Maternal obesity is linked to heightened metabolic disease risk in offspring, but the mediators of this intergenerational effect remain unclear. Using a diet-induced obesity (DIO) mouse model, we showed that maternal circulating small extracellular vesicles (sEVs) crossed the placenta and delivered obesity-associated miRNAs to the fetal liver, with lasting consequences for insulin sensitivity in male offspring. Among these miRNAs, miR-29a-3p was pathologically elevated and targeted both DNA methyltransferases and demethylases, thereby reshaping the DNA methylation landscape. This included hypomethylation of the Pgc-1α locus, a key regulator of gluconeogenesis, which resulted in premature activation of hepatic gluconeogenesis that contributed to the persistent metabolic dysfunction in adulthood in male offspring. These findings identify a transplacental sEV-miRNA-epigenetic axis that perturbs fetal metabolic programming and may represent a conserved mechanism underlying the developmental origins of metabolic disease. The study identifies a transplacental sEV–miRNA–epigenetic axis as a mediator of maternal obesity. Maternal plasma sEVs transfer miR-29a-3p to the fetal liver, epigenetically reprogramming glucose metabolism and driving adult insulin resistance.

BACKGROUND:The circuit of scar-related upper loop macroreentry atrial flutter (AFL) surrounding the superior vena cava (SVC) has been described by prior case reports. However, the correlation between the circuit and arrhythmogenic substrates and the corresponding optimized ablation strategy need to be further investigated. OBJECTIVE:We aimed to identify the electrophysiologic substrate and corresponding ablation strategies of SVC-AFL using high-resolution mapping. METHODS:From June 1, 2017, to May 1, 2023, consecutive patients with macroreentrant atrial tachycardias (ATs) from 7 institutions were retrospectively evaluated. Patients with SVC-AFL were enrolled and analyzed. RESULTS:Of 1282 patients with macroreentrant ATs, 16 patients (1.2%; median age, 60.9 years; 8 male) had SVC-AFL (mean cycle length, 281.0 ± 55.1 ms), all identified during high-resolution activation mapping. All patients had prior cardiac surgery (14 [87.5%]) or catheter ablation (8 [50.0%]). A longitudinal surgical incision/scar extending from the SVC to the right atrium was observed in all patients, enabling macroreentry. SVC-AFLs with shorter circuits (<180 mm) had more slow conduction areas than those with longer circuits (>180 mm; 3.0 [2.0-4.0] vs 1.0 [1.0-1.5]; P = .023]. All ATs were terminated by ablating the channel between the surgical incision/scar and anatomic barriers. Cavotricuspid isthmus block was achieved in all patients. During a 21-month follow-up, all patients were free of atrial arrhythmias except for 4 patients experiencing short-lived paroxysmal ATs that did not require further ablation. CONCLUSION:A surgical incision/scar extending from the SVC to right atrium promotes the development of SVC-AFL. Substrate-based linear lesions along with prophylactic cavotricuspid isthmus ablation afford favorable clinical outcomes.

Extracellular vesicles (EVs) have clinically emerged as promising biocompatible vesicles for delivering therapeutic siRNAs to the central nervous system. Among targeting strategies, the rabies virus glycoprotein (RVG) peptide is the most commonly used modification on the EV surface to enable efficient systemic delivery of EVs. Although RVG is widely believed to facilitate blood-brain barrier (BBB) through receptor interactions, the underlying mechanism remains indirect and equivocal. Similarly, cell-penetrating peptide (CPP) modifications have been used to enhance BBB transport of various vehicles, such as CPP.16, which improves the brain delivery efficiency of adeno-associated virus 9 capsids. However, whether CPP.16 retains its delivery efficacy when applied to EVs remains unclear, raising concerns about carrier-specific limitations. In this study, we investigate the mechanisms underlying the transcytosis and delivery efficiency of RVG- and CPP.16-modified small EVs (sEVs) loaded with siRNAs. Using an in vitro BBB model, we found that these modifications do not alter the internalization of siRNAs by endothelial cells. Instead, these modifications appear to divert sEVs and siRNAs into transcytotic pathways, enabling their release into abluminal cells and subsequent target gene silencing. Moreover, RVG-sEVs primarily interact with the receptor and are internalized via clathrin-mediated endocytosis, leading to more efficient BBB penetration compared with CPP.16-sEVs. Consistently, in vivo studies demonstrate that RVG-sEVs deliver siRNAs more efficiently to both neurons and astrocytes compared with unmodified or CPP.16-sEVs. Our findings support the clinical potential of BBB-targeting peptides and provide critical insights for the rational selection of guiding peptides in central nervous system drug delivery.

