H19X-encoded miR-424(322)/-503 cluster: emerging roles in cell differentiation, proliferation, plasticity and metabolism.

Using circulating miRNAs as biomarkers has gained tremendous research interests [ 128 , 129 ]. Many cell types, such as reticulocyte, dendritic cell, B cell, T cell, mast cell, epithelial cell, as well as tumor cell, release miRNAs. They are incorporated into exosomes/extracellular vesicles (EVs) and transferred to body fluids, such as plasma, urine and saliva. Exosomes/EVs carry and deliver mRNAs and miRNAs into recipient cells and exert profound physiological and pathological functions [ 130 , 131 ]. Meantime, these circulating RNAs constitute a new category of non-invasive disease markers. Circulating miR-15/-16 show correlation with several cancer types, including glioma, esophageal adenocarcinoma, cervical cancer and breast cancer [ 132 – 135 ]. They exhibit prognostic value in melanoma and acute heart failure [ 136 , 137 ]. miR-424(322)/-503, especially miR-424, often constitute miRNA signatures with high predictive power for disease outcomes. Bye et al. assayed 179 miRs in the serum of 112 healthy participants who either suffered from fatal AMI within 10 years or remained healthy. They established a model for predicting future AMI consisting of miR-106a-5p, miR-424-5p, let-7g-5p, miR-144-3p and miR-660-5p, with 74.1% and 81.8% correct classification for men and women, respectively [ 138 ]. de Andrade et al. showed in 39 ALS patients/39 controls that miR-424 and miR-206 were higher in patient plasma and the baseline levels were associated with clinical deterioration [ 139 ]. Two groups independently demonstrated the prognostic value of miR-424 in non-small cell lung cancers. One model includes four miRNAs (miR-200c, miR-424, miR-29c and miR-124), whereas the other includes six miRNAs (miR-29a, miR-542-5p, miR-502-3p, miR-376a, miR-500a, miR-424), each holding prognostic value for overall survival [ 140 , 141 ]. A 3-miRNA signature (miR-199a, miR-29c and miR-424) was found to distinguish breast cancer patients from controls [ 142 ]. We have summarized recent reports about miR-424(322)/-503 as circulating biomarkers in a variety of diseases (Table  2 ). Though there are many caveats regarding using circulating miRNA as biomarkers, it is clear that miR-424 is one of the best candidates that may be vetted in larger cohorts. Table 2 Detection of miR-424(322)/-503 as biomarkers in body fluids miRNA Disease or physiological status Sample Expression change Method Refs. miR-424 Breast cancer Serum + SdM-RT-PCR [ 142 ] miR-424 Advanced NSCLC Blood + miRNA microarray [ 141 ] miR-424 FXTAS Blood + miRNA microarray and sequencing [ 143 ] miR-424 ALS Skeletal muscle, plasma + miRNA microarray [ 139 ] miR-503 Vertebral fractures Serum − qRT-PCR [ 144 ] miR-503 Postmenopausal osteoporosis Blood − miRNA microarray [ 145 ] miR-424 PH Serum + qRT-PCR [ 146 ] miR-424-5p Type 1 diabetes Serum + qRT-PCR [ 147 ] miR-424-5p DVT Plasma + qRT-PCR array [ 148 ] miR-424-5p AMI Serum + qRT-PCR array [ 138 ] miR-424-5p Heart failure Plasma − qRT-PCR array [ 149 ] miR-503 CAD Plasma − qRT-PCR [ 150 ] miR-424-3p, miR-424-5p Aerobic exercise Serum + qRT-PCR [ 151 ] sdM-RT-PCR serum-direct multiplex detection assay based on RT-PCR, NSCLC non-small cell lung cancer, FXTAS fragile X-associated tremor/ataxia syndrome, ALS amyotrophic lateral sclerosis, PH pulmonary hypertension, DVT deep-vein thrombosis, AMI acute myocardial infarction, CAD coronary artery disease Detection of miR-424(322)/-503 as biomarkers in body fluids sdM-RT-PCR serum-direct multiplex detection assay based on RT-PCR, NSCLC non-small cell lung cancer, FXTAS fragile X-associated tremor/ataxia syndrome, ALS amyotrophic lateral sclerosis, PH pulmonary hypertension, DVT deep-vein thrombosis, AMI acute myocardial infarction, CAD coronary artery disease A major challenge is that the source of the circulating miRNAs is often unknown. It is critical to distinguish whether they are from a primary lesion such as cancer, or a secondary and reactive source, such as lymphocytes or muscles. Technical issues include the lack of a housekeeping circulating RNA control to normalize among individuals and different diseases, and variations introduced by different extraction and quantification methods. The importance of addressing these challenges cannot be overestimated; it likely constitutes a major milestone in diagnostics.

