Micrornas Role In Diabetes Biology Essay


Diabetes is a serious global health problem in developing countries including India, annually some 76,000 children aged under 15 year develop diabetes worldwide. It is seen that long term diabetic patients develop several secondary complications such as, cardiovascular diseases, kidney diseases and endothelial dysfunction. The emergence of small noncoding RNAs called as microRNA showed their significant role in combating various complex diseases like cancer, heart diseases, liver diseases and neurodegenerative diseases. microRNA also been shown to regulate production, secretion and action of insulin. This review highlights the involvement of miRNAs in several diseases with a major focus on their role in diabetes.

Diabetes is a chronic health related problem that has been developed in the low and middle income countries. It is caused when the body has a shortage of insulin or decreased ability to use insulin, a hormone that allows glucose to enter into cells for production of energy. There are two types of diabetes: Type 1 Diabetes Mellitus (T1DM) and Type 2 Diabetes Mellitus (T2DM). T1DM is also sometimes called as insulin dependent, immune-mediated or juvenile onset diabetes. It is caused by destruction of β cells of islets of Langerhans of pancreas, typically due to an autoimmune reaction where they are attacked by body's defence system, so a little or no insulin is produced by β cells. T2DM is characterized by insulin resistance or insulin deficiency as the pancreatic cells don't produce enough insulin. It occurs after age of 40. This form of diabetes is associated with older age, obesity, family history of diabetes, physical inactivity and ethnicity. It is seen that both of the two types of diabetes are related to various secondary complications such as cardiovascular diseases, kidney diseases [1], peripheral vascular disease [2], oxidative stress and endothelial dysfunction [3] etc. Over the past 30 years the status of diabetes has been changed i.e. from mild disorder of elderly it has enveloped as major cause of morbidity and mortality in youths and middle aged. According to Diabetes Atlas 2009, published by International Diabetes Federation it was seen that annually some 76,000 children aged under 15 year develop T1DM worldwide. Of estimated 480,000 children with T1DM, 24% come from South-East Asia.

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The discovery of microRNA and their role in regulation of post transcriptional protein expression has also enlightened their role in control of glucose homeostasis. This review will discuss the potentials of miRNA in various diseases with a major emphasis on their role in diabetes.

MicroRNA (mi-RNAs)

MicroRNAs (miRNAs) are noncoding RNAs that are ~18-25 nucleotides long. The discovery of these miRNA was done in 1993 in nematode Caenorhabditis elegans [4]. The scientists reported that the lin-4 gene encodes a small RNA which was found to be complementary to segments in the 3' untranslated region (UTR) of a specific mRNA encoding protein LIN-14. The binding of this small RNA to the complementary mRNA during larval development blocks the translation to the next stage of development [5]. The second discovery was that of ~21 nucleotide species called let-7, another small RNA involved in development of C.elegans [6]. Its conservation throughout evolution suggested that these small RNA's have important and conserved role in gene regulation. The miRNA are predicted to directly regulate the expression of at least 30% of all human protein-encoding genes [7]. MicroRNAs have linked themselves to a variety of pathways and processes such as cell proliferation, development, cell death (apoptosis), morphogenesis, viral infection and also in diseases such as cancer, neurodegenerative diseases and in cardiovascular diseases [8].


miRBase of  University of Manchester is the central online database for miRNA nomenclature, sequence data, annotation and target prediction. miRBase is available at http://www.mirbase.org. It was seen that there were as many as 3000 new hairpin sequences and more than 4000 new mature sequences [9].The miRBase database is a searchable database of all published miRNA sequences and annotation. The entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript and also the information about the location and sequence of the mature miRNA sequence. Both hairpin and mature sequences are available for searching and browsing.


Since miRNAs are produced by the same processing machinery to that responsible for formation of small interfering RNA (siRNA) these two could be considered as "cousins". Both of these two, miRNAs and siRNA act to silence expression of cytoplasmic mRNAs. However there are certain differences in two:

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miRNA are purposely expressed products of an organism own genome and are endogenous. But siRNA are derived from double stranded product of virus or transposable elements and target same transcripts from which they arose.

Second difference is that miRNA are processed from incomplete double stranded RNA whereas siRNA are obtained from complementary double stranded character RNA [10].