Cold has been a long-term survival challenge in the evolutionary process of mammals. In response to cold stress, in addition to brown adipose tissue (BAT) dissipating energy as heat through glucose and lipid oxidation to maintain body temperature, cold stimulation can strongly activate thermogenesis and energy expenditure in beige fat cells, which are widely distributed in the subcutaneous layer. However, the effects of cold stimulation on other tissues and systemic lipid metabolism remain unclear. Our previous research indicated that, under cold stress, BAT not only produces heat but also secretes numerous exosomes to mediate BAT-liver crosstalk. Whether subcutaneous fat has a similar mechanism is still unknown. Therefore, this study aimed to investigate the alterations in lipid metabolism across various tissues under cold exposure and to explore whether subcutaneous fat regulates systemic glucose and lipid metabolism via exosomes, thereby elucidating the regulatory mechanisms of lipid metabolism homeostasis under physiological stress. RT-qPCR, Western blot, and H&E staining methods were used to investigate the physiological changes in lipid metabolism in the serum, liver, epididymal white adipose tissue, and subcutaneous fat of mice under cold stimulation. The results revealed that cold exposure significantly enhanced the thermogenic activity of subcutaneous adipose tissue and markedly increased exosome secretion. These exosomes were efficiently taken up by hepatocytes, where they profoundly influenced hepatic lipid metabolism, as evidenced by alterations in the expression levels of key genes involved in lipid synthesis and catabolism pathways. This study has unveiled a novel mechanism by which subcutaneous fat regulates lipid metabolism through exosome secretion under cold stimulation, providing new insights into the systemic regulatory role of beige adipocytes under cold stress and offering a theoretical basis for the development of new therapeutic strategies for obesity and metabolic diseases.

Cardiovascular disease remains the leading cause of death worldwide, with atherosclerosis (AS) serving as a critical underlying pathological process and major risk factor. Regular physical exercise is widely recognized as an effective strategy to reduce the risks and severity of AS, yet the precise molecular mechanisms through which exercise exerts its protective effects are still not fully understood. MicroRNAs (miRNAs), key regulators of gene expression, play integral roles in the progression of AS by influencing vascular function, lipid metabolism, and inflammation. The exercise-induced improvement of AS is a complex process, with miRNAs playing essential roles not only within cells and tissues but also circulating stably in the bloodstream as novel signaling molecules. These circulating miRNAs mediate communication between organs and tissues, acting as potential biomarkers that could provide deeper, systemic insights into the metabolic benefits of exercise. In this review, we explore recent advancements in our understanding of how exercise affects both intracellular and circulating miRNAs. We emphasize how exercise-regulated miRNAs contribute to endothelial function, promote lipid metabolism across various metabolic organs, and reduce monocyte-mediated systemic inflammation, while also addressing their role in alleviating frailty. Circulating miRNAs, which dynamically reflect tissue-specific responses to exercise, hold great promise as diagnostic and prognostic biomarkers for AS. Moreover, we discuss the challenges and future directions in this field, aiming to uncover how exercise-induced miRNA modulation could offer innovative therapeutic strategies for the prevention and treatment of AS.

Inflammatory bowel disease (IBD) is a group of chronic relapsing diseases associated with inflammatory disorders and microbial dysbiosis of the intestine. The use of traditional Chinese medicine (TCM) to treat colitis has the advantage of fewer side effects, but the molecular mechanism is not clear. Recently, miRNAs have been recognized as novel functional small molecules in plants that have regulatory effects on biological activities. This study mainly investigated the mechanism of action of MIR2911 from Honeysuckle, the main component of TCM preparations for colitis. The results demonstrated that MIR2911 can be absorbed through the diet and secreted within host small extracellular vesicles (sEVs), acting directly on intestinal bacteria, reducing the abundance of Escherichia-Shigella, and improving colitis symptoms. This study provides a new theoretical basis for the molecular mechanism of TCM therapy and identifies a potential new drug a new drug target for the treatment of colitis.