Consistent with a role in adapting cells to changed environment, miR-424 has been shown to regulate major metabolic switches. TGF-β or PDGF-induced CAF formation is accompanied by metabolic switch from oxidative phosphorylation to aerobic glycolysis [ 104 ]; miR-424 plays a critical role in the process. TGF-β upregulates the expression of miR-424, and miR-424 directly targets isocitrate dehydrogenase 3a, an enzyme catalyzing the conversion from isocitrate to α-ketoglutarate (α-KG) in the tricarboxylic acid (TCA) cycle. Upregulated miR-424 causes a drop in α-KG, which contributes to the stability of HIF-1α. Proteasome-mediated HIF-1α degradation requires HIF-1α hydroxylation on two proline residues, P402 and P564; the activity of the responsible enzyme, proline hydroxylase, requires oxygen and α-KG. Structural analogs of α-KG, such as succinate and fumarate, inhibit proline hydroxylase activity [ 123 – 125 ]. Increased miR-424 effectively drops the ratio between α-KG and succinate/fumarate, and reduces HIF-1α degradation [ 104 ]. This is another mechanism that miR-424 uses to stabilize HIF-1α (Fig.  5 ). Stabilized HIF-1α transactivates genes involved in glycolysis. Diabetic levels of glucose significantly drive down miR-424 expression in breast cancer cells [ 126 ]. Lowered miR-424 levels lead to higher expression of its target gene, CDC42. CDC42 induces the expression of transcription factor PRDM14, which is associated with poor prognosis in breast cancer patients [ 126 ]. Wang et al. reported decreased miR-150, miR-146a and miR-424 in peripheral blood mononuclear cells from type I diabetic patients, and the decrease is associated with ongoing autoimmunity of pancreatic islet [ 127 ]. However, it is not clear how miR-424 is suppressed under hyperglycemic conditions, and how this change contributes to adaptive or pathological alterations. Research in this area is not yet sufficient to build a unitary framework, but miR-424(322)/-503 apparently influences metabolic pathways, which serve as bridges linking many processes discussed so far.

The expression of miR-424(322)/-503 is significantly altered during several hormone-controlled processes. Endometriosis is a benign gynecological disease among women in reproductive age, characterized by the presence of endometrial glands and stroma in locations other than the uterine cavity. Several groups reported the downregulation of miR-424 or miR-503 in endometrial tissues vs. controls, or in ectopic vs. eutopic endometrial tissues [ 88 – 90 ]. There is an inverse correlation between miR-424 and the levels of VEGF-A [ 89 ]. Higher VEGF-A levels may be responsible for elevated angiogenic activity in endometriotic lesions. Ovary granulosa cells support the growth and maturation of follicles, and they undergo constant morphological and functional changes. miR-424/-503 are highly expressed in granulosa cells, with varying expression levels during the menstrual cycle [ 91 – 93 ]. miR-424/-503 regulate proliferation of granulosa cells, but how they affect follicle growth and maturation is unknown [ 91 , 94 ]. Two groups have reported that expression of miR-424(322)/-503 is responsive to estrogen in MCF-7 breast epithelial cells. One group captured the temporal profiles of miRNAs and identified both miR-424 and miR-503 as among the most upregulated by E2 [ 95 ]. The other group captured the temporal profiles of mRNAs following E2 exposure and reversely predicted miRNAs that may be regulated. miR-424 was among the top findings and experimentally vetted [ 96 ]. Though these studies support the close association of miR-424 and -503 with estrogen, it is unknown if an estrogen receptor element is present on the upstream regulatory region and essential for H19X transcription. Efforts in this area will provide new insights into hormone-related disease mechanisms. Further, as miR-424(322)/-503 emerge as a critical regulator of muscle differentiation, growth and metabolism [ 42 , 43 , 97 ], their hormone link may help explain sex differences in muscle physiology and diseases.