But a recent breakthrough is the demonstration that miRNAs can act as siRNA. Many plant miRNA have a high complementarity to their target mRNA and these miRNA act as siRNA by cleaving their mRNA targets. This shows that miRNA directed endonuclease similar to siRNA directed RISC endonuclease is present in miRNP's and its target shows its function i.e. if binding is partial, translational of target is repressed and if binding is severe target mRNA is cleaved [30-32] . Most of the miRNA are encoded by individual genes, but many of them exist in clusters, also many of miRNAs are transcribed from introns and/or exons [11] and even from 3'untranslated regions (3' UTR) of proteins [12] .The transcription of miRNA occurs by RNA polymerase II in humans and animals [13] but in case of viral encoded miRNA they are transcribed by RNA polymerase III [14,15] .The initial product of genes transcription is a large molecule of approximately 200 nucleotide to several kb in length (the pri-miRNA) .Like usual mRNA they also have a 5' 7- methyl guanosine cap (m7G) and 3' poly A tail [16]. The steps included in biogenesis and mechanism of miRNA is:

In the first step, within the nucleus the pri- miRNA is cleaved by a multi-protein complex (microprocessor) RNAIII enzyme, Drosha liberating a ~70 nucleotide long miRNA stem loop precursor pre-miRNA. This pre-miRNA has a 3' overhang at one end [12,17] .The pre miRNA is then exported to cytoplasm by exportin-5 (a carrier protein) and Ran-GTP which binds selectively to pre-miRNA [18,19] . This binding protects it against exo-nucleolytic digestion [20].

In second step, the pre-miRNA is further cleaved by a cellular RNase III endonuclease enzyme Dicer [19]. This forms a ~21 nucleotide long miRNA duplex having 3'overhang ends at both ends .Out of 2 strands, guide strand of which 5'termini is stable associates with a protein complex similar to RISC i.e. miRNA induce silencing complex (miRISC) while other strand is released and degraded [21,22].

This mature miRNA then binds to complementary sequences. This results in cleavage of than RNA and its degradation [23] but if there is not perfect complementarily the translational is suppressed. There is a seed region i.e. at 2-7 or 8 nucleotide of miRNA at 5' region i.e. important for miRNA targeting [24, 25]. Thus, for miRNA the primary mechanism of action is repression of translation, although it can be accompanied by degradation of message [26].

Other than this an alternate pathway for intron-derived miRNA is identified in humans [27]. In this pathway the enzyme Drosha is not involved .These introns are called as mitrons and they have structural features as pre-miRNA and enters miRNA pathway after the first step .These mitrons have splicing machinery for maturation [28]. miRNA basically binds partially to the 3' UTR's regulatory elementary on mRNA [29] and act as transcription repressors .This allows miRNAs to target multiple 3'UTR's.

miRNA and diseases.

Identification and characterization of miRNAs is a growing area of research, as they regulate a variety of processes such as development, cell proliferation and death .Their excess and deficiencies have also involved them in a number of important diseases like cancer, diabetes, metabolic diseases, heart diseases, liver diseases [33-37].


Cancer is found to be a leading cause of death in all the developed countries, thus it is a field of concern in present and future, until a significant breakthrough occurs in the line of its treatment. Cancer has a number of characteristics such as differentiation, proliferation and apoptosis (programmed cell death).

Calin et al for the first time studies the role of miRNA in cancer. They found out that the cluster of two miRNA i.e. miRNA-15 and miRNA-16 which is located in genomic region of chromosome 13q14 was deleted in majority of Chronic lymphocytic leukaemia (CLL) cases [38] .So, these were not expressed in CLLs leading to increase level of oncogenetic targets. These two miRNA basically down regulate the anti -apoptotic protein Bcl-2 and this protein was found expressed at increase level of CLL [39].

