RESUMO
microRNAs (miRNAs) are key regulators of both physiological and pathophysiological mechanisms in diabetes and gastrointestinal (GI) dysmotility. Our previous studies have demonstrated the therapeutic potential of miR-10a-5p mimic and miR-10b-5p mimic (miR-10a/b mimics) in rescuing diabetes and GI dysmotility in murine models of diabetes. In this study, we elucidated the safety profile of a long-term treatment with miR-10a/b mimics in diabetic mice. Male C57BL/6 mice were fed a high-fat, high-sucrose diet (HFHSD) to induce diabetes and treated by five subcutaneous injections of miR-10a/b mimics for a 5 month period. We examined the long-term effects of the miRNA mimics on diabetes and GI dysmotility, including an assessment of potential risks for cancer and inflammation in the liver and colon using biomarkers. HFHSD-induced diabetic mice subcutaneously injected with miR-10a/b mimics on a monthly basis for 5 consecutive months exhibited a marked reduction in fasting blood glucose levels with restoration of insulin and significant weight loss, improved glucose and insulin intolerance, and restored GI transit time. In addition, the miR-10a/b mimic-treated diabetic mice showed no indication of risk for cancer development or inflammation induction in the liver, colon, and blood for 5 months post-injections. This longitudinal study demonstrates that miR-10a/b mimics, when subcutaneously administered in diabetic mice, effectively alleviate diabetes and GI dysmotility for 5 months with no discernible risk for cancer or inflammation in the liver and colon. The sustained efficacy and favorable safety profiles position miR-10a/b mimics as promising candidates in miRNA-based therapeutics for diabetes and GI dysmotility.
Assuntos
Diabetes Mellitus Experimental , MicroRNAs , Neoplasias , Masculino , Animais , Camundongos , Diabetes Mellitus Experimental/genética , Estudos Longitudinais , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Inflamação , Fígado , Insulina , ColoRESUMO
BACKGROUND & AIMS: Constipation is commonly associated with diabetes. Serotonin (5-HT), produced predominantly by enterochromaffin (EC) cells via tryptophan hydroxylase 1 (TPH1), is a key modulator of gastrointestinal (GI) motility. However, the role of serotonergic signaling in constipation associated with diabetes is unknown. METHODS: We generated EC cell reporter Tph1-tdTom, EC cell-depleted Tph1-DTA, combined Tph1-tdTom-DTA, and interstitial cell of Cajal (ICC)-specific Kit-GCaMP6 mice. Male mice and surgically ovariectomized female mice were fed a high-fat high-sucrose diet to induce diabetes. The effect of serotonergic signaling on GI motility was studied by examining 5-HT receptor expression in the colon and in vivo GI transit, colonic migrating motor complexes (CMMCs), and calcium imaging in mice treated with either a 5-HT2B receptor (HTR2B) antagonist or agonist. RESULTS: Colonic transit was delayed in males with diabetes, although colonic Tph1+ cell density and 5-HT levels were increased. Colonic transit was not further reduced in diabetic mice by EC cell depletion. The HTR2B protein, predominantly expressed by colonic ICCs, was markedly decreased in the colonic muscles of males and ovariectomized females with diabetes. Ca2+ activity in colonic ICCs was decreased in diabetic males. Treatment with an HTR2B antagonist impaired CMMCs and colonic motility in healthy males, whereas treatment with an HTR2B agonist improved CMMCs and colonic motility in males with diabetes. Colonic transit in ovariectomized females with diabetes was also improved significantly by the HTR2B agonist treatment. CONCLUSIONS: Impaired colonic motility in mice with diabetes was improved by enhancing HTR2B signaling. The HTR2B agonist may provide therapeutic benefits for constipation associated with diabetes.
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Colo/efeitos dos fármacos , Constipação Intestinal/prevenção & controle , Complicações do Diabetes/prevenção & controle , Motilidade Gastrointestinal/efeitos dos fármacos , Indóis/farmacologia , Células Intersticiais de Cajal/efeitos dos fármacos , Complexo Mioelétrico Migratório/efeitos dos fármacos , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Tiofenos/farmacologia , Animais , Sinalização do Cálcio , Colo/metabolismo , Colo/fisiopatologia , Constipação Intestinal/etiologia , Constipação Intestinal/metabolismo , Constipação Intestinal/fisiopatologia , Complicações do Diabetes/metabolismo , Complicações do Diabetes/fisiopatologia , Modelos Animais de Doenças , Feminino , Genes Reporter , Células Intersticiais de Cajal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovariectomia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Serotonina/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
BACKGROUND & AIMS: Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans. METHODS: We developed an inducible, EC cell-specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell-depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell-depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG). RESULTS: Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate. CONCLUSIONS: The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG.
