miRNA inherited disease, DNA repair, cancer, alcoholism, obesity,heart disease

Disease

Just as miRNA is involved in the normal functioning of eukaryotic cells, so has dysregulation of miRNA been associated with disease.[140] A manually curated, publicly available database, miR2Disease, documents known relationships between miRNA dysregulation and human disease.[141]

Inherited diseases

A mutation in the seed region of miR-96, causes hereditary progressive hearing loss.[142]

A mutation in the seed region of miR-184, causes hereditary keratoconus with anterior polar cataract.[143]

Deletion of the miR-17~92 cluster, causes skeletal and growth defects.[144]

Cancer

Role of miRNA in a cancer cell

The first human disease known to be associated with miRNA deregulation was chronic lymphocytic leukemia.[46] Many other miRNAs also have links with cancer[46] and accordingly are sometimes referred to as “oncomirs“. In malignant B cells miRNAs participate in pathways fundamental to B cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches and the production and class-switching of immunoglobulins. MiRNAs influence B cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells.[46]

A study of mice altered to produce excess c-Myc — a protein with mutated forms implicated in several cancers — shows that miRNA affects the cancer development. Mice engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days.[46] Leukemia can be caused by the insertion of a viral genome next to the 17-92 array of microRNAs, leading to increased expression of this microRNA.[46]

Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off.[46]

By measuring activity among 217 genes encoding miRNAs, patterns of gene activity that can distinguish types of cancers were identified. miRNA profiling can determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer.[46]

A novel miRNA-profiling-based screening assay for the detection of early-stage colorectal cancer is undergoing a clinical trial. Early results showed that blood plasmasamples collected from patients with early, resectable (Stage II) colorectal cancer could be distinguished from those of sex-and age-matched healthy volunteers. Sufficient selectivity and specificity could be achieved using small (less than 1 mL) samples of blood.[145][146]

Another role for miRNA in cancers is to use their expression level for prognosis. For example, one study on NSCLC samples found that low miR-324a levels could serve as an indicator of poor survival.[147] Either high miR-185 or low miR-133b levels may correlate with metastasis and poor survival in colorectal cancer.[148]

Furthermore, specific miRNAs may be associated with certain histological subtypes of colorectal cancer. For instance, expression levels of miR-205 and miR-373 have been shown to be increased in mucinous colorectal cancers and mucin-producing Ulcerative Colitis-associated colon cancers, but not in sporadic colonic adenocarcinoma that lack mucinous components.[149] In-vitro studies suggested that miR-205 and miR-373 may functionally induce different features of mucinous-associated neoplastic progression in intestinal epithelial cells.[149]

Hepatocellular carcinoma cell proliferation may arise from miR-21 interaction with MAP2K3, a tumor repressor gene.[150] Optimal treatment for cancer involves accurately identifying patients for risk-stratified therapy. Those with a rapid response to initial treatment may benefit from truncated treatment regimens, showing the value of accurate disease response measures. Cell-free miRNA are highly stable in blood, are overexpressed in cancer and are quantifiable within the diagnostic laboratory. In classical Hodgkin lymphoma, plasma miR-21, miR-494, and miR-1973 are promising disease response biomarkers.[151] Circulating miRNAs have the potential to assist clinical decision making and aid interpretation of positron emission tomography combined with computerized tomography. They can be performed at each consultation to assess disease response and detect relapse.

A 2009 study explored miR-205 targeted for inhibiting the metastatic nature of breast cancer.[152] Five members of the microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) are down-regulated in tumour progression of breast cancer.[153]

The specific microRNA, miR-506 has been found to work as a tumor antagonist in several studies.[154] In a 2014 study, a significant number of cervical cancer samples were found to have down-regulated expression of miR-506. Additionally, studies found that miR-506 works to promote apoptosis of cervical cancer cells, through its direct target hedgehog pathway transcription factor, Gli3.[155]

A 2015 study used a triple helix of three miRNAs embedded in a dextran aldehyde/dendrimer gel in a mouse model of triple negative breast cancer. mir-205 and mir-212 targeted specific RNAs, while the other miRNA stabilized the others. The treatment reduced tumor sizes by 90% with survival times of 75 days.[156][157]

MicroRNAs have the potential to be used as targets for treatment of different cancers. The specific microRNA, miR-506 has been found to work as a tumor antagonist in several studies. In a 2014 study, a significant number of cervical cancer samples were found to have downregulated expression of miR-506. Additionally, studies found that miR-506 works to promote apoptosis of cervical cancer cells, through its direct target hedgehog pathway transcription factor, Gli3.[154][155]

DNA repair and cancer

DNA damage is considered to be the primary underlying cause of cancer.[158] If DNA repair is deficient, damage can accumulate. Such damage can cause mutationalerrors during DNA replication due to error-prone translesion synthesis. Accumulated damage can also cause epigenetic alterations due to errors during DNA repair.[159][160] Such mutations and epigenetic alterations can give rise to cancer (see malignant neoplasms).