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Some miRNA's are thought to have oncogenic activity while others have tumour suppressive activity. Oncogenic miRNA are upregulated in cancer like for e.g: 9-miR-17-92 cluster i.e. made up of 6 miRNAs [34] is encoded by c13orf25 gene located at 13q31, a genomic locus i.e. amplified in case of B- cell lymphomas [40]. Over expression of miR-17-92 cluster was found to co-operate with C-myc to accelerate tumour development [41]. The tumour suppressor action could be seen by miR15a and miR-16-1 that showed as tumour suppressor action by inhibiting Bcl-2 function [42]

Table 1: showing involvement of different miRNAs in different types of cancers [43]


It is seen that miRNA can confer host immunity against viral infection but at the same time they can also be utilized by virus for their replication.

In case of Herpes simplex virus-1(HSV-1) an important gene is LAT gene (latency associated transcript) which code for a protein [44]. It was found that the anti- apoptotic activity of LAT was achieved by miRNA entrapped in LAT (mi-LAT).This down regulates transforming growth factor (TGF-β) and SMAD-3. Former prevent cell proliferation and induce cell death while later is a mediator of signalling pathway induced by TGF-β [45].

An example that shows host immunity against viral infection is of miR-32 retroviruses primate foaming viral infection (PFV) in human embryonic kidney cell line 293T which shows the miR-32 inhibits PFV replication by impairing translational of viral mRNA target sequences [46].


miRNA have also evolved as regulators of heart disease. They have found to play important role in cardiac function and dysfunction, including myocyte growth integrity of ventricular wall, contractility, gene expression, and maintenance of cardiac rhythm [47-50]. Rooji in his experiment showed that cardiac specific over expression of miRNA -195 which is upregulated in rodent results in dilated cardiomyopathy and heart failure in mice as easily as 2 weeks of age [51]. A widely conserved miRNA displaying cardiac and skeletal muscle specific expression during development and in adult is miR-133 and thus is critically studied.


Neurodegenerative diseases are a result of progressive deterioration, dysfunction and extensive loss of neurons in central and/or peripheral nervous system. It has been seen that dysregulation of miRNA is implicated in development and onset of human neurodegenerative diseases [52, 53].

The example of upregulation of miRNA [54] can be seen in Alzheimer's disease where miR-128 a was found to regulate Cochaperone BAG-2 that lead to pathway of degradation for microtubule associated tau proteins with a propensity for misfolding. BAG 2 reduces levels of sarkosyl insoluble- proteins by directing tau towards ubiquitin-induced pathway. Thus upregulation of miR-128 revealed tau inclusions in neurodegenerative diseases [55]. Whereas many miRNAs such as miR-29 a/b-1, miR-298, miR-328 are found to be downregulated in Alzheimer's disease [56, 57].

Table: showing involvement of different miRNAs in neurological diseases [58]


miRNAs involvement has also been seen in various liver diseases such as viral hepatitis, fatty liver due to alcohol or metabolic syndrome, drug induced liver disease and by autoimmune processes. Some of miRNA's are found to be upregulated and some are found to be downregulated. For e.g.: miR-122 was first liver- specific cellular miRNA that was identified. This miRNA enhances replication of Hepatitis C virus (HCV) by targeting viral 5' non coding region [59]. It appeared that HCV replication associates itself with expression of cholesterol biosynthesis gene that are regulated by miR-122 and are considered as targets for antiviral interventions [60]. Recently Kota et al. [61] showed the activity of miR-26a which is under-expressed in hepatitis cellular carcinoma cells and downregulates cyclin D2 and E2, he administered it using adeno associated virus which resulted in inhibition of cancer cells, proliferation, induction of apoptosis and protection from disease progression without toxicity in mouse model of hepatitis cellular carcinoma.

Table: showing involvement of different miRNAs in liver diseases [36]


miRNAs are found to be associated with various complications that are secondary effects of diabetes [37]. These also play a direct role in development of pancreatic islet [62], in insulin secretion [63] and are also found to indirectly control glucose and lipid metabolism [64].

miRNA in secretion of insulin from pancreatic β -cells

miRNA as we had seen can be both upregulated as well as downregulated. Thus maintenance of appropriate glucose level in the blood is necessary; this balance helps in regulation of insulin levels. If the level of insulin is not adequate diabetes of type 1 and type 2 can occur. miR-375 was found to be expressed in pancreatic endocrine cell lines. Poy et al, in 2004 [63] found out that miR-375 if over expressed resulted in suppression of glucose-stimulated insulin secretion while its inhibition by anti-miRNA oligonucleotides (AMO's) designated as antagomirs enhanced insulin secretion. One of the target gene of miR-375 was identified as Myotrophin (Mtpn) - that had a role in release of neurotransmitter catecholamines. It was found out that over expression of miR-375 led to decrease levels of Mtpn and its inhibition by use of antagomirs like 2'-o-methyl-375 that inhibits miRNA could increase levels of Mtpn [63].

