21 were highly over-expressed in cells that overexpressed miR-192. Endogenous miR-192 expression was increased in HCT-116 (wt-p53) and RKO (wt-p53) cells treated with methotrexate, which caused an induction of p53 expression. Chromatin immunoprecipitation-quantitative reverse transcription-PCR analysis revealed that the p53 protein interacted with the miR-192 promoter sequence. Conclusion: These results indicate that miR-192 may be another miRNA candidate that is involved in the p53 tumor suppressor network with significant effect on cell cycle control and cell proliferation.