Dissection of Microrna-30Ds Function Roles

Dissection of Microrna-30Ds Function RolesDISSECTION OF MICRORNA-30DS FUNCTION ROLES IN MAMMALIAN PANCREATIC-BETA CELLSByYiping MaoABSTRACTMicroRNAs (miRNAs) argon a group of scurvy non- code RNAs (about 21-22 nucleotides long) that picturesque tune marker protein output through messenger RNA abjection or inhibition of its comment. recent studies showing that miRNAs and their routine comp acents respond to electric cellular stress to maintain regular(a) state physiology of the cells. Since there atomic number 18 thousands of microRNAs real in all sympathetic of cells, their useful characterization during the normal states or stress conditions is not amply addressed yet.In this thesis, important aspects of pancreatic -cell function under normal or stress condition much(prenominal) as apoptosis, proliferation, insulin intersection and release and their regulation by the miRNA were explored. Pancreatic -cell is a group of insulin producing cells and plays critical role in maintaining glucose he aboutasis. By combining reverse and cell line brokertic approaches, spicy-throughput deep sequencing, a list of cell assays and molecular techniques, we have shed light on the novel roles of miR-30d, one highly expressed miRNA when -cell responding to high glucose stimulus, in regulating -cell passel on the middle aged mice. We present that everyplaceexpress of miR-30d deteriorated glucose gross profit superpower of the mice with or without high fat diet treatment by signifi advisetly reducing the -cell mass with less insulin production. Additionally, we exhibit that the reduced -cell mass is because both apoptosis road and proliferation pathway have been effected by miR-30d by targeting variety of protein factors expression. BCL2 interacting protein 3 (BNIP3) and cyclin E2 (CCNE2) have been respectively confirmed as miR-30ds targets and the effect of their regulation by miR-30d in pancreatic -cell proliferation or apoptosis aspects have been address ed as advantageously. Further much, we could show that silencing of miR-30d in MIN6 cell (-cell mimicking cell line) by CRISPR-CAS9 ingredient redact system promotes the insulin secernment, which is through potentiated expression of MAFA, an insulin ar setment factor. These studies uncovered novel functional roles of the miR-30d pathway in mediating -cell function and fate. Further dissection of these pathways shall uncover some(prenominal) mechanisms by which the -cells undertake to maximize their efficiency during disease states such as T2D.CHAPTER 1 INTRODUCTION AND BACKGROUND1.1 The Brief History of Diabetes and ResearchLong in front terming diabetes, which means to pass through by Greek Apollonius of Memphis in 250 BCE and the re-discovery of honey-like-urine (glycosuria) by Thomas Willis, who included the term mellitus, Diabetes has discovered its primary saying rough 1500 B.C. in an Egyptian manuscript. It was perceived as a disease link with too great emptying of t he urine (polyuria). Later, important discoveries includes Matthew Dobsons front evidence of elevated urine and blood glucose levels (hyperglycemia) in people with diabetes (Dobson, 1776). In 1889, Joseph von Mering and Oskar Minkowski were the first to give evidence that pancreas removal in dogs shitd diabetes, proposing that pancreas functional link to glucose levels. Afterward, Edward Albert Sharpey-Schafer proposed that diabetes could be brought on by losing a pancreatic chemical, which he named as insulin in 1910 (Polonsky, 2012). taken together, diabetes is currently well recognized as a gathering of heterogeneous disorders characterized by hyperglycemia because of loss of insulin or its effectiveness. genuine worldwide trends demonstrate a surprising 382 million individuals have diabetes and this is predicted to grow to 592 million by the year 2035 (IDF Diabetes Atlas, 2013). The cases of diabetes complications, including diabetic retinopathy, cardiovascular disease a nd renal failure ar constantly rising and the death rate because of those are worsen every year.Claude Bernards identification of liver as the major glucose production organ led to the first concept of homeostasis which has been termed and expanded by Walter Bradford in the mid-nineteenth century, to describe the maintaining of steady-state physiology of the cells (Robin, 1979). This gave the notion that actually the disturbed glucose homeostasis is one of the important events of the diabetes progression. presumptuousness the evidences of insulin is involved in maintaining the glucose homeostasis, Frederick banteng and Charles Best integrated a serial of scientific approaches, and were able to purify the insulin from the pancreas. Moreover, they successfully treated the patients who suffer from