Also miR-375 binds to the 3' UTR of Mtpn and overexpression of miR-375 causes down regulation of Mtpn, indicating it to be a target [63]. Myotropin is basically involved in changing actin network and thus affecting docking and fusion [65, 66]. In an experiment when homologous deletion of miR-375 was done it caused hyperglycaemia in mice due to decrease in pancreatic β cell mass and insulin levels [64].

miR-375 on Mtpn expression and insulin secretion may be because of transcription factor nuclear factor- kappaB (NF-kappaB) [65]. Myotropin act as transcription activator of NF-kappaB as increased level of NF-kappaB leads to secretion of glucose dependent insulin [71] while inactivation results in decrease insulin secretion from β cells [65].

Ouaamari et al in his experiment presented a new candidate PDK1 (3' phosphoinositide - dependent protein kinase 1) [67]. PDK1 is known as a important component of PI3K/ protein kinase B signal cascade and plays a key role in mediating action of insulin on cell growth and development [78]. These scientists used computational algorithms and identified a binding site on 3'UTR of PDK1 to which miR-375 binds. It was seen that the elevated expression of miR-375 in pancreatic islets decreases the level of PDK1 protein leading to downstream signalling including that of protein kinase B and glycogen synthase kinase-3 phosphorylation and decreased β cell no. But miR-375 inhibitor caused increased level of PDK1 and elevated glucose dependent insulin mRNA and β cell proliferation [67]. Thus, miR-375 i.e. highly expressed in pancreatic islets is required for normal glucose homeostasis.

Other than miR-375, miR-124a is also found abundantly in pancreatic β cells. A study done by Baroukh et al showed the miRNA expression profiling at 2 key stages of mouse embryonic pancreas development, e14.5 and e18.5. It was seen that miR-124a2 expression was increased at e-18.5 as compared with e-14.5, suggesting possible role of miR-124a in β cell differentiation [70]. Foxa2 (Forkhead box protein A2) was identified as the gene target to play a important role in β cell differentiation. This Foxa2 is a transcription factor i.e. found important for differentiation of β cells, glucose metabolism, pancreatic development and insulin secretion [71-73]. miR-124a was overexpressed or downregulated in MIN6 β cells that caused change in Foxa2 levels and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 is known as a master regulator of genes involved in glucose metabolism and insulin secretion, including Kir-6.2 (inwardly rectifying potassium channel) and Sur-1 (sulfonylurea receptor) . Thus overexpression of miR-124a decreases and anti-miR 124a increased Kir 6.2 and Sur-1 mRNA levels [70].

Plaisance et al [74] in his experiment found out a role of miR-9 in insulin secretion other than it had in brain specificity [75]. It was seen that overexpression of miR-9 in insulin secreting cells causes reduction in exocytosis. miR-9 acts by dimishing the expression of transcription factor onecut-2. Onceut-2 in turn repress expression of Granuphilin/S1p4, is a Rab GTPase effector i.e. associated with secretory granules and have a negative control on insulin release [74].

In an experiment conducted by Tang et al 61 glucose regulated miRNAs were found out of 108 miRNA in mouse insulinoma cell lines, MIN6 [76]. Of these some were downregulated like miR-296, miR-184 and miR-160 by high glucose treatment and some were upregulated like miR-124a, miR-107 and miR-30d in presence of high glucose.

miRNAs in Development of Pancreas

Certain mouse models were generated by intentionally blocking miRNA generation by deletion of a conditional Dicer allele [81]. It was seen that miRNA has a critical role during pancreas development when Dicer was deleted at early stage of pancreas development using Pdx-1 promoter, as serious developmental defects were observed. When Dicer was deleted later in development using insulin promoter, a little effect was observed on pancreas morphology and on β cell maintenance.