The incidence of type 2 diabetes mellitus (T2DM) induced by obesity is rapidly increasing. Although there are many synthetic drugs for treating T2DM, they have various side effects. Here, we report that miR8175, a plant miRNA from burdock root, has effective antidiabetic activity. After administration of burdock decoction or synthetic miR8175 by gavage, both burdock decoction and miR8175 can significantly improve the impaired glucose metabolism of diabetic mice induced by a high-fat diet (HFD). Our results demonstrate that burdock decoction and miR8175 enhance the insulin sensitivity of the hepatic insulin signaling pathway by targeting Ptprf and Ptp1b, which may be the reason for the improvement in metabolism. This study provides a theoretical basis for the main active component and molecular mechanism of burdock to improve insulin resistance. And the study also suggests that plant miRNA may be an indispensable nutrient for maintaining human health.

The increasing prevalence and persistence of nanoplastics (NPs) have become critical environmental concerns. These particles have the potential to enter the food chain and accumulate in living organisms, which exerts their adverse effects on human health. The release of nanoparticles from feeding bottles raises concerns about potential health issues, especially for newborns exposed to NPs at the neonatal stage. In this study, we examined the impacts of neonatal exposure to polystyrene nanoplastics (PS-NPs) on neurodevelopment. Our study demonstrates that exposure to PS-NPs in newborn mice impairs microglial autophagic function and energy metabolism, leading to the disruption of microglia-mediated synaptic pruning during early neurodevelopment. These mice subsequently develop social behavioral defects in adulthood, suggesting the long-lasting effects of neonatal PS-NP exposure on brain development and behavior. Together, these data provide insights into the mechanism by which PS-NPs affect early neurodevelopment, thus emphasizing the crucial need to address plastic pollution globally.

Adipose tissue is traditionally classified into two main types based on their functions: brown adipose tissue (BAT) and white adipose tissue (WAT). Each type plays a distinct role in the body's energy metabolism. Additionally, a third type, beige adipose tissue, can develop within subcutaneous WAT (including inguinal WAT, iWAT) in response to specific stimuli and exhibits characteristics of both BAT and WAT. Extracellular vesicles (EVs) are crucial for intercellular communication, carrying a diverse array of biomolecules such as proteins, lipids, and nucleic acids. While the functional diversity and endocrine roles of adipose tissues are well-documented, a comparative analysis of the functions of EVs released by different adipose tissues from mice housed at room temperature has not been thoroughly explored. MicroRNAs (miRNAs), which are highly enriched in small extracellular vesicles (sEVs), offer a promising avenue for investigating the complex functions and unique roles of various adipose tissues. In this study, we isolated sEVs from different adipose tissues under basal conditions and performed a comprehensive analysis of their miRNA content. By comparing miRNA profiles across different adipose tissues, we aim to elucidate the potential roles of sEV-derived miRNAs in mediating intercellular communication and the distinct physiological functions of adipose tissues. Understanding the molecular features of miRNAs in adipose tissue EVs could reveal new aspects of adipose tissue biology and lay the groundwork for further research into their physiological significance.

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multi-pass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N -glycosylated, but the precise site-specific N -glycosylation information and its functional contribution remain unclear. In this study, we employ high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N -glycosites and quantify the N -glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N -linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N -glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system, with potential implications for therapeutic applications.