the diabetes, with their purified insulin (Banting Best, 1922 Banting et al., 1922). This landmark finding set the stage for treating the severe diabetes with insulin. However, it has bee n almost a century now since the first time insulin was discovered and purified, diabetes remains the incurable disease, requires life-time attention and treatment because of its complexity.Diabetes has been classified as a couple diametric types nowadays, the major dickens types are cognize as the Type I Diabetes (T1D) or Insulin Dependent Diabetes Mellitus (IDDM) and Type II Diabetes (T2D) or Non-Insulin Dependent Diabetes Mellitus (NIDDM). Surprisingly, these two major types has been notified as early as 100-200 B.C by Indian physicians (Kahn, 1994). However, the first scientific evidence was brought by Wilhelm Falta and Harold Himsworth afterwards they set the standardized glucose/insulin tolerance test in human to distinguish the insulin sensitive from non-sensitive patients (Himsworth, 1936). Insulin dependent T1D is feature in insufficient insulin production due to autoimmune response to the pancreatic beta-cells and it in the main affects young kids. T2D is more descri bed as a metabolic syndrome with emergence of insulin resistance and obesity etc, and it is more common among adults (Moller, 2001). There is a complex network of several insulin responding tissues contributes to the maintenance of glucose homeostasis, and of course any constipation in this network induces the progression of T2D. Besides the metabolic relevant tissues, the gene-interactions overly play critical roles in the instruction of T2D and obesity such kind of metabolic diseases (Doria et al., 2008).1.2 The Islet Architecture, -cell Fate determinationThe islets of Langerhans are superfluousized endocrinal part of pancreas and are the only part producing the secreting hormones. They basically comprise of unhomogeneous cell sorts named , , , PP, and that secret the islet hormones glucagon, insulin, somatostatin, polypeptide Y, and ghrelin individually, and these hormones are required to maintain the glucose homeostasis at normal or stress state. Furthermore, the islets are known to have high-power and plastic architecture that is proposed to be adjusted over the time of development (Steiner et al., 2010). During the progression of insulin resistance, pregnancy or T2D, the islets extend in size to apply up for expanded insulin requirement (Weir and Bonner-Weir, 2004 Kim et al., 2009). But afterwards, there is a vast loss of -cell mass because of environmental or genetic factors, inducing the undecomposed hyperglycemia in cause of insulin deficiency.While islets count to just 1-2% of the intact pancreas, the insulin producing -cells represent 65-80% of the islet mass, constituting to roughly 2% of pancreatic weight. Furthermore, the rest otherwise cell types are considered as non -cells of the islets (Weir and Bonner-Weir, 2013). During the development, these distinctive cell types are known to emerge from a single progenitor cell that producing Neurogenin3 or Ngn3, a transcription factor that decides endocrine cell destiny (Edlund, 2002). Af terwards, other transcription factors, for example, Pdx1, Pax4, Nkx2.2, Nkx6.1, MafA, and Foxo1 help the -cell fate determination (Ziv et al., 2013). While it had been learned as that -cells proliferate by self-duplication from old -cells instead of differentiation from stem cell (Dor et al., 2004), near other study suggested that multipotent cells at heart the pancreas could differentiate into -cells as well (Xu et al., 2008). The latter study is also confirmed by explorees showing how expressing the -cell particularized transcription factors in non -cell could trigger a -cell lineage in mice (Collombat et al., 2009 Al-Hasani et al., 2013). Recent studies additionally showing that non- cells, for example, -and -cells could experience transdifferentiation into -like cells when the mice is suffering from significant -cell loss (Thorel et al., 2010 Chera et al., 2014). On the other hand, a few researches demonstrate that the -cells can likewise fall behind their fate or dediffere ntiate into non- or progenitor cells when losing any of the previously mentioned -cell particular transcription factor (Ziv et al., 2013). Moreover, a current study present that human -cells are capable of converting into -cells with no genetic modification (Spijker et al., 2013). Taken together, all these researches have showed that the dynamic plasticity of islet cells.1.3 Glucose Stimulate Insulin secernmentOne unique and significant feature of -cells is to detect the blood glucose changes and mask insulin into extracellular milieu in response to keeping the glucose levels within the range of 4-8mM (Weir and Bonner-Weir, 2013). This