Over a decade of research has accumulated a large body of knowledge related to the miR-424(322)/-503 cluster. Individually or together, these miRNAs are involved in placenta, heart and skeletal muscle development during embryogenesis. They regulate core cellular processes including cell cycle control and EMT. They are the most dynamic in responding to a range of cellular stresses, including hypoxia and ischemia, and help restore homeostasis. Hormonal regulation over the biogenesis of these miRNAs and their involvement in physiological and pathological processes of female reproduction organs prompt an important question, are they one of the deciding factors of sex differences? The identification of miR-424(322)/-503 as biomarkers in many human diseases, especially the frequent detection in plasma samples, poses exciting clinical opportunities. However, this is still a new research subject; there remain many challenges awaiting exploration. Many important hypotheses are not validated. The connection to H19 has not been supported by genetic evidence in animal models. Does miR-424(322)/-503 overexpression stall placental development, like in H19 transgenic mouse? Is the H19X locus paternally imprinted? Genetic ablation of H19 or miR-424(322)/-503 causes very mild systemic phenotypes: H19 KO animal has mild overgrowth [ 29 ], whereas miR-424(322)/-503 KO animal has mild white fat accumulation [ 62 ]. Will double-knockout animal display more pronounced phenotypes related to organism growth or homeostasis? Answering these questions would provide important insights into ncRNA regulatory mechanisms of growth control. It remains difficult to rank the importance of molecular targets of miRNAs in a biological process. Most studies rely on computation programs or transcriptome survey to predict miRNA targets, and select one or two for additional investigation. Such strategies have intrinsic weaknesses: miRNAs are known to target many genes simultaneously, and mainly through affecting protein abundance, not mRNA abundance. With the advancement of technologies such as reverse phase protein array (RPPA), we may include protein arrays into the toolbox of miRNA target identification. This note is especially important for miR-424(322)/-503, as they are highly dynamic and their identified targets sometimes occupy opposite sides of signaling pathways. Developing therapeutic interfering strategies require understanding of the redundancy and coordination between miR-424(322)/-503 and other miR-15/107 family miRNAs. It is a challenging task to inhibit the activities of miR-424(322)/-503, because other miR-15 family members may be parallel or compensatory. Thus, developing strategies that target all the miRNAs sharing the same seed sequence would provide conclusive evidence for the function of miR-424(322)/-503 in important processes, and also form the base for therapy development. Finally, building connections among the currently separated processes is critical. For instance, under cellular stress, cell proliferation, plasticity and metabolism may be well orchestrated, and miR-424(322)/-503 may coordinate the expression of a network of genes and help cells adapt and regain homeostasis. Systemic dissection of these processes, especially at protein and organism levels, would likely yield important insights that are currently unavailable.

MicroRNAs (miRNAs) are small non-coding RNAs 18–24 nucleotides (nt) in length that regulate posttranscriptional gene expression [ 1 ]. miRNA genes are transcribed by RNA polymerase II to produce pri-miRNA, which is cleaved by Drosha to give rise to hairpin-structured pre-miRNA, ~ 60–100 nt in length. Exportin transports pre-miRNA into the cytoplasm, where it is processed by Dicer to produce a ~ 22 nt double-stranded intermediate comprising the mature miRNA strand and its complementary strand. The mature miRNA is loaded into the RNA-induced silencing complex (RISC) where it binds to the 3′ UTR of target mRNAs by partial sequence complement in the “seed” region, causing degradation of the mRNA transcript or inhibition of its translation. The miR-15/107 family of microRNAs shares the “AGCAGC” sequence within the “seed” region, starting at either the first or the second nucleotide from the 5′ end [ 2 ]. They are critical regulators of cell division, apoptosis, stress response and metabolism, and involved in cancer, cardiovascular and neurodegenerative disorders. miR-424 (ortholog of rodent miR-322) and miR-503 are mammal-specific members of the miR-15/107 family. They are encoded as one cluster by H19X, located in human Xq26.3 [ 3 ]. Their expression is more dynamic and tissue restrictive than other miR-15/107 family members. miR-424(322)/-503 regulate fundamental processes such as cell cycle, epithelial-to-mesenchymal transition and hypoxia, drive tissue differentiation and remodeling, and paradoxically participate in tumor initiation and progression. Here, we summarize a decade of literature and provide a primer for future investigation concerning miR-424(322)/-503.

