cancers Review MicroRNA Regulation of Epigenetic Modifiers in Breast Cancer Brock Humphries 1,* , Zhishan Wang 2 and Chengfeng Yang 2,3,* 1 Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, MI 48109; USA 2 Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, KY 40536, USA 3 Center for Research on Environment Disease, College of Medicine, University of Kentucky, Lexington, KY 40536; USA * Correspondence: [email protected] (B.H.); [email protected] (C.Y.) Received: 24 May 2019; Accepted: 24 June 2019; Published: 27 June 2019 Abstract: Epigenetics refers to the heritable changes in gene expression without a change in the DNA sequence itself. Two of these major changes include aberrant DNA methylation as well as changes to histone modification patterns. Alterations to the epigenome can drive expression of oncogenes and suppression of tumor suppressors, resulting in tumorigenesis and cancer progression. In addition to modifications of the epigenome, microRNA (miRNA) dysregulation is also a hallmark for cancer initiation and metastasis. Advances in our understanding of cancer biology demonstrate that alterations in the epigenome are not only a major cause of miRNA dysregulation in cancer, but that miRNAs themselves also indirectly drive these DNA and histone modifications. More explicitly, recent work has shown that miRNAs can regulate chromatin structure and gene expression by directly targeting key enzymes involved in these processes. This review aims to summarize these research findings specifically in the context of breast cancer. This review also discusses miRNAs as epigenetic biomarkers and as therapeutics, and presents a comprehensive summary of currently validated epigenetic targets in breast cancer. Keywords: non-coding RNAs; microRNAs; epigenetics; breast cancer 1. Non-coding RNAs (ncRNAs) and MicroRNAs (miRNAs) The discovery that the majority of the human genome is not transcribed into protein [1], expanded our initial definition of a gene to include non-coding RNAs. Unlike the traditional definition of a gene, a DNA sequence that is transcribed into RNA then translated to produce a functional protein product, non-coding RNAs (ncRNAs) are functional RNA molecules that are not translated into protein. While the function of many of the ncRNAs in the genome is not yet determined, ncRNAs can generally be separated into two different groups: infrastructural and regulatory. Infrastructural ncRNAs are constitutively expressed and function within many cell homeostatic processes, such as splicing and translation, and include species such as transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA). On the other hand, regulatory ncRNAs elicit effects on other RNA molecules, and can be further classified based upon their size into short (<200 nt), which includes microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs), and long (lncRNA, >200 nt) ncRNAs. MicroRNAs (miRNAs) are a large family of ~21–22 nucleotide RNAs that negatively regulate protein-coding gene expression post-transcriptionally in both metazoans and plants [2]. Canonically, miRNAs are transcribed in the nucleus by RNA polymerase II [3] or III [4] into primary miRNA (pri-miRNA), which range from hundreds to thousands of nucleotides long. Intronic miRNAs, located Cancers 2019, 11, 897; doi:10.3390/cancers11070897 www.mdpi.com/journal/cancers Cancers 2019, 11, x 2 of 24 Canonically, miRNAs are transcribed in the nucleus by RNA polymerase II [3] or III [4] into Cancersprimary2019 miRNA, 11, 897 (pri-miRNA), which range from hundreds to thousands of nucleotides 2long. of 25 Intronic miRNAs, located within a host gene, are transcribed using the same promoter as the withinprimary a hosttranscript, gene, are while transcribed intergenic using miRNAs the same rely promoter on their asown the promoters primary transcript, [5–7]. After while transcription intergenic miRNAsby RNA relypolymerase, on their ownpri-miRNAs promoters are [5 –then7]. Afterpolyadenylated transcription and by capped. RNA polymerase, Pri-miRNA pri-miRNAs transcripts arecontain then a polyadenylatedstem-loop structure and that capped. contains Pri-miRNA the mature transcripts miRNA sequences, contain a stem-loopwhich are cleaved structure via that an containsRNase III the type mature enzyme, miRNA Drosha, sequences, and its binding which are partner cleaved DiGeorge via an RNasesyndrome III type critical enzyme, region Drosha, gene 8 and(DGCR8) its binding [8–10]. partner The resultant DiGeorge stem-loop syndrome intermed criticaliates region are termed gene 8 precursor (DGCR8) miRNA [8–10]. (pre-miRNA). The resultant stem-loopEach pre-miRNA intermediates is then are exported termed precursorto the cytosol