Germ line mutations in DNA repair genes cause only 2–5% of colon cancer cases.[161] However, altered expression of microRNAs, causing DNA repair deficiencies, are frequently associated with cancers and may be an important causal factor.

Among 68 sporadic colon cancers with reduced expression of the DNA mismatch repair protein MLH1, most were found to be deficient due to epigenetic methylation of the CpG island of the MLH1 gene.[162] However, up to 15% of MLH1-deficiencies in sporadic colon cancers appeared to be due to over-expression of the microRNA miR-155, which represses MLH1 expression.[163]

In 29–66%[164][165] of glioblastomas, DNA repair is deficient due to epigenetic methylation of the MGMT gene, which reduces protein expression of MGMT. However, for 28% of glioblastomas, the MGMT protein is deficient, but the MGMT promoter is not methylated.[164] In glioblastomas without methylated MGMT promoters, the level of microRNA miR-181d is inversely correlated with protein expression of MGMT and the direct target of miR-181d is the MGMT mRNA 3’UTR (the three prime untranslated region of MGMT mRNA).[164] Thus, in 28% of glioblastomas, increased expression of miR-181d and reduced expression of DNA repair enzyme MGMT may be a causal factor.

HMGA proteins (HMGA1a, HMGA1b and HMGA2) are implicated in cancer, and expression of these proteins is regulated by microRNAs. HMGA expression is almost undetectable in differentiated adult tissues, but is elevated in many cancers. HMGA proteins are polypeptides of ~100 amino acid residues characterized by a modular sequence organization. These proteins have three highly positively charged regions, termed AT hooks, that bind the minor groove of AT-rich DNA stretches in specific regions of DNA. Human neoplasias, including thyroid, prostatic, cervical, colorectal, pancreatic and ovarian carcinomas, show a strong increase of HMGA1a and HMGA1b proteins.[166] Transgenic mice with HMGA1 targeted to lymphoid cells develop aggressive lymphoma, showing that high HMGA1 expression is associated with cancers and that HMGA1 can act as an oncogene.[167] A 2003 study[168] showed that HMGA1 protein binds to the promoter region of DNA repair gene BRCA1 and inhibits BRCA1 promoter activity. They also showed that while only 11% of breast tumors had hypermethylation of the BRCA1 gene, 82% of aggressive breast cancers have low BRCA1 protein expression, and most of these reductions were due to chromatin remodeling by high levels of HMGA1 protein.

HMGA2 protein specifically targets the promoter of ERCC1, thus reducing expression of this DNA repair gene.[169] ERCC1 protein expression was deficient in 100% of 47 evaluated colon cancers (though the extent to which HGMA2 was involved is not known).[170] A 2012 study[171] showed that in normal tissues, HGMA1 and HMGA2 genes are targeted (and thus strongly reduced in expression) by miR-15, miR-16, miR-26a, miR-196a2 and Let-7a. However, each of these HMGA-targeting miRNAs are drastically reduced in almost all human pituitary adenomas studied, when compared with the normal pituitary gland. Consistent with the down-regulation of these HMGA-targeting miRNAs, an increase in the HMGA1 and HMGA2-specific mRNAs was observed. Three of these microRNAs (miR-16, miR-196a and Let-7a)[172][173]have methylated promoters and therefore low expression in colon cancer. For two of these, miR-15 and miR-16, the coding regions are epigenetically silenced in cancer due to histone deacetylase activity.[174] When these microRNAs are expressed at a low level, then HMGA1 and HMGA2 proteins are expressed at a high level. HMGA1 and HMGA2 target (reduce expression of) BRCA1 and ERCC1 DNA repair[175] genes. Thus DNA repair can be reduced, likely contributing to cancer progression.[158]

In contrast to the previous example, where under-expression of miRNAs indirectly caused reduced expression of DNA repair genes, in some cases over-expression of certain miRNAs may directly reduce expression of specific DNA repair proteins. A 2011 study[176] referred to 6 DNA repair genes that are directly targeted by the miRNAs indicated: ATM (miR-421), RAD52 (miR-210, miR-373), RAD23B (miR-373), MSH2 (miR-21), BRCA1 (miR-182) and P53 (miR-504, miR-125b). More recently, A 2014 study[177] listed multiple DNA repair genes directly targeted by these additional miRNAs: ATM (miR-100, miR18a, miR-101), DNA-PK (miR-101), ATR (mir-185), Wip1 (miR-16), MLH1MSH2MSH6 (miR-155), ERCC3ERCC4 (miR-192) and UNG2 (miR-16, miR-34c). Among these miRNAs, miR-16, miR-18a, miR-21, miR-34c, miR-101, miR-125b, miR-155, miR-182, miR-185, miR-192 and miR-373 were identified[173] as over-expressed in colon cancer through epigenetic hypomethylation. Over expression of any one of these miRNAs can cause reduced expression of its target DNA repair gene.