The miR-15/107 family includes ten miRNAs based on the presence of “AGCAGC” in the “seed” region situated at positions 2–7 from the 5′ end of mature microRNAs [ 2 ]. However, there is no consensus regarding the criteria of miRNA family classification; distinct classifications were proposed for these ten miRNAs [ 4 – 9 ]. We have adopted the classification by Finnerty et al. [ 2 ], but this is likely to change with new functional data accumulated and new miRNAs identified (Fig.  1 a). Fig. 1 Members of the miR-15/107 microRNA family. a 10 microRNAs sharing “AGCAGC” within the “seed” region. b A phylogenetic tree of miR-15/107 family members from Homo sapiens ( hsa ), Monodelphis domestica ( mdo ), Macaca mulatta ( mml ), Mus musculus ( mmu ), Ornithorhynchus anatinus ( oan ), Gallus gallus ( gga ) and Xenopus tropicalis ( xtr ). miR-103 and -107 are the least closely related, and omitted in the plot. miR-424(322) is most closely related to miR-15c in mdo , oan , gga and xtr , while miR-503 is most closely related to miR-16c in gga , oan and xtr Members of the miR-15/107 microRNA family. a 10 microRNAs sharing “AGCAGC” within the “seed” region. b A phylogenetic tree of miR-15/107 family members from Homo sapiens ( hsa ), Monodelphis domestica ( mdo ), Macaca mulatta ( mml ), Mus musculus ( mmu ), Ornithorhynchus anatinus ( oan ), Gallus gallus ( gga ) and Xenopus tropicalis ( xtr ). miR-103 and -107 are the least closely related, and omitted in the plot. miR-424(322) is most closely related to miR-15c in mdo , oan , gga and xtr , while miR-503 is most closely related to miR-16c in gga , oan and xtr miR-15/107 family members are only expressed in chordates, with several being mammal specific (miR-195, -497, -503, -424 and -646) [ 2 ]. Genes of miR-15/107 family members are genomically associated with protein-coding genes or lncRNAs. They also show conserved tandem organization: miR-15 with miR-16, miR-424(322) with miR-503, and miR-497 with miR-195. To explore the evolutionary relation of miR-424(322)/-503 to others, we have updated a phylogenetic tree originally built by Necsulea et al. [ 3 ], using “stem-loop” pre-miRNA sequences in Homo sapiens , Monodelphis domestica , Macaca mulatta , Mus musculus , Ornithorhynchus anatinus , Gallus gallus and Xenopus tropicalis extracted from miRBase. miR-424 (together with miR-322 and miR-15c) and miR-503 (together with miR-16c) represent two distinct subfamilies related to miR-15 and miR-16, respectively (Fig.  1 b). Supporting a common phylogenetic origin, miR-15/107 family members have similar expression patterns and functions. miR-15a, -15b, -16, -322 and -503 are dynamically upregulated during serum starvation and contact inhibition, with miR-503 showing the highest fold change [ 10 ]. miR-15a, -15b, -16 and -497 are essential for the switch from expansion to differentiation in precursor B lymphocytes [ 11 ]. On the other side, loss of either miR-15a/-16-1 or miR-15b/-16-2 by genomic deletion causes B cell chronic lymphocytic leukemia [ 12 – 14 ]. miR-15/-16 family members also drive NK cell maturation, by targeting Myb [ 15 ]. miR-15/107 family members inhibit cell proliferation in many tissue types. They are broadly upregulated after birth and cause cardiomyocyte mitotic arrest in rodents [ 16 , 17 ]. Additionally, miR-15/107 family members respond to cellular stresses such as hypoxia, ischemia, ultraviolet, environmental toxin, etc., and induce adaptive changes in angiogenesis and cellular metabolism [ 18 – 23 ]. As will be detailed in the rest of this essay, the function of miR-424(322)/-503 overlaps with other family members, but is under unique temporal and spatial regulations and works in distinct processes. Necsulea et al. surveyed eight organs in 11 tetrapod species for the expression profiles of ncRNAs [ 3 ]. They have identified approximately 400 lncRNA genes that are at least 300 million years old. These lncRNAs evolve rapidly in terms of sequence and expression levels, but conserve tissue specificity. One evolutionarily conserved co-expression network is predicted to regulate placenta development. This network comprises H19 and the lncRNA that encodes the miR-424(322)/-503 cluster, which was hence named H19X. H19 is best known as the imprinting paradigm [ 24 – 29 ]: except under