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Esvaziamento Gástrico/genética , Trânsito Gastrointestinal/genética , Serotonina/deficiência , Animais , Linhagem Celular , Células Enterocromafins/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Triptofano Hidroxilase/metabolismoRESUMO
BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) and pancreatic ß cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. METHODS: We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. RESULTS: miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of ß cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Krüppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. CONCLUSIONS: miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus/prevenção & controle , Esvaziamento Gástrico , Trânsito Gastrointestinal , Gastroparesia/prevenção & controle , Células Secretoras de Insulina/metabolismo , Células Intersticiais de Cajal/metabolismo , MicroRNAs/metabolismo , Adulto , Idoso , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Feminino , Gastroparesia/genética , Gastroparesia/metabolismo , Gastroparesia/fisiopatologia , Células HEK293 , Humanos , Células Secretoras de Insulina/patologia , Células Intersticiais de Cajal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Adulto JovemRESUMO
The gastrointestinal (GI) tract in mammals is comprised of dozens of cell types with varied functions, structures, and histological locations that respond in a myriad of ways to epigenetic and genetic factors, environmental cues, diet, and microbiota. The homeostatic functioning of these cells contained within this complex organ system has been shown to be highly regulated by the effect of microRNAs (miRNA). Multiple efforts have uncovered that these miRNAs are often tightly influential in either the suppression or overexpression of inflammatory, apoptotic, and differentiation-related genes and proteins in a variety of cell types in colorectal cancer (CRC). The early detection of CRC and other GI cancers can be difficult, attributable to the invasive nature of prophylactic colonoscopies. Additionally, the levels of miRNAs associated with CRC in biofluids can be contradictory and, therefore, must be considered in the context of other inhibiting competitive endogenous RNAs (ceRNA) such as lncRNAs and circRNAs. There is now a high demand for disease treatments and noninvasive screenings such as testing for bloodborne or fecal miRNAs and their inhibitors/targets. The breadth of this review encompasses current literature on well-established CRC-related miRNAs and the possibilities for their use as biomarkers in the diagnoses of this potentially fatal GI cancer.
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Neoplasias Colorretais , MicroRNAs , RNA Longo não Codificante , Animais , Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Redes Reguladoras de Genes , Mamíferos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/genéticaRESUMO
Following the discovery of nucleic acids by Friedrich Miescher in 1868, DNA and RNA were recognized as the genetic code containing the necessary information for proper cell functioning. In the years following these discoveries, vast knowledge of the seemingly endless roles of RNA have become better understood. Additionally, many new types of RNAs were discovered that seemed to have no coding properties (non-coding RNAs), such as microRNAs (miRNAs). The discovery of these new RNAs created a new avenue for treating various human diseases. However, RNA is relatively unstable and is degraded fairly rapidly once administered; this has led to the development of novel delivery mechanisms, such as nanoparticles to increase stability as well as to prevent off-target effects of these molecules. Current advances in RNA-based therapies have substantial promise in treating and preventing many human diseases and disorders through fixing the pathology instead of merely treating the symptomology similarly to traditional therapeutics. Although many RNA therapeutics have made it to clinical trials, only a few have been FDA approved thus far. Additionally, the results of clinical trials for RNA therapeutics have been ambivalent to date, with some studies demonstrating potent efficacy, whereas others have limited effectiveness and/or toxicity. Momentum is building in the clinic for RNA therapeutics; future clinical care of human diseases will likely comprise promising RNA therapeutics. This review focuses on the current advances of RNA therapeutics and addresses current challenges with their development.