is primarily accomplished by the take-up of extracellular glucose by the glucose conveyor belt 2 (Glut2 Bell et al., 1990) at the plasma membrane. Upon uptake, the intracellular glucose sensor, glucokinase (Gck), subjects glucose moieties to quick metamorphosis system by glycolysis (Matschinsky and Ellerman, 1968). This brings about the producin g of three carbon products pyruvate, which takes part in the tricarboxylic corrosive (TCA) hertz inside the mitochondrion to eventually create adenosine triphosphates (adenosine triphosphate) by means of the electron transport chain system. The ATP therefore leads to the emergence of ATP/ADP proportion in the cytoplasm, activating the closure of the ATP sensitive potassium (KATP) channel. Vitally, mutation in the kir6.2 subunit of this channel were demonstrated to induce neonatal diabetes in both mice and human because of loss of insulin secretion as a consequence of constitutively open KATP channel (Koster et al., 2000 Gloyn et al., 2004). It has long been agnise that glucose stimulates the closure of these KATP bring thus leading the slow menbrane depolarization (Ashcroft et al., 1984). This promotes extracellular calcium influx by voltage dependent calcium channels and potentiates the releasing of insulin (Matschinsky et al., 1998). Insulin is secreted in an oscillatory deme anor because of the blood glucose level and triggers downstream insulin signaling cascade in insulin-responsive tissues for the taking up glucose. It has well been shown that islets can be entrained to nonaged changes in glucose and thus the plasma insulin has high relative frequency of oscillation. However, this capacity of entrainment of the islets is disturbed in patients with T2D (Mao et al., 1999). It exhibits -cell malfunction because of loss of insulin secretion is a major issue during the clinical indication of T2D.1.4 Compensatory Islet Expansion During Insulin oppositeDuring the state of insulin resistance or over-weight, elevated plasma insulin levels (named hyperinsulinemia) has been found in polygenic mouse models showing insulin resistance and human subjects because of increasing of insulin secretion (Yalow and Berson, 1960 Polonsky et al., 1988 Brning et al., 1997). It has been suggested later that both in rodents and people, this improved insulin secretion is on the face of it because of an expansion in -cell mass by either -cell proliferation (Steil et al., 2001) or -cell hypertrophy (Weir and Bonner-Weir, 2004). On the other hand, -cell failure because of different genetic or environmental variables, is known to cause declined plasma insulin levels in diabetics (Maclean and Ogilvie, 1955). It has been showed that lessened levels of insulin are often associated with a noteworthy loss of -cell mass because of -cell apoptosis (Butler et al., 2003 Rhodes, 2005).Other than the diabetes perspective, it has been demonstrated that matured -cells have long life-span and low proliferative rates at steady state. This is because of a potential limitation of the entry of matured -cells into cell cycle (Teta et al., 2005 Kushner, 2013). Other than this perception, later study suggested that adult -cells do have the ability to proliferate (Stolovich-Rain et al., 2012). In light of these findings on -cell proliferation, a few research groups have reveal ed various proteins essential for assisting -cell proliferation on knockout or transgenic mouse. know cell cycle controllers including Cyclins D1 and D2, Cyclin subordinate kinase 4 (Cdk4), Cdk inhibitors (CKIs) such as Cip/ catch some Zs and INK4, transcription factors Retinoblastoma (Rb) and p53, have been proved on genetic mouse models as regulators of -cell proliferation and survival (Heit et al., 2006).Although transient high glucose has been considered as the result of insufficient insulin secretion or insulin resistance, it has also been revealed to promote the compensate -cell mass expansion (Bonner-Weir et al., 1989). This speculation was further supported recently by another observation, that it is the glucose metabolism, instead the glucose itself that triggers compensatory -cell proliferation in vivo (Porat et al., 2011). Some other attentions have been centered on the effect of energizing of insulin/IRS2 pathway on driving -cell proliferation. The components of the pat hway including IRS2 (Withers et al., 1998) and AKT (Bernal-Mizrachi et al., 2001) were demonstrated to be implicit in(p) for -cell survival. Moreover, study has shown that the impact of insulin in -cell proliferation is even stronger when with hyperglycemia (Paris et al., 2003). It is notable that when in the state of severe insulin resistance, the pancreatic islets adjust themselves to meet the expanding requirement for producing and secretion more insulin by