TY - JOUR
T1 - Epigenetic silencing of miR-340-5p in multiple myeloma
T2 - Mechanisms and prognostic impact
AU - Li, Zhenhai
AU - Wong, Kwan Yeung
AU - Calin, George A.
AU - Chng, Wee Joo
AU - Chan, Godfrey Chi Fung
AU - Chim, Chor Sang
N1 - Publisher Copyright:
© 2019 The Author(s).
PY - 2019/5/7
Y1 - 2019/5/7
N2 - Background: miR-340-5p, localized to 5q35, is a tumor suppressor miRNA implicated in multiple cancers. As a CpG island is present at the putative promoter region of its host gene, RNF130, we hypothesized that the intronic miR-340-5p is a tumor suppressor miRNA epigenetically silenced by promoter DNA methylation of its host gene in multiple myeloma. Results: By pyrosequencing-confirmed methylation-specific PCR, RNF130/miR-340 was methylated in 8/15 (53.3%) myeloma cell lines but not normal plasma cells. Methylation correlated inversely with the expression of both miR-340-5p and RNF130. In completely methylated WL-2 and RPMI-8226R cells, 5-AzadC treatment led to demethylation and re-expression of miR-340-5p. In primary samples, RNF130/miR-340 methylation was detected in 4 (22.2%) monoclonal gammopathy of undetermined significance, 15 (23.8%) diagnostic myeloma, and 7 (23.3%) relapsed myeloma. RNF130/miR-340 methylation at diagnosis was associated with inferior overall survival (median 27 vs. 68 months; P = 3.944E-5). In WL-2 cells, overexpression of miR-340-5p resulted in reduced cellular proliferation [MTS, P = 0.002; verified in KMS-12-PE (P = 0.002) and RPMI-8226R (P = 2.623E-05) cells], increased cell death (trypan blue, P = 0.005), and enhanced apoptosis by annexin V-PI staining. Moreover, by qRT-PCR, overexpression of miR-340-5p led to repression of both known targets (CCND1 and NRAS) and bioinformatically predicted targets in MAPK signaling (MEKK1, MEKK2, and MEKKK3) and apoptosis (MDM4 and XIAP), hence downregulation of phospho-ERK1/2 and XIAP by Western blot. Furthermore, by qRT-PCR, in CD138-sorted primary samples (n = 37), miR-340-5p and XIAP were inversely correlated (P = 0.002). By luciferase assay, XIAP was confirmed as a direct target of miR-340-5p via targeting of the distal but not proximal seed region binding site. Conclusions: Collectively, tumor-specific methylation-mediated silencing of miR-340-5p is likely an early event in myelomagenesis with adverse survival impact, via targeting multiple oncogenes in MAPK signaling and apoptosis, thereby a tumor suppressive miRNA in myeloma.
AB - Background: miR-340-5p, localized to 5q35, is a tumor suppressor miRNA implicated in multiple cancers. As a CpG island is present at the putative promoter region of its host gene, RNF130, we hypothesized that the intronic miR-340-5p is a tumor suppressor miRNA epigenetically silenced by promoter DNA methylation of its host gene in multiple myeloma. Results: By pyrosequencing-confirmed methylation-specific PCR, RNF130/miR-340 was methylated in 8/15 (53.3%) myeloma cell lines but not normal plasma cells. Methylation correlated inversely with the expression of both miR-340-5p and RNF130. In completely methylated WL-2 and RPMI-8226R cells, 5-AzadC treatment led to demethylation and re-expression of miR-340-5p. In primary samples, RNF130/miR-340 methylation was detected in 4 (22.2%) monoclonal gammopathy of undetermined significance, 15 (23.8%) diagnostic myeloma, and 7 (23.3%) relapsed myeloma. RNF130/miR-340 methylation at diagnosis was associated with inferior overall survival (median 27 vs. 68 months; P = 3.944E-5). In WL-2 cells, overexpression of miR-340-5p resulted in reduced cellular proliferation [MTS, P = 0.002; verified in KMS-12-PE (P = 0.002) and RPMI-8226R (P = 2.623E-05) cells], increased cell death (trypan blue, P = 0.005), and enhanced apoptosis by annexin V-PI staining. Moreover, by qRT-PCR, overexpression of miR-340-5p led to repression of both known targets (CCND1 and NRAS) and bioinformatically predicted targets in MAPK signaling (MEKK1, MEKK2, and MEKKK3) and apoptosis (MDM4 and XIAP), hence downregulation of phospho-ERK1/2 and XIAP by Western blot. Furthermore, by qRT-PCR, in CD138-sorted primary samples (n = 37), miR-340-5p and XIAP were inversely correlated (P = 0.002). By luciferase assay, XIAP was confirmed as a direct target of miR-340-5p via targeting of the distal but not proximal seed region binding site. Conclusions: Collectively, tumor-specific methylation-mediated silencing of miR-340-5p is likely an early event in myelomagenesis with adverse survival impact, via targeting multiple oncogenes in MAPK signaling and apoptosis, thereby a tumor suppressive miRNA in myeloma.
KW - DNA methylation
KW - Multiple myeloma
KW - Overall survival
KW - XIAP
KW - miR-340-5p
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U2 - 10.1186/s13148-019-0669-2
DO - 10.1186/s13148-019-0669-2
M3 - Article
C2 - 31064412
AN - SCOPUS:85065503631
SN - 1868-7075
VL - 11
JO - Clinical epigenetics
JF - Clinical epigenetics
IS - 1
M1 - 71
ER -