Assuntos
MicroRNAs , Nanopartículas , Ácidos Nucleicos , Humanos , MicroRNAs/genética , Nanopartículas/uso terapêutico , RNA Interferente Pequeno/genética , RNA não Traduzido/genéticaRESUMO
Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα+ cells. However, the cellular origin for this gene expression is controversial as it is also known to be expressed in intestinal macrophages. We found that Adamdec1 mRNAs were selectively expressed in colonic mucosal subepithelial PDGFRα+ cells. ADAMDEC1 protein was mainly released from PDGFRα+ cells and accumulated in the mucosal layer lamina propria space near the epithelial basement membrane. PDGFRα+ cells significantly overexpressed Adamdec1 mRNAs and protein in DSS-induced colitis mice. Adamdec1 was predominantly expressed in CD45- PDGFRα+ cells in DSS-induced colitis mice, with only minimal expression in CD45+ CD64+ macrophages. Additionally, overexpression of both ADAMDEC1 mRNA and protein was consistently observed in PDGFRα+ cells, but not in CD64+ macrophages found in human colonic mucosal tissue affected by Crohn's disease. In summary, PDGFRα+ cells selectively express ADAMDEC1, which is localized to the colon mucosa layer. ADAMDEC1 expression significantly increases in DSS-induced colitis affected mice and Crohn's disease affected human tissue, suggesting that this gene can serve as a diagnostic and/or therapeutic target for intestinal inflammation and Crohn's disease.
Assuntos
Proteínas ADAM , Colite , Doença de Crohn , Doenças Inflamatórias Intestinais , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Animais , Biomarcadores , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colo/citologia , Colo/metabolismo , Doença de Crohn/metabolismo , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Ulva lactuca (U. lactuca) is a green alga distributed worldwide and used as a food and cosmetic material. In our previous study, we determined the effects of U. lactuca methanol extracts on the UVB-induced DNA repair. In the present study, we fractionated U. lactuca methanol extracts to identify the effective compound for the DNA repair. MTT assay demonstrated that (+)-epiloliolide showed no cytotoxicity up to 100 µM in BJ-5ta human dermal fibroblast. Upon no treatment, exposure to UVB 400 J/m2 decreased cell viability by 45%, whereas (+)-epiloliolide treatment for 24 h after UVB exposure significantly increased the cell viability. In GO and GESA analysis, a number of differentially expressed genes were uniquely expressed in (+)-epiloliolide treated cells, which were enriched in the p53 signaling pathway and excision repair. Immunofluorescence demonstrated that (+)-epiloliolide increased the nuclear localization of p53. Comet assay demonstrated that (+)-epiloliolide decreased tail moment increased by UVB. Western blot analysis demonstrated that (+)-epiloliolide decreased the levels of p-p53, p21, Bax, and Bim, but increased that of Bcl-2. Reverse transcription PCR (RT-PCR) demonstrated that (+)-epiloliolide decreased the levels of MMP 1, 9, and 13, but increased that of COL1A1. These results suggest that (+)-epiloliolide regulates p53 activity and has protective effects against UVB.
Assuntos
Benzofuranos/farmacologia , Fibroblastos/efeitos dos fármacos , Envelhecimento da Pele , Proteína Supressora de Tumor p53/efeitos dos fármacos , Ulva , Organismos Aquáticos , Humanos , Fitoterapia , Raios UltravioletaRESUMO
The cellular microenvironment composition and changes therein play an extremely important role in cancer development. Changes in the extracellular matrix (ECM), which constitutes a majority of the tumor stroma, significantly contribute to the development of the tumor microenvironment. These alterations within the ECM and formation of the tumor microenvironment ultimately lead to tumor development, invasion, and metastasis. The ECM is composed of various molecules such as collagen, elastin, laminin, fibronectin, and the MMPs that cleave these protein fibers and play a central role in tissue remodeling. When healthy cells undergo an insult like DNA damage and become cancerous, if the ECM does not support these neoplastic cells, further development, invasion, and metastasis fail to occur. Therefore, ECM-related cancer research is indispensable, and ECM components can be useful biomarkers as well as therapeutic targets. Colorectal cancer specifically, is also affected by the ECM and many studies have been conducted to unravel the complex association between the two. Here we summarize the importance of several ECM components in colorectal cancer as well as their potential roles as biomarkers.
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Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Animais , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas da Matriz Extracelular/genética , HumanosAssuntos
Trânsito Gastrointestinal , Tato , Envelhecimento , Animais , Digestão , Humanos , Canais Iônicos/metabolismo , Mecanotransdução Celular , CamundongosRESUMO
PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3'-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.