Severe morphological defects were also noted when knockdown of miR-375 was done in pancreatic islets in zebra fish. This revealed the dual function of miR-375 in mediating insulin secretion as well as in development of pancreatic islets [78]. Other miRNAs that were islets specific were miR-7, miR-9 and miR-376 which are found to be expressed at high levels during human pancreatic islet development [79].

miRNAs in lipid metabolism

Insulin Resistance can occur by abnormalities of triglyceride storage and lipolysis in insulin sensitive tissues. The discovery of miRNA has shown a role in regulation of lipid metabolism. It was found that miR-14 play an evident role in fat metabolism in Drosophila [81]. Animals with miR-14 deletion have increase amount of circulating fat and increased peripheral lipid droplets. Another miRNA is miR-278. It is a miRNA that is expressed most abundantly in Drosophila adipose tissue. It was found that miR-278 mutants have increase insulin production and also low level of adipose. But these species were also found to have high circulating glucose levels due to increased glycogen mobilisation and decreased insulin sensitivity [80].Also t no exact human orthologs to Drosophila miR-14 and miR-278 are yet known. But some of human miRNA shows sequence homology to Drosophila miRNA like human miR-511 and miR-620 resembles Drosophila miR-14 and human miR-658 and miR-583 resembles Drosophila miR-278[82].

miR-103(1), miR-103(2) and miR-107 are three known human miRNA paralogs [82] that may play a role in human metabolism. miR-103 genes encode two identical miRNAs and a third paralog miR-107 differs at a single nucleotide miRNA exist within introns in the genes that encodes for PANK (pantothenate kinase) enzymes. It is seen that miR-103(1) reside in PANK3, miR-103(2) in PANK2 and miR-107 in PANK1. PANKs catalyses rate limit step of pantothenate phosphorylation during generation of Coenzyme A (CoA) that is a cofactor in reactions involved in metabolism. These reactions include steps in metabolism and synthesis of fatty acids, amino acids, cholesterol, glucose, Krebs cycle etc. Biogenetics predict that miR-103/7 regulate metabolism with emphasis on acetyl CoA and lipid metabolism.

Esau et al also release the role of miR-122 a liver specific miRNA as a significant regulator of hepatic lipid metabolism. In his study he injected miR-122 antagonist (an antisense oligonucleotide with 2'-o-methoxyethyl phosphororothiate) into mice. The injection of this antagonist resulted in decrease in plasma cholesterol levels, hepatic fatty acid and cholesterol synthesis. The circulatory cholesterol levels were also found to be reduced, indicated that miR-122 inhibition play a significant role decreasing plasma cholesterol level that was found to be elevated in several metabolic diseases [83].

miRNA and secondary diabetic complications.

Diabetes is characterised by development of various secondary severe complications such as heart disease, cardiac hypertrophy i.e. characterized by thicking of myocardial wall, in renal glomerulus, retina and in peripheral nerve. miRNA appears to play a role in establishment of diabetic complications.miR-133 was a miRNA that was found to change its level in diabetic heart. Changes were associated with type 2 of and consequences were cardiac hypertrophy and a long QT syndrome (LQTS) [37]. Human ether-a-go-go related genes (HERG) was identified as a LQTs gene that encodes cardiac K+ channel responsible for rapid delayed rectifier K+ current[84]. miR-133 was reported to be overexpressed in heart of diabetic rabbits that was accompanied by increased in expression of serum response factor (SRF). Delivery of exogenous miR-133 into rabbit myocytes and cell lines produced post- transcriptional repression of ERG, this lead to down regulation of ERG protein level without altering its transcript level and caused decrease of I(Kr) an effect that was abrogated by miR-133 anti-sense inhibitor. Functional inhibition of SRF down regulated miR-133 expression and increased I (Kr) density. Repression of ERG by miR-133 causes depression of I (Kr) and contributes to repolarisation slowing thereby QT prolongation and associated arrhythmia in diabetic heart [37].

miR-192 is a microRNA that is highly expressed in kidney. In case of diabetic nephropathy i.e. a progressive kidney disease and causes kidney failure in patients with a long term diabetes mellitus [66]. It is seen that one of the gene smad-interacting protein 1'(SIP 1) is the target of miR-192.It was seen that in diabetes nephropathy there is accumulation of extracellular matrix proteins such as collagen 1-α 1and -2 (col 1a 1 and -2) the key regulator of these genes, transforming growth factor β1 (TGF-β) was found to be increased in the mesangial cell in this disease.

