DNA Methylation Inhibitors Upregulate MiRNA Expression in Patients with Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia is a disease with a variable outcome. Since early treatment does not result in a survival benefit, clinicians often wait for evidence of progressive disease before offering systemic chemotherapy. We recently reported on the contribution of DNA methylation to disease progression. CLL patients with higher than age-expected global DNA methylation demonstrated a requirement for chemotherapy within 12 months as compared to patients with lower than age expected levels. We hypothesized that treatment of patients with early Rai Stage CLL can delay progression of disease by re-expression of tumor suppressor genes. We used two different DNA methylation inhibitors: a DNA methyltransferase enzyme inhibitor, 5-azacytidine, and a methyl substrate inhibitor, 2-chloro-2-deoxyadenosine. We demonstrate increased microRNA expression in six patients treated with 5-azacytidine and the one patient treated with 2-chloro-2-deoxyadenosine. In 6 out of 7 patients, DNA methylation globally decreased by 8% after treatment. However, consistent microRNA upregulation was seen in mir-17-3p, mir-21, mir-29a, mir-29b, mir-29c, mir-30e, mir-104, mir-126, mir-128a, mir-130a, mir-141, mir-142-3p, mir-148a, mir-151, mir-199a, mir-199a*, and mir-301 by real-time PCR using the Early Access Human Panel from Applied Biosystems. Using the University of California Santa Cruz genome database, mir-17, mir-126, mir-128a, mir-148a, mir-151, mir-199a, and mir-301 are sequence conserved in at least 5 mammalian species and are associated with a CpG island upstream from the predicted transcriptional start site. All but mir-148a are embedded within a known gene. No changes were seen in bcl-2, p53, mir-15, or mir-16. Overexpression of mir-199-a and mir-199a* has been shown to induce cell cycle arrest. Mir-17-3p and mir-21 have anti-apoptotic properties. MiRNAs in patients with CLL are likely regulated by DNA promoter methylation.