miR-1250-5p is a novel tumor suppressive intronic miRNA hypermethylated in non-Hodgkin’s lymphoma: novel targets with impact on ERK signaling and cell migration

Abstract

Background: miR-1250 is localised to the second intron of AATK at chromosome 17q25. As a CpG island is present at the putative promoter region of its host gene, AATK, we postulated that the intronic miR-1250-5p is a tumor suppressor miRNA co-regulated with its host gene, AATK, by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL).

Methods: AATK/miR-1250 methylation was studied in healthy controls, including ten normal peripheral blood buffy coats and eleven normal tonsils, ten lymphoma cell lines, and 120 primary lymphoma samples by methylation-specific PCR (MSP). The expression of miR-1250-5p and AATK was investigated by quantitative real-time PCR. Tumor suppressor properties of miR-1250-5p were demonstrated by over-expression of precursor miR-1250-5p in lymphoma cells. The target of miR-1250-5p was verified by luciferase reporter assay.

Results: AATK/miR-1250 methylation was absent in healthy peripheral blood and tonsils, but detected in five (50%) NHL cell lines. AATK/miR-1250 methylation correlated with repression of miR-1250-5p and AATK in NHL cell lines. In completely methylated SU-DHL-6 and SUP-T1 cells, treatment with 5-AzadC led to promoter demethylation and re-expression of both miR-1250-5p and AATK. In primary lymphoma samples, AATK/miR-1250 was frequently methylated in B-cell lymphoma (n = 41, 44.09%) and T-cell lymphoma (n = 9, 33.33%) with a comparable frequency (P = 0.318). In SU-DHL-6 and SU-DHL-1 cells, restoration of miR-1250-5p resulted in decreased cellular proliferation by MTS assay, increased cell death by trypan blue staining and enhanced apoptosis by annexin V-PI assay. Moreover, MAPK1 and WDR1 were verified as direct targets of miR-1250-5p by luciferase assay. In 39 primary NHLs, miR-1250-5p expression was shown to be inversely correlated with each of MAPK1 (P = 0.05) and WDR1 (P = 0.031) by qRT-PCR. Finally, in SU-DHL-1 cells, overexpression of miR-1250-5p led to repression of MAPK1 and WDR1 at both transcript and protein levels, with downregulation of phospho-ERK2 by Western-blotting and inhibition of SDF-1-dependent cell migration by transwell assay.

Conclusions: miR-1250-5p is a novel tumor suppressive intronic miRNA co-regulated and silenced by promoter DNA methylation of its host gene AATK in NHL. MAPK1 and WDR1 are novel miR-1250-5p direct targets rendering inhibition of MAPK/ERK signaling and SDF-1-dependent cell migration, hence implicated in survival and dissemination of lymphoma.