miRNA via (pre-miRNA). exportin-5 [11–13], Each pre-miRNA and once in is thenthe cytosol exported is tosubjected the cytosol to viaanother exportin-5 microprocessing [11–13], and event once incarr theied cytosol out by is subjectedthe RNase to anotherIII enzyme microprocessing Dicer. Dicer eventcleaves carried the pre-miRNA out by the RNase into an III enzymeRNA duplex Dicer. (~22 Dicer nucleotides) cleaves the pre-miRNAcontaining intothe mature an RNA miRNA duplex (~22sequence nucleotides) [14–18]. containing In most cases, the mature one strand miRNA of sequencethis duplex [14 –is18 preferentially]. In most cases, degraded, one strand while of thisthe duplexother is isselected preferentially to be loaded degraded, into the while RNA the induced other is silencing selected complex to be loaded (RISC) into [19,20]. the RNA Once induced loaded silencinginto the RISC, complex the (RISC)mature [ 19miRNA,20]. Once provides loaded the into binding the RISC, specificity the mature for the miRNA complex, provides and the the complex binding specificityelicits the fornegative the complex, regulation and of the the complex target mR elicitsNA. the A negativefigure graphically regulation describing of the target the mRNA. canonical A figuremiRNA graphically biogenesis describing pathway theis shown canonical in miRNAFigure 1. biogenesis It should pathwayalso be noted is shown that in recent Figure research1. It should has alsoidentified be noted several that recentmiRNAs research that hasare identifiedformed by several bypassing miRNAs key that steps are in formed the canonical by bypassing miRNA key stepsbiogenesis in the pathway canonical [21,22]. miRNA Howeve biogenesisr, these pathway non-canonical [21,22]. However, miRNAs these still non-canonicalfunction like the miRNAs canonical still functionmiRNAs. like the canonical miRNAs. FigureFigure 1.1. CanonicalCanonical miRNAmiRNA biogenesisbiogenesis pathway.pathway. miRNAs are transcribed in the nucleusnucleus viavia theirtheir ownown promoterspromoters oror hosthost genegene promoterspromoters byby RNARNA polymerasepolymerase IIII oror III,III, formingforming thethe primaryprimary miRNAmiRNA transcriptstranscripts whichwhich can can range range from from hundreds hundreds to thousands to thousands of nucleotides of nucleotides long. Pri-miRNA long. Pri-miRNA transcripts aretranscripts polyadenylated are polyadenylated and capped, an thend capped, subjected then to subjected a microprocessing to a microprocessing cleavage event cleavage by an RNase event IIIby typean RNase enzyme, III type Drosha, enzyme, and its Drosha, binding and partner its binding DiGeorge partner syndrome DiGeorge critical syndrome region critical gene 8 (DGCR8)region gene to form8 (DGCR8) a 60–120 to nucleotideform a 60–120 long nucleotide precursor miRNAlong prec transcriptursor miRNA (pre-miRNA). transcript After (pre-miRNA). the cleavage After event, the pre-miRNAscleavage event, are pre-miRNAs then exported are out then of the exported nucleus ou byt of exportin-5 the nucleus to cytoplasm by exportin-5 and againto cytoplasm subjected and to aagain microprocessing subjected to eventa microprocessing by another RNase event II by enzyme, another Dicer, RNase to II form enzyme, a miRNA Dicer, duplex. to form Unwinding a miRNA ofduplex. the miRNA Unwinding duplex of occurs the miRNA and one duplex strand occurs is usually and degraded, one strand while is usually the other degraded, is loaded while into thethe RNA induced silencing complex (RISC). Once loaded, the RISC searches for targets of the miRNA in other is loaded into the RNA induced silencing complex (RISC). Once loaded, the RISC searches for the genome. Once bound to a target mRNA, the RISC may induce negative expression of the mRNA targets of the miRNA in the genome. Once bound to a target mRNA, the RISC may induce negative by three ways: 1) mRNA destabilization and degradation, 2) mRNA translational inhibition, or 3) expression of the mRNA by three ways: 1) mRNA destabilization and degradation, 2) mRNA mRNA cleavage. The path at which the mRNA is regulated depends upon multiple factors of the translational inhibition, or 3) mRNA cleavage. The path at which the mRNA is regulated depends mature miRNA. upon multiple factors of the mature miRNA. Although exceptions have been found, miRNAs typically elicit their inhibitory effects by base pairing with the 30 untranslated regions (30-UTR) of target mRNAs through their seed sequence. The seed sequence is the second to eighth nucleotide region at the 50 end of the mature miRNA, and is Cancers 2019, 11, 897 3 of 25 primarily responsible for determining the specificity of the miRNA to its mRNA. Although the seed sequence is