MIR129-2 has been shown to be a tumor suppressor microRNA hypermethylated in epithelial cancers. Patients and methods Epigenetic inactivation of MIR129-2 was studied by methylation-specific PCR (MSP) in 13 cell lines (eight myeloma and five lymphoma), 15 normal controls and 344 primary samples including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM) at diagnosis, MM at relapse/progression, and monoclonal gammopathy of undetermined significance (MGUS). Expression of MIR129 and its target, SOX4 , in cell lines was measured before and after hypomethylating treatment and MIR129 overexpression. MIR129 expression was correlated with MIR129-2 methylation status in primary lymphoma samples. Tumor suppressor function of MIR129 was demonstrated by MTT and trypan blue exclusion assay after MIR129 overexpression. Results The sensitivity of the methylated-MSP was one in 10 3 . Different MSP statuses, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. All five lymphoma and seven of eight myeloma cell lines showed complete and partial MIR129-2 methylation. In primary samples, MIR129-2 methylation was absent in AML and CML, but detected in 5% ALL, 45.9% CLL, 49.5% MM at diagnosis, and 59.1% NHL. In CLL, MIR129-2 methylation adversely impacted on survival (p=0.004). In MM, MIR129-2 methylation increased from 27.5% MGUS to 49.5% MM at diagnosis and 41.5% at relapse/progression (p=0.023). In NHL, MIR129-2 methylation was associated with MIR124-1 and MIR203 methylation (p<0.001), and lower MIR129 expression (p=0.009). Hypomethylation treatment of JEKO-1, homozygously methylated for MIR129-2 , led to MIR129-2 demethylation and MIR129 re-expression, with downregulation of SOX4 mRNA. Moreover, MIR129 overexpression in both mantle cell lines, JEKO-1 and GRANTA-519, inhibited cellular proliferation and enhanced cell death, with concomitant SOX4 mRNA downregulation. Conclusions MIR129-2 is a tumor suppressive microRNA frequently methylated in lymphoid but not myeloid malignancies, leading to reversible MIR129-2 silencing. In CLL, MIR129-2 methylation was associated with an inferior survival. In MM, MIR129-2 methylation might be acquired during progression from MGUS to symptomatic MM. In NHL, MIR129-2 methylation might collaborate with MIR124-1 and MIR203 methylation in lymphomagenesis.
Wonget al. Journal of Hematology & Oncology2013,6:16 http://www.jhoonline.org/content/6/1/16
R E S E A R C H
JOURNAL OF HEMATOLOGY & ONCOLOGY
Open Access
Epigenetic inactivation of theMIR1292in hematological malignancies 1†1†31 2 KwanYeung Wong , Rita LokHay Yim , YokLam Kwong , ChungYing Leung , PakKwan Hui , 4 1 5 1* Florence Cheung , Raymond Liang , DongYan Jin and ChorSang Chim
Abstract Background:MIR1292has been shown to be a tumor suppressor microRNA hypermethylated in epithelial cancers. Patients and methods:Epigenetic inactivation ofMIR1292was studied by methylationspecific PCR (MSP) in 13 cell lines (eight myeloma and five lymphoma), 15 normal controls and 344 primary samples including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), nonHodgkin’s lymphoma (NHL), multiple myeloma (MM) at diagnosis, MM at relapse/progression, and monoclonal gammopathy of undetermined significance (MGUS). Expression ofMIR129and its target,SOX4, in cell lines was measured before and after hypomethylating treatment andMIR129overexpression.MIR129expression was correlated withMIR1292methylation status in primary lymphoma samples. Tumor suppressor function of MIR129was demonstrated by MTT and trypan blue exclusion assay afterMIR129overexpression. 3 Results:The sensitivity of the methylatedMSP was one in 10 . Different MSP statuses, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. All five lymphoma and seven of eight myeloma cell lines showed complete and partialMIR1292 methylation. In primary samples,MIR1292methylation was absent in AML and CML, but detected in 5% ALL, 45.9% CLL, 49.5% MM at diagnosis, and 59.1% NHL. In CLL,MIR1292methylation adversely impacted on survival (p=0.004). In MM,MIR1292methylation increased from 27.5% MGUS to 49.5% MM at diagnosis and 41.5% at relapse/ progression (p=0.023). In NHL,MIR1292methylation was associated withMIR1241andMIR203methylation (p<0.001), and lowerMIR129expression (p=0.009). Hypomethylation treatment of JEKO1, homozygously methylated forMIR1292, led toMIR1292demethylation andMIR129reexpression, with downregulation ofSOX4mRNA. Moreover,MIR129overexpression in both mantle cell lines, JEKO1 and GRANTA519, inhibited cellular proliferation and enhanced cell death, with concomitantSOX4mRNA downregulation. Conclusions:MIR1292is a tumor suppressive microRNA frequently methylated in lymphoid but not myeloid malignancies, leading to reversibleMIR1292silencing. In CLL,MIR1292methylation was associated with an inferior survival. In MM,MIR1292methylation might be acquired during progression from MGUS to symptomatic MM. In NHL,MIR1292methylation might collaborate withMIR1241andMIR203methylation in lymphomagenesis. Keywords:microRNA, Tumor suppressor, Hypermethylation,MIR129, Hematological cancers
* Correspondence: jcschim@hku.hk † Equal contributors 1 Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Hong Kong Full list of author information is available at the end of the article