Assuntos
Células Intersticiais de Cajal/metabolismo , Intestino Delgado/metabolismo , Estágio Paquíteno/genética , RNA Interferente Pequeno/genética , Testículo/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Regulação da Expressão Gênica , Intestino Delgado/citologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Testículo/citologia , TranscriptomaRESUMO
Glucagon-like peptide-1 (GLP-1) is an incretin hormone known for its pivotal role in enhancing insulin secretion and reducing glucagon release from the pancreas. Diabetic nephropathy, which is characterized by albuminuria, represents a significant microvascular complication of diabetes. Most of the previous studies mainly focused on the therapeutic renal protective effect in clinical trials after the administration of GLP-1 receptor agonists (GLP-1 RAs), rather than before administration. Hence, this study aimed to investigate the association between fasting plasma GLP-1 levels and albuminuria before GLP-1 RA administration. A cross-sectional study was designed to evaluate the association between fasting plasma GLP-1 levels and albuminuria in patients with type 2 diabetes mellitus (T2DM). A cohort of 68 participants with T2DM was analyzed using data collected at Wonkwang University Hospital in Iksan, Korea. Logistic regression analysis was employed to determine the odds ratio (OR) and 95% confidence interval (CI) of the incidence of albuminuria between two groups categorized by fasting GLP-1 levels, low (Group L) and high GLP-1 (Group H). The OR (95% CI) for the incidence of albuminuria comparing Group L with Group H of fasting plasma GLP-1 levels was 3.41 (1.16-10.02), p = 0.03 after adjustment for relevant variables including age, gender, fasting plasma glucose, HbA1c, C-peptide, creatinine, and medication use [angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs), sodium-glucose cotransporter-2 (SGLT-2) inhibitors, and dipeptidyl peptidase-4 (DPP-4) inhibitors]. When analyzed as a continuous variable, each 1 pmol/L reduction in fasting plasma GLP-1 levels was associated with an OR (95% CI) of 1.67 (1.17-1.87), p = 0.02, following full adjustment. These results highlight a negative association between fasting plasma GLP-1 levels and the incidence of albuminuria in Korean patients with T2DM, before GLP-1 RA administration. These findings suggest that endogenous GLP-1 may have a beneficial impact in mitigating albuminuria.
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Mammalian genomes encode a large number of small noncoding RNAs (sncRNAs) that play regulatory roles during development and adulthood by affecting gene expression. Several sncRNA species, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), endogenous small interfering RNAs (endo-siRNAs), and small nucleolar RNAs (snoRNAs), are abundantly expressed in the testis and required for normal testicular development and spermatogenesis. To evaluate global changes in sncRNA expression, the next-generation sequencing (NGS)-based sncRNA transcriptomic analysis has become routine, because it allows rapid determination of the small RNA transcriptome of a particular testicular cell type. However, annotation of small RNA NGS reads can be challenging due to the volume of reads obtained, which is usually in the millions. Therefore, we developed a computer-assisted sncRNA annotation protocol that could identify not only known sncRNAs but also previously uncharacterized ones. Using this protocol, we annotated NGS reads of a Sertoli cell sncRNA library, and we report to our knowledge the first comprehensive annotation of the sncRNA transcriptome of immature murine Sertoli cells. Moreover, the computer-assisted sncRNA annotation pipeline that we report is applicable for annotating NGS reads derived from other cell types and/or sequencing platforms.
Assuntos
Regulação da Expressão Gênica/fisiologia , Pequeno RNA não Traduzido/metabolismo , RNA/metabolismo , Células de Sertoli/metabolismo , Transcriptoma , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA/genética , Pequeno RNA não Traduzido/genética , Alinhamento de Sequência , Análise de Sequência de RNA/métodos , SoftwareRESUMO
BACKGROUND/AIM: Diabetes has substantive co-occurrence with disorders of gut-brain interactions (DGBIs). The pathophysiological and molecular mechanisms linking diabetes and DGBIs are unclear. MicroRNAs (miRNAs) are key regulators of diabetes and gut dysmotility. We investigated whether impaired gut barrier function is regulated by a key miRNA, miR-10b-5p, linking diabetes and gut dysmotility. METHODS: We created a new mouse line using the Mb3Cas12a/Mb3Cpf1 endonuclease to delete mir-10b globally. Loss of function studies in the mir-10b knockout (KO) mice were conducted to characterize diabetes, gut dysmotility, and gut barrier dysfunction phenotypes in these mice. Gain of function studies were conducted by injecting these mir-10b KO mice with a miR-10b-5p mimic. Further, we performed miRNA-sequencing analysis from colonic mucosa from mir-10b KO, wild type, and miR-10b-5p mimic injected mice to confirm (1) deficiency of miR-10b-5p in KO mice, and (2) restoration of miR-10b-5p after the mimic injection. RESULTS: Congenital loss of mir-10b in mice led to the development of hyperglycemia, gut dysmotility, and gut barrier dysfunction. Gut permeability was increased, but expression of the tight junction protein Zonula occludens-1 was reduced in the colon of mir-10b KO mice. Patients with diabetes or constipation- predominant irritable bowel syndrome, a known DGBI that is linked to leaky gut, had significantly reduced miR-10b-5p expression. Injection of a miR-10b-5p mimic in mir-10b KO mice rescued these molecular alterations and phenotypes. CONCLUSIONS: Our study uncovered a potential pathophysiologic mechanism of gut barrier dysfunction that links both the diabetes and gut dysmotility phenotypes in mice lacking miR-10b-5p. Treatment with a miR-10b-5p mimic reversed the leaky gut, diabetic, and gut dysmotility phenotypes, highlighting the translational potential of the miR-10b-5p mimic.
