Epigenetic regulation and functional characterization of microRNA-142 in mesenchymal cells.
The transcripts encoded by the microRNA mir-142 gene are highly active in hematopoietic cells, but expressed at low levels in many other cell types. Treatment with the demethylating agent 5-Aza-2'-deoxycytidine increased both the 1,636 nucleotide primary transcript and mature miR-142-5p/3p in mesenchymal cells, indicating that mir-142 is epigenetically repressed by DNA methylation. The transcription start site was determined to be located 1,205 base pairs upstream of the precursor sequence within a highly conserved CpG island. In addition, a second CpG island overlapped with the precursor. A TATA-box, several promoter-proximal elements and enrichment of conserved transcription factor binding sites within the first 100 base pairs upstream of the transcription start site, suggests that this region represents the core/proximal mir-142 promoter. Moreover, both CpG islands were heavily methylated in mesenchymal cells, having low levels of miR-142-5p/3p, and unmethylated in hematopoietic cells where both miRNAs were abundantly expressed. We show that treatment with 5-Aza-2'-deoxycytidine significantly reduced the DNA methylation of the upstream CpG island, which led to increased expression, and that in vitro DNA methylation of the upstream region of the mir-142 precursor repressed its transcriptional activity. When overexpressed, miR-142-5p/3p reduced proliferation of cells with epigenetic silencing of endogenous mir-142. This finding is interesting as miR-142-5p/3p have been reported to be deregulated in tumors of mesenchymal origin. We provide the first experimental evidence that transcription of mir-142 is directly repressed by DNA methylation. In addition, we discovered that the antisense strand of mir-142 might act as a precursor for functional mature antisense miRNAs. Thus, our study expands the current knowledge about the regulation of mir-142 and function of miR-142-5p/3p, and adds novel insight into the rapidly increasing field of microRNA regulation.
Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
Magne
M
Skårn
Tale
T
Barøy
Eva Wessel
EW
Stratford
Ola
O
Myklebost
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D020029:Base Pairing; D001483:Base Sequence; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D018899:CpG Islands; D019175:DNA Methylation; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000187:drug effects; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012322:RNA Precursors / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics; D014158:Transcription, Genetic; D014410:Tumor Stem Cell Assay
C555159:MIRN142 microRNA, human; D035683:MicroRNAs; D012322:RNA Precursors; D016372:RNA, Antisense; D000077209:Decitabine; D001374:Azacitidine