rare pathological conditions, the H19 gene is only expressed from the maternal allele, while the adjacent IGF2 gene is only expressed from the paternal allele. Imprinting provides an important mechanism of gene dose control, allowing expression from only one allele, while the other is epigenetically silenced. Many imprinted genes are involved in placenta development; H19 regulates placenta growth in late gestation, via its “spinoff” miR-675 [ 30 ]. Overexpression of H19 causes embryonic and perinatal lethality [ 31 ]. H19 is quickly downregulated in most tissues except skeletal muscles after birth [ 32 , 33 ]. H19X resembles H19 in several ways. First, H19X may be imprinted (see following section). Second, miR-424 is downregulated by hypoxia in trophoblasts, and higher miR-424 levels are associated with fetal growth restriction, indicating a role in placenta growth regulation [ 34 , 35 ]. Third, expression of H19X is striated muscle restricted during embryogenesis. In addition to these suggestive evidences, it will be important to know if H19 and H19X are mutually regulatory, and if they cooperate with or compensate for each other in genetic models. Addressing these questions may provide insights into the intricate mechanisms of gene dose control and coordination in embryonic development. The human H19X locus encodes seven non-coding RNAs, including a number of microRNAs (miR-424, 503, 542, 450-1, 450-2, and 450b) and a long non-coding RNA (miR503HG), spanning a region of ~ 7 kb pairs on Xq26.3 (Fig.  2 ). The microRNAs are highly conserved in mammals. The lncRNA has similar expression patterns as miR-424(322)/-503, agreeing with being the host gene of the miRNA cluster. The ENCODE project has identified clustered H3K27Ac and DNAse I signals, while the GeneHancer project has identified a high-confidence cluster of regulatory elements in the upstream regulatory region and gene bodies of miR-424 [ 36 ], miR-503 and miR503HG, suggesting that the H19X locus is actively transcribed and intricately regulated. Fig. 2 Schematic diagram of the H19X locus. miR-424, -503, -542, -450b, -450a1 and -450a1, and at least one lncRNA, miR503HG, are encoded in the H19X locus. H19X spans approximately 7 k nucleotides on the minus strand of Xq26.3. It is unknown if other adjacent ncRNAs, such as linc00629, are related to H19X. In the upstream regulatory regions as well as gene bodies, active epigenetic marks including H3K27Ac and DNAse I-hypersensitive clusters were identified by the ENCODE project; a high-confidence enhancer/promoter cluster was identified by the GeneHancer project. Information obtained from UCSC Genome Browser Schematic diagram of the H19X locus. miR-424, -503, -542, -450b, -450a1 and -450a1, and at least one lncRNA, miR503HG, are encoded in the H19X locus. H19X spans approximately 7 k nucleotides on the minus strand of Xq26.3. It is unknown if other adjacent ncRNAs, such as linc00629, are related to H19X. In the upstream regulatory regions as well as gene bodies, active epigenetic marks including H3K27Ac and DNAse I-hypersensitive clusters were identified by the ENCODE project; a high-confidence enhancer/promoter cluster was identified by the GeneHancer project. Information obtained from UCSC Genome Browser In human and mouse genomes, H19X is situated between PLAC1 and HPRT on the 5′ and 3′ ends, respectively. PLAC1 is a placenta-specific protein whose expression is restricted in the trophoblast lineage. PLAC1 is paternally imprinted, and deficiency of it causes placentomegaly [ 37 ]. In marsupials, the downstream gene of H19X is RNA-on-the-silent X (Rsx), an Xist-like lncRNA that drives X-inactivation [ 38 ]. Collectively, the genomic location and indication in placenta growth suggest that H19X is probably imprinted. The precursor sequence of the miR-424(322)/-503 cluster encodes both -5p and -3p mature miRNAs. According to expression levels, miR-322-5p, miR-424-5p and miR-503-5p are predominant over their -3p counterparts. The -3p miRNAs are not related to the miR-15/107 family. Throughout this manuscript, miR-322, miR-424 and miR-503 refer to -5p mature miRNAs. There are few studies about the function of -3p miRNAs encoded by the miR-424(322)/-503 cluster; how these -3p miRNAs contribute to described phenotypes is completely unknown.