Heart disease

The global role of miRNA function in the heart has been addressed by conditionally inhibiting miRNA maturation in the murine heart. This revealed that miRNAs play an essential role during its development.[178][179] miRNA expression profiling studies demonstrate that expression levels of specific miRNAs change in diseased human hearts, pointing to their involvement in cardiomyopathies.[180][181][182] Furthermore, animal studies on specific miRNAs identified distinct roles for miRNAs both during heart development and under pathological conditions, including the regulation of key factors important for cardiogenesis, the hypertrophic growth response and cardiac conductance.[179][183][184][185][186][187] miRNA’s in animal models have also been linked to cholesterol metabolism and regulation.[188]

miRNA-712

Murine microRNA-712 is a potential biomarker (i.e. predictor) for atherosclerosis, a cardiovascular disease of the arterial wall associated with lipid retention and inflammation.[189] Non-laminar blood flow also correlates with development of atherosclerosis as mechanosenors of endothelial cells respond to the shear force of disturbed flow (d-flow).[175] A number of pro-atherogenic genes including matrix metalloproteinases (MMPs) are upregulated by d-flow ,[175] mediating pro-inflammatory and pro-angiogenic signals. These findings were observed in ligated carotid arteries of mice to mimic the effects of d-flow. Within 24 hours, pre-existing immature miR-712 formed mature miR-712 suggesting that miR-712 is flow-sensitive.[175] Coinciding with these results, miR-712 is also upregulated in endothelial cells exposed to naturally occurring d-flow in the greater curvature of the aortic arch.[175]

Gene origin

Pre-mRNA sequence of miR-712 is generated from the murine ribosomal RN45s gene at the internal transcribed spacer region 2 (ITS2).[175] XRN1 is an exonuclease that degrades the ITS2 region during processing of RN45s.[175] Reduction of XRN1 under d-flow conditions therefore leads to the accumulation of miR-712.[175]

Mechanism

MiR-712 targets tissue inhibitor of metalloproteinases 3 (TIMP3).[175] TIMPs normally regulate activity of matrix metalloproteinases (MMPs) which degrade the extracellular matrix (ECM). Arterial ECM is mainly composed of collagen and elastin fibers, providing the structural support and recoil properties of arteries.[190] These fibers play a critical role in regulation of vascular inflammation and permeability, which are important in the development of atherosclerosis.[191] Expressed by endothelial cells, TIMP3 is the only ECM-bound TIMP.[190] A decrease in TIMP3 expression results in an increase of ECM degradation in the presence of d-flow. Consistent with these findings, inhibition of pre-miR712 increases expression of TIMP3 in cells, even when exposed to turbulent flow.[175]

TIMP3 also decreases the expression of TNFα (a pro-inflammatory regulator) during turbulent flow.[175]  Activity of TNFα in turbulent flow was measured by the expression of TNFα-converting enzyme (TACE) in blood. TNFα decreased if miR-712 was inhibited or TIMP3 overexpressed,[175] suggesting that miR-712 and TIMP3 regulate TACE activity in turbulent flow conditions.

Anti-miR-712 effectively suppresses d-flow-induced miR-712 expression and increases TIMP3 expression.[175] Anti-miR-712 also inhibits vascular hyperpermeability, thereby significantly reducing atherosclerosis lesion development and immune cell infiltration.[175]

Human homolog microRNA-205

The human homolog of miR-712 was found on the RN45s homolog gene, which maintains similar miRNAs to mice.[175] MiR-205 of humans sshare similar sequences with miR-712 of mice and is conserved across most vertebrates.[175] MiR-205 and miR-712 also share more than 50% of the cell signaling targets, including TIMP3.[175]

When tested, d-flow decreased the expression of XRN1 in humans as it did in mice endothelial cells, indicating a potentially common role of XRN1 in humans.[175]

