MicroRNAs (miRNAs) and Epigenetics

MicroRNAs (miRNAs)

• miRNAs are small noncoding RNAs, approximately 18-25 nucleotides in length
• They are a major regulatory gene family in eukaryotes
• They affect protein levels of target mRNAs without changing gene sequences
• miRNAs can be regulated by epigenetic modifications like DNA methylation, RNA modification, and histone modifications

miRNA-Epigenetic Feedback Loop

• The reciprocal actions of miRNAs and epigenetic pathways
• It has an extensive influence on gene expression proliferation
• Dysregulation can interfere with physiological and pathological processes, contributing to various diseases

Biogenesis and RNAi Mechanism of miRNAs

• miRNA biogenesis is initiated by RNA polymerase II
• It transcribes miRNA genes as primary miRNAs (pri-miRNAs)
• Pri-miRNAs are recognized by a microprocessor complex containing Drosha and DGCR8 in the nucleus
• This complex cleaves pri-miRNAs into precursor miRNAs (pre-miRNAs) of 60–100 nucleotides
• Pre-miRNAs are transported to the cytoplasm by Exportin-5
• In the cytoplasm, pre-miRNAs are processed by Dicer into 18-25 nucleotide double-strand RNA
• The duplex is loaded into the RNA-induced silencing complex (RISC); one strand degrades, and the other guides RISC to the mRNA target
• miRNAs can also be produced by a Dicer-independent pathway with Ago catalysis
• miRNAs usually target sites in the 3'-UTR of mRNA
• RISC hybridization mainly causes degradation or repression of the target mRNA
• miRNAs can also bind to the 5'-UTR or coding region and activate mRNA translation
• miR-10a interacts with the 5'-UTR of many mRNAs encoding ribosomal proteins and enhances mRNAs translation

Epigenetic Regulation of miRNAs

• miRNAs play an important role in biological processes like cell proliferation, differentiation, apoptosis, and hematopoiesis
• miRNA expression profiles can function as biomarkers of diseases
• Aberrant miRNAs are known as epigenetic modulation
• miRNA expression is under epigenetic regulation: DNA methylation, RNA modification, and post-translational modification of histones

DNA Methylation-Mediated Regulation of miRNAs

• DNA methylation is predominantly found in cytosine of CpG islands
• Hypermethylation of CpG islands within miRNA gene promoters causes gene silencing and tumor suppressor properties
• Methylation of CpG islands within miRNA promoters inhibits transcription factors and RNA polymerase from binding to DNA, repressing miRNA gene expression
• Hypomethylation of CpG islands activates gene expression and promotes cancer procession
• Hypermethylation of the promoter CpG islands of miR-424 in glioma reduced miR-424 expression, associated with cell invasion and migration and promotes cell apoptosis
• When the promoter was treated with 5-aza-2'-dexycytidine (5-AZA), the expression level of miR-424 was increased
• HOXA10 was overexpressed in gastric tumors and positively correlated with miR-196b-5p expression levels
• Demethylation of the HOXA10 promoter induced the overexpression of HOXA10 and miR-196b-5p, promoting gastric cancer proliferation and invasion in vitro
• Hypermethylation of the miR-10b promoter suppressed the expression of miR-10b and promoted the progression of clear-cell renal cell carcinoma (ccRCC)
• Approximately half of miRNA genes are associated with CpG islands, suggesting they could be subjected to regulation of DNA methylation

Histone Modification-Mediated Regulation of miRNAs

• In addition to DNA methylation, histone modification may activate or suppress miRNA expression
• Histone amino-terminal regions can undergo modification, such as methylation, acetylation, ubiquitylation, phosphorylation, sumoylation, biotinylation, and ADP-ribosylation
• Acetylated lysine may relax chromatin structure and active gene-transcription, whereas histone deacetylation may lead to more compact chromatin state and gene-transcription suppression
• Treatment of HL60 leukemic cell line with panobinostat (LBH589), a histone deacetylase (HDAC) inhibitor, revealed miR-124 upregulation, with expression inhibition of downstream targets CDK4, CDK6 and EZH2
• Inhibition or RNA interference against HDAC1 in chronic lymphocytic leukemia (CLL) can induce upregulation of miRNAs targeting Bruton tyrosine kinase (BTK), subsequently suppressing downstream signaling and causing cell death
• Treating prostate cancer cell lines with OBP-801, an HDAC inhibitor, suppressed androgen receptor activity via a posttranscriptional upregulation of miRNA, repressing prostate tumorigenesis
• Lysine methylation at different positions causes different effects on gene expression, such as methylation at K36, H3K4, and K79 (transcriptional activation), whereas methylation at H3K27, H3K9, and H4K20 is associated with gene suppression
• Treating with 3-deazaneplanocin-A (DZNep), an EZH2 inhibitor, downregulated 52.3% of miRNAs and activated the expression of more than 50 miRNAs in MIA PaCa-2
• Using GSK-J4, which selectively targets the H3K27 demethylase subfamily to increase the trimethylation of H3K27, Wang et al. downregulated the expression of miR-145 in 293T cells
• Treating H1299 cells with BIX01294, an inhibitor of G9a methyltransferase, the downregulation of miR-106b-3p and miR-151a-3p was observed in lung cancer development