Assuntos
Diabetes Mellitus , Síndrome do Intestino Irritável , MicroRNAs , Humanos , Camundongos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , FenótipoRESUMO
BACKGROUND & AIMS: Smooth muscle cells (SMCs) change phenotypes under various pathophysiological conditions. These changes are largely controlled by the serum response factor (SRF), a transcription factor that binds to CC (A/T)6 GG (CArG) boxes in SM contractile genes. MicroRNAs (miRNA) regulate transitions among SMC phenotypes. The SMC miRNA transcriptome (SMC miRNAome) and its regulation by SRF have not been determined. METHODS: We performed massively parallel sequencing to identify gastrointestinal (GI) SMC miRNA transcriptomes in mice and humans. SMC miRNA transcriptomes were mapped to identify all CArG boxes, which were confirmed by SRF knockdown and microarrays. Quantitative polymerase chain reaction was used to identify SMC-phenotypic miRNAs in differentiated and proliferating SMCs. Bioinformatics and target validation analysis showed regulation of SMC phenotype by SRF-dependent, SMC-phenotype miRNAs. RESULTS: We cloned and identified GI miRNA transcriptomes using genome-wide analyses of mouse and human cells. The SM miRNAome consisted of hundreds of unique miRNAs that were highly conserved among both species. We mapped miRNAs CArG boxes and found that many had an SRF-dependent signature in the SM miRNAome. The SM miRNAs CArG boxes had several distinct features. We also identified approximately 100 SMC-phenotypic miRNAs that were induced in differentiated or proliferative SMC phenotypes. We showed that SRF-dependent, SMC-phenotypic miRNAs bind and regulate Srf and its cofactors, myocadin (Myocd) and member of ETS oncogene family Elk1. CONCLUSIONS: The GI SMC phenotypes are controlled by SRF-dependent, SMC-phenotypic miRNAs that regulate expression of SRF, MYOCD, and ELK1.
Assuntos
Trato Gastrointestinal/metabolismo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Biologia Computacional , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genótipo , Proteínas de Fluorescência Verde/genética , Humanos , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cadeias Pesadas de Miosina/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Interferência de RNA , Fator de Resposta Sérica/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Functional gastrointestinal disorders (FGIDs) have been re-named as disorders of gut-brain interactions. These conditions are not only common in clinical practice, but also in the community. In reference to the Rome IV criteria, the most common FGIDs, include functional dyspepsia (FD) and irritable bowel syndrome (IBS). Additionally, there is substantial overlap of these disorders and other specific gastrointestinal motility disorders, such as gastroparesis. These disorders are heterogeneous and are intertwined with several proposed pathophysiological mechanisms, such as altered gut motility, intestinal barrier dysfunction, gut immune dysfunction, visceral hypersensitivity, altered GI secretion, presence and degree of bile acid malabsorption, microbial dysbiosis, and alterations to the gut-brain axis. The treatment options currently available include lifestyle modifications, dietary and gut microbiota manipulation interventions including fecal microbiota transplantation, prokinetics, antispasmodics, laxatives, and centrally and peripherally acting neuromodulators. However, treatment that targets the pathophysiological mechanisms underlying the symptoms are scanty. Pharmacological agents that are developed based on the cellular and molecular mechanisms underlying pathologies of these disorders might provide the best avenue for future pharmaceutical development. The currently available therapies lack long-term effectiveness and safety for their use to treat motility disorders and FGIDs. Furthermore, the fundamental challenges in treating these disorders should be defined; for instance, 1. Cause and effect cannot be disentangled between symptoms and pathophysiological mechanisms due to current therapies that entail the off-label use of medications to treat symptoms. 2. Despite the knowledge that the microbiota in our gut plays an essential part in maintaining gut health, their exact functions in gut homeostasis are still unclear. What constitutes a healthy microbiome and further, the precise definition of gut microbial dysbiosis is lacking. More comprehensive, large-scale, and longitudinal studies utilizing multi-omics data are needed to dissect the exact contribution of gut microbial alterations in disease pathogenesis. Accordingly, we review the current treatment options, clinical insight on pathophysiology, therapeutic modalities, current challenges, and therapeutic clues for the clinical care and management of functional dyspepsia, gastroparesis, irritable bowel syndrome, functional constipation, and functional diarrhea.