Kidney disease

Targeted deletion of Dicer in the FoxD1-derived renal progenitor cells in a murine model resulted in a complex renal phenotype including expansion of nephronprogenitors, fewer renin cells, smooth muscle arterioles, progressive mesangial loss and glomerular aneurysms.[192] High throughput whole transcriptome profiling of the FoxD1-Dicer knockout mouse model revealed ectopic upregulation of pro-apoptotic gene, Bcl2L11 (Bim) and dysregulation of the p53 pathway with increase in p53 effector genes including BaxTrp53inp1, Jun, Cdkn1aMmp2, and Arid3a. p53 protein levels remained unchanged, suggesting that FoxD1 stromal miRNAs directly repress p53-effector genes. Using a lineage tracing approach followed by Fluorescent-activated cell sorting, miRNA profiling of the FoxD1-derived cells not only comprehensively defined the transcriptional landscape of miRNAs that are critical for vascular development, but also identified key miRNAs that are likely to modulate the renal phenotype in its absence. These miRNAs include miRs‐10a, 18a, 19b, 24, 30c, 92a, 106a, 130a, 152, 181a, 214, 222, 302a, 370, and 381 that regulate Bcl2L11 (Bim) and miRs‐15b, 18a, 21, 30c, 92a, 106a, 125b‐5p, 145, 214, 222, 296‐5p and 302a that regulate p53-effector genes. Consistent with the profiling results, ectopic apoptosis was observed in the cellular derivatives of the FoxD1 derived progenitor lineage and reiterates the importance of renal stromal miRNAs in cellular homeostasis.[192]

Nervous system

miRNAs appear to regulate the development and function of the nervous system.[193] Neural miRNAs are involved at various stages of synaptic development, including dendritogenesis (involving miR-132, miR-134 and miR-124), synapse formation[194] and synapse maturation (where miR-134 and miR-138 are thought to be involved).[195] Some studies find altered miRNA expression in schizophrenia, as well as bipolar disorder and major depression and anxiety disorders.[196][197][198]

Alcoholism

The vital role of miRNAs in gene expression is significant to addiction, specifically alcoholism.[199] Chronic alcohol abuse results in persistent changes in brain function mediated in part by alterations in gene expression.[199] miRNA global regulation of many downstream genes deems significant regarding the reorganization or synaptic connections or long term neuroadaptions involving the behavioral change from alcohol consumption to withdrawal and/or dependence.[200] Up to 35 different miRNAs have been found to be altered in the alcoholic post-mortem brain, all of which target genes that include the regulation of the cell cycleapoptosiscell adhesionnervous system development and cell signaling.[199] Altered miRNA levels were found in the medial prefrontal cortex of alcohol-dependent mice, suggesting the role of miRNA in orchestrating translational imbalances and the creation of differentially expressed proteins within an area of the brain where complex cognitive behavior and decision making likely originate.[201]

miRNAs can be either upregulated or downregulated in response to chronic alcohol use. miR-206 expression increased in the prefrontal cortex of alcohol-dependent rats, targeting the transcription factor brain-derived neurotrophic factor (BDNF) and ultimately reducing its expression. BDNF plays a critical role in the formation and maturation of new neurons and synapses, suggesting a possible implication in synapse growth/synaptic plasticity in alcohol abusers.[202] miR-155, important in regulating alcohol-induced neuroinflammation responses, was found to be upregulated, suggesting the role of microglia and inflammatory cytokines in alcohol pathophysiology.[203] Downregulation of miR-382 was found in the nucleus accumbens, a structure in the basal forebrain significant in regulating feelings of reward that power motivational habits. miR-382 is the target for the dopamine receptor D1 (DRD1), and its overexpression results in the upregulation of DRD1 and delta fosB, a transcription factor that activates a series of transcription events in the nucleus accumbens that ultimately result in addictive behaviors.[204] Alternatively, overexpressing miR-382 resulted in attenuated drinking and the inhibition of DRD1 and delta fosB upregulation in rat models of alcoholism, demonstrating the possibility of using miRNA-targeted pharmaceuticals in treatments.[204]

Obesity

miRNAs play crucial roles in the regulation of stem cell progenitors differentiating into adipocytes.[205] Studies to determine what role pluripotent stem cells play in adipogenesis, were examined in the immortalized human bone marrow-derived stromal cell line hMSC-Tert20.[206] Decreased expression of miR-155,miR-221,and miR-222, have been found during the adipogenic programming of both immortalized and primary hMSCs, suggesting that they act as negative regulators of differentiation. Conversely, ectopic expression of the miRNAs 155,221, and 222 significantly inhibited adipogenesis and repressed induction of the master regulators PPARγ and CCAAT/enhancer-binding protein alpha (CEBPA).[207] This paves the way for possible genetic obesity treatments.

Another class of miRNAs that regulate insulin resistanceobesity, and diabetes, is the let-7 family. Let-7 accumulates in human tissues during the course of aging.[208]When let-7 was ectopically overexpressed to mimic accelerated aging, mice became insulin-resistant, and thus more prone to high fat diet-induced obesity and diabetes.[209] In contrast when let-7 was inhibited by injections of let-7-specific antagomirs, mice become more insulin-sensitive and remarkably resistant to high fat diet-induced obesity and diabetes. Not only could let-7 inhibition prevent obesity and diabetes, it could also reverse and cure the condition.[210] These experimental findings suggest that let-7 inhibition could represent a new therapy for obesity and type 2 diabetes.

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