increasing their -cell mass (Weir and Bonner-Weir, 2004). Other than the involvement of protein coding genes, a few non-coding RNAs (ncRNAs), mostly microRNAs and long ncRNAs (lncRNAs), have been shown in the diabetogenes list, adding on to the complex genetic architecture of human diabetes.2.1 The Brief History of MicroRNAsThe most recent decade has seen huge attention regarding a new and special class of little ncRNAs such as microRNAs (miRNAs) in regulating the social structure and function of -cells. With the first obser vation of a miRNA, lin-4 in C. elegans, researchers showed how a gene product encodes two little RNAs, instead of a protein. Besides, they demonstrated that these small RNAs binds to the compensatory sites at the 3 end of untranslated region (UTR) of lin-14, a development related to heterochronic gene. This interaction is appeared to negatively regulate the expression of lin-14 by blocking its variant (Lee et al., 1993 Wightman et al., 1993), proposing miRNAs as negative regulators of gene expression.Even since the discovery of microRNAs, a conduct of related research results have updated the mechanism of gene regulation to a novel level. miRNAs now are known as a group of small ncRNAs of 21-22 nucleotides long that can complementary or non-complementary base-pairing the template RNA of protein coding genes, thus to regulate their expression at post-transcriptional level (Bartel, 2004 and 2009). Actually after the identification of lin-4, another miRNA called let-7 was revealed like lin-4 in both biogenesis and function level. Soon after, there starts a prevail in discovering new microRNASs mostly by high throughput sequencing technologies in research area and surprisingly, about 30,000 miRNA over about 200 species have been identified, which includes about 2,500 mature human miRNAs. (Kozomara and Griffiths-Jones, 2014). umpteen computational methods have also been produced to predict the potential targets of miRNAs based on the stable miRNA-target mRNA binding model (Lewis et al., 2003 Krek et al., 2005).2.2 MicroRNA Induced Gene SilencingThe intercellular gene silencing mechanism, termed as RNA silencing (RNAi) or post-transcriptional gene silencing (PTGS) is currently well known to be led by a group of small RNAs, for example, short interfering RNA (siRNA), piwi interacting RNA (piRNA), or the miRNAs. Basically, their working mechanisms are similar and the difference exist mostly in their biogenesis inside the cells (Ender and Meister, 2010). Mature mi RNA producing has been through several timbers transcribed from DNA by RNA polymerase II, primary miRNA (pri-miRNA) has much longer sequence. at a time transcribed, the pri-miRNA is further touch to precursor miRNA (pre-miRNA) about 60 nucleotides long by enzyme Dorsa and DGCR8 protein complex (Lee et al., 2003). Once pre-miRNAs are produced inside the core group, it will be export out of the nucleus by protein Exportin 5 to the cytoplasm in a Ran-GTP dependent manner (Lund et al., 2004). Another important enzyme, which is also critical for mouse development, Dicer would recognize the pre-miRNA and service it to about 22 nucleotides long mature miRNA duplex form (Bernstein et al.,2003). single one of stands of the duplex will be transported to miRNA-induced silencing complex (RISC) by Dicer, and the other strand, termed miRNA* is usually degraded in the end (Schwarz et al., 2003 Ender Meister, 2010).There are several components on the RISC, and one key protein component is AGO family. There are four well characterized members of AGO family in human AGO1, AGO2, AGO3 and AGO4. And AGO2 is expressed more often than other forms (Su et al., 2009 Wang et al., 2012). All the AGO proteins have the ability to diagonal mRNA because of their PAZ and PIWI cleavage domains under the guidance of miRNA sequence. Each miRNA has an important generator region, typically from 2nd-7th nucleotides, that could fully or partially bind to the mRNA 3 UTR sequence. The base pairing condition between the miRNA and target mRNA determines the target recognition and binding of miRISC, but also the fate of the mRNA- to be cleaved or to be repressed in translation. In animal system, the miRNA does not fully complementary bind to the 3 UTR of the target mRNA, through blocking the translation machinery, miRNA silencing the gene expression, without interference of the target stability. However, recent studies on the miRNA mediated gene silencing in mammalian cells reveals that miRNA may act through two step modes at first, repressing the translation, then deadenylation and destabilization of target mRNA 27qualifier. The deadenylation has been suggested to induce mRNA degradation 48 and translation inhibition is probably the requisite of mRNA degradation in mammalian cells 49.