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Unsupervised clustering of single-cell RNA sequencing data (scRNA-seq) is important because it allows us to identify putative cell types. However, the large number of cells (up to millions), the high-dimensionality of the data (tens of thousands of genes), and the high dropout rates all present substantial challenges in single-cell analysis. Here we introduce a new method, named single-cell Clustering using Autoencoder and Network fusion (scCAN), that can overcome these challenges to accurately segregate different cell types in large and sparse scRNA-seq data. In an extensive analysis using 28 real scRNA-seq datasets (more than three million cells) and 243 simulated datasets, we validate that scCAN: (1) correctly estimates the number of true cell types, (2) accurately segregates cells of different types, (3) is robust against dropouts, and (4) is fast and memory efficient. We also compare scCAN with CIDR, SEURAT3, Monocle3, SHARP, and SCANPY. scCAN outperforms these state-of-the-art methods in terms of both accuracy and scalability. The scCAN package is available at https://cran.r-project.org/package=scCAN . Data and R scripts are available at http://sccan.tinnguyen-lab.com/.
Assuntos
Comunicação Celular , Análise de Célula Única , Análise por Conglomerados , Perfilação da Expressão Gênica , Análise de Sequência de RNA , Análise de Célula Única/métodos , Sequenciamento do ExomaRESUMO
Maturity-Onset Diabetes of the Young (MODY) is a rare form of diabetes which affects between 1% and 5% of diagnosed diabetes cases. Clinical characterizations of MODY include onset of diabetes at an early age (before the age of 30), autosomal dominant inheritance pattern, impaired glucose-induced secretion of insulin, and hyperglycemia. Presently, 14 MODY subtypes have been identified. Within these subtypes are several mutations which contribute to the different MODY phenotypes. Despite the identification of these 14 subtypes, MODY is often misdiagnosed as type 1 or type 2 diabetes mellitus due to an overlap in clinical features, high cost and limited availability of genetic testing, and unfamiliarity with MODY outside of the medical profession. The primary aim of this review is to investigate the genetic characterization of the MODY subtypes. Additionally, this review will elucidate the link between the genetics, function, and clinical manifestations of MODY in each of the 14 subtypes. In providing this knowledge, we hope to assist in the accurate diagnosis of MODY patients and, subsequently, in ensuring they receive appropriate treatment.
RESUMO
BACKGROUND: Specialized enterochromaffin (EC) cells within the mucosal lining of the gut synthesize and secrete almost all serotonin (5-hydroxytryptamine, 5-HT) in the body. Significantly lower amounts of 5-HT are made by other peripheral tissues and serotonergic neurons within the enteric nervous system (ENS). EC cells are in close proximity to 5-HT receptors in the ENS, and the role of 5-HT as a modulator of gut motility, particularly colonic motor complexes, has been well defined. However, the relative contribution of neuronal 5-HT to this process under resting and stimulus-evoked conditions is unclear. METHODS: In this study, we combined the use of the selective serotonin transporter (SERT) inhibitor, fluoxetine, with two models of mucosal 5-HT depletion-surgical removal of the mucosa and our Tph1Cre/ERT2 ; Rosa26DTA mouse line-to determine the relative contribution of neuronal and mucosal 5-HT to resting and distension-evoked colonic motility. KEY RESULTS: Fluoxetine significantly reduced the frequency of colonic migrating complexes (CMCs) in flat-sheet preparations with the mucosa present and in intact control Tph1-DTA colons in which EC cells were present. No such effect was observed in mucosa-free preparations or in intact Tph1-DTA preparations lacking EC cell 5-HT. CONCLUSIONS AND INFERENCES: We demonstrate that mucosal 5-HT release plays an important role in distension-evoked colonic motility, and that SERT inhibition no longer alters gut motility when EC cells are absent, thus demonstrating that ENS 5-HT does not play a role in regulating gut motility.