RNA Modification Mediated Regulation of miRNAs

• RNA modification also plays important roles in the expression of miRNAs, termed RNA epigenetics or epitranscriptomics
• More than 140 different forms of RNA modification have been found in mammalian systems, such as m6A, m5C, m1A, pseudourylation (Ψ) and deamination (A-to-I RNA editing)
• m6A is the best understood and most frequent mark of mRNA, located in the pri-miRNAs and marking them for DGCR8 recognition and processing
• Depletion of METTL3 reduced DGCR8 binding to pri-miRNAs, reducing mature miRNAs and accumulating pri-miRNAs
• HNRBPA2B1 promotes pri-miRNA processing interacted with DGCR8
• Knockdown of m6A demethylase FTO results in altered steady state levels of several miRNAs

miRNAs Affect Epigenetic Expression

• miRNAs can affect the expressions of components of the epigenetic machinery by targeting epigenetics-associated enzymes
• Epigenetic-related enzymes (DNMTs, TETs, HDACs, and EZH) can be epigenetically regulated by miRNAs and are called epi-miRNAs
• The significant regulation of miRNAs on epigenetic expression, including DNA methylation, RNA modification, and histone modification, has been increasingly recognized in recent years

miRNAs Regulate DNA Methylation and RNA Modification

• DNA methylation and demethylation are mainly regulated by DNMTs and TETs
• miRNAs can regulate the expression level of DNA methylation by influencing the expression of DNA methylation related enzymes, thereby affecting the whole genome methylation profile
• miR-29b targets DNMTs and TETs, influencing DNA methylation
• miR-29b inhibitor increased DNA methylation levels of global genome by upregulating DNMT3A/B and TET1 and downregulating TET2/3 during porcine early embryo development
• Suppression of miR-29 by ATDC led to DNMT3A upregulation and genome methylation, accompanied by the silencing of the tumor suppressor PTEN
• miR-101 is complementary to the 3'-UTR of DNMT3A mRNA. Overexpression of miR-101 could silence the expression of DNMT3A and suppress lung cell proliferation and S/G2 translation, eventually causing the apoptosis of the lung cells
• miRNAs can also target DNMT1
• miR-377 targeted the 3'-UTR of DNMT1 mRNA and regulated promoter methylation levels of skin aging-associated genes
• m6A could be regulated by miRNAs via a sequence pairing mechanism which indicates the reciprocity relationship between miRNAs and m6A

miRNAs Regulate Histone Modification

• In addition to the DNA methyltransferases, miRNAs can also regulate the histone modification
• Bioinformatics analyses have identified histone modifications, including H3K4me1, H3K27me3, H3K27ac, H3K9ac, H3K4me3, and H2AZ that are regulated by miRNAs during mammalian spermatogenesis
• HDAC1 is regulated by miR-34a via binding to the 3'UTR of HDAC1 mRNA in the foam cells
• Overexpression of miR-34a repressed HDAC1 expression and increased H3K9ac acetylation levels, eventually causing aberrant lipid accumulation in the foam cell
• KDM5B was regulated by miR-138 in breast cancer: Low-expression of miR-138 and overexpression of KDM5B were found in breast cancer cells
• Restoring miR-138 resulted in reduced KDM5B and inhibited breast cancer cell proliferation and migration
• 14 overexpressed miRNAs and 16 downregulated miRNAs were involved in the regulation of H3K9 methylation and related with the development of HCC

Crosstalk Between miRNAs and Epigenetic Regulators

• The same miRNAs that are influenced by epigenetic machinery can inversely provide epigenetic regulation
• Few miRNAs have been found to be regulated by epigenetic regulators and affect the epigenetic machinery
• miR-137 is commonly downregulated due to the hypermethylation of miR-137 CpG islands in colorectal cancer (CRC)
• Transfection of miR-137 precursor into CRC cell lines reduced cell proliferation
• Lysine (K)-specific demethylase 1A (LSD1) was discovered as the target of miR-137
• LSD1 is extensively influenced the global DNA methylation through the demethylation of DNMT1 by increasing the stability of DNMT1
• The expression of miR-137 was inhibited by the hypermethylation of CpG islands and inversely correlated with the level of the global DNA methylation
• Both miR-24 and miR-221 were repressed by protein arginine methylation (PRMT-7) which upregulated the methylation level of histone marks at the miRNAs promoter in mouse embryonic stem cells (ESCs)
• The two miRNAs and PRMT7 form a negative feedback loop

Conclusion and Future Prospective

• The discussed evidence indicates the reciprocal regulation between miRNAs and epigenetic machinery
• Epigenetic regulator drugs which can regulate the miRNA-epigenetic feedback loop,have become a potential powerful therapeutic strategy for various diseases
• Utilize synthetic miRNA mimics and miRNA sponges which contain complementary binding sites to the miRNA of interest to control the effector of epigenetic enzymes and further influence the expression of a broad range of proteins would provide a promising therapeutic strategy for associated diseases
• In the future, miRNA-epigenetic feedback loops in clinical diagnosis and therapy