Thus, upregulation of miR-192 by TGF-β resulted in down regulation of SIP 1 through translational repression. TGF-β was also found to be down regulated the transcription factor SEF 1 (δ- crystalline enhance binding protein). Consequently down regulation of SIP 1 and SEF 1 enhances expression of col 1a 2 by derepressing E- box elements located on col 1a2 promoter .It was also seen that miR-192 levels enhanced significantly in glomeruli isolated from Streptozotocin injected diabetic mice as well as diabetic db/db mice relative to non diabetic controls with parallel increase of TGF-βand col1 a 2 levels. These findings suggested a role of miR-192 in kidney and diabetic neuropathy development. [85].


There are various strategies that are developed to exploit the sequence directed properties of the miRNA that are:

It is the most simplest and a direct therapeutic strategy evolved to redirect the miRNA against its target .Their is a need to deliver the synthetic mature or engineered miRNA to its known mRNA target [86.87] so that it can bind to the RISC complex and recognizes its target ,there by blocking the translation of mRNA, leading to its reduced stability. This leads to decrease in the synthesis of proteins by that target [88]. To achieve this, the strategy relies upon understanding the association of the genes on the target mRNA and to know its physiology. A recent publication reported use of "miRNA replacement therapy" that aimed at restoring miR26a expression in hepatocellular carcinoma. It was seen that when miR26a was delivered in a mouse model using adeno-associated virus, this miRNA was able to suppress proliferation and induce apoptosis, that resulted in inhibition of hepatocellular carcinoma cancer.[88,89]

Antisense inhibition of mature miRNA

This strategy involves the inhibition of the endogenous functions of miRNA by base pairing it with a complementary nucleic acid analogues.

Modified anti-miRNA oligonucleotides (AMOs) also designated as "antagomirs" are used for inhibition of miRNA. 3 major types of AMOs are oligonucleotides with modified 2-OH residues of ribose by 2'-O-methyl (2'-OMe) , 2'-O-methoxyethyl (2'-MOE) and locked nucleic acid (LNA) that were found to be successfully inhibiting mature miRNA in cell culture [90,91], flies [91,92] and mice [93]. One of the example of this in vivo inhibition of miR-122 miRNA using a 15 nucleotide LNA bearing analogues [94]. miR-122 is a liver specific miRNA and is implicated in cholesterol and lipid metabolism and linked to replication of hepatitis C virus (HCV) in vitro [95]. It was seen that reduction of mature miR-122 level in liver was accompanied by substantial and reversible reduction in plasma cholesterol, without inducing any sign of toxicity or damage to tissues [94]. Thus this can be targeted in chronically infected HCV that could in turn reduce associated risk of carcinoma. Various other targeted miRNAs include miR-16, miR-21, miR34a, b, c, miR29b etc. Those are found to be cancer relevant [96].


Considering that each miRNA can target several genes and each gene can be regulated by several miRNAs, the story of miRNAs is very complicated [97]. Recent advances in miRNA research have provided us more insights and improved understanding about the biogenesis and function of these miRNAs in various diseases including cancer, heart diseases, liver diseases, neurodegenerative diseases. Since majority of human miRNA have been identified the major challenge is now to identify the function of these miRNAs in various tissues. The search for distinguishing characteristics of animal miRNAs precursor also continues.

Moreover there is now the need to understand the signatures of these miRNA in susceptible individuals and even in miRNA target variants (like in single nucleotide polymorphism). Thus there is a need to develop "miRNA based individual specific therapy" for diagnosis and treatment of various diseases. Also the emerging evidence of miRNA in production, secretion and action of insulin has shown their significant role in diabetes. Due to diabetes there has been a change in miRNA expression profiles that is noticed in many tissues, like muscle, liver and pancreas. All this shows that miRNA plays a very critical role in diabetes progression and pathogenesis and their altered expression offers a valuable tool for diagnosis and treatment of diabetes.