Machado-Joseph disease, also known as spinocerebellar ataxia type 3 (MJD/SCA3), is a fatal autosomal dominant hereditary ataxia characterized by cerebellar ataxia resulting from the abnormal expansion of CAG repeats in exon 10 of the ATXN3 gene. At present, there is no effective treatment for SCA3. Small interfering RNAs (siRNAs) are emerging as potential therapeutic strategies to target the disease-causing mutant ATXN3 (mATXN3) protein specifically. However, the efficiency of delivery of siRNAs remains a major obstacle for clinical application, particularly in brain disorders.The aim of this study was to develop a synthetic biology strategy to reprogram the host liver as a tissue chassis to induce and deliver in vivo self-assembled siRNAs to target the ATXN3 gene. A synthetic construct directed by a cytomegalovirus promoter was designed to encode a neuron-targeting rabies virus glycoprotein tag and mATXN3-siRNA. After intravenous injection, the synthetic construct was taken up by mouse livers, which were then reprogrammed to enable the self-assembly, production and secretion of small extracellular vesicles encapsulating mATXN3-siRNA. The small extracellular vesicle-encapsulated mATXN3-siRNA was transported through the endogenous circulating system of small extracellular vesicles, crossing the blood-brain barrier and reaching the cerebellar cortex and spinal cerebellar tract, where they silenced the ATXN3 gene.Treatment with the synthetic construct for 8 or 12 weeks led to significant improvements in motor balance ability and reduction of cerebellar atrophy in YACMJD84.2 transgenic mice. The number of Purkinje cells in the cerebellar cortex was significantly increased, and the loss of myelin basic protein was reduced. Moreover, the quantity of neurotoxic nuclear inclusion bodies and the expression of glial fibrillary acidic protein, which promotes neuroinflammation in activated astrocytes, were decreased significantly.The synthetic construct facilitated the generation and delivery of in vivo self-assembled siRNA to the cerebellar cortex and spinal cerebellar tract, thereby inhibiting the expression of mATXN3 protein. This treatment successfully addressed motor impairments, alleviated neuropathological phenotypes and mitigated neuroinflammation in YACMJD84.2 transgenic mice. Our strategy effectively overcomes the primary challenges associated with siRNA therapy for cerebellar ataxia, offering a promising avenue for future clinical treatments. Machado-Joseph disease (MJD) is caused by CAG expansion in ATXN3. Li et al. used a synthetic biology approach to drive the self-assembly of small extracellular vesicles carrying siRNAs targeting mutant ATXN3. The vesicles delivered siRNAs to the cerebellar cortex and spinal cerebellar tract, silencing ATXN3 and reducing pathology in MJD mice.

Background and Objective: Aortic aneurysm and dissection are serious life-threatening cardiovascular emergencies, and their pathogenesis includes vascular inflammation, extracellular matrix remodeling and matrix metalloproteinases, phenotype switch of vascular smooth muscle cells and apoptosis, but the specific mechanisms have not been fully elucidated. As gene expression regulators, microRNAs are also key molecules in vascular function. This article not only describes the role of microRNAs in the pathogenesis and progression of aortic aneurysm and dissection, but also further illustrates the molecular mechanism of aortic aneurysm and dissection, which is of great significance for their prevention and treatment. In addition, we discuss miRNAs as a clinical biomarker for the diagnosis and monitoring of aortic aneurysms and dissection, as well as the possibility of developing new effective therapeutic targets. Methods: As of October 8, 2023, relevant publications containing miRNAs involvement in aortic aneurysms and dissection were systematically searched in the PubMed database. Key Content and Findings: Many miRNAs are involved in the pathogenesis of aortic aneurysm and dissection, including vascular inflammation, extracellular matrix remodeling, and homeostasis regulation of vascular smooth muscle cells. Among these miRNAs, some candidates have become potential biomarkers for the early diagnosis and long-term prognosis of aortic aneurysm and dissection due to their high sensitivity, specificity and stability. In addition, miRNAs are also becoming important targets for drug discovery. We have summarized miRNAs with clinical application prospects in aortic aneurysm and dissection. Conclusions: MiRNAs play a vital part in the pathogenesis of aortic aneurysm and dissection. The research on miRNAs is moving from the laboratory to the clinical, and miRNAs are expected to be used for the diagnosis and treatment of aortic aneurysm and dissection in the future.

Reactive astrocytes play an important role in neurological diseases, but their molecular and functional phenotypes in epilepsy are unclear. Here, we show that in patients with temporal lobe epilepsy (TLE) and mouse models of epilepsy, excessive lipid accumulation in astrocytes leads to the formation of lipid-accumulated reactive astrocytes (LARAs), a new reactive astrocyte subtype characterized by elevated APOE expression. Genetic knockout of APOE inhibited LARA formation and seizure activities in epileptic mice. Single-nucleus RNA sequencing in TLE patients confirmed the existence of a LARA subpopulation with a distinct molecular signature. Functional studies in epilepsy mouse models and human brain slices showed that LARAs promote neuronal hyperactivity and disease progression. Targeting LARAs by intervention with lipid transport and metabolism could thus provide new therapeutic options for drug-resistant TLE.

During cold exposure, activated brown adipose tissue (BAT) takes up a large amount of circulating glucose to fuel non-shivering thermogenesis and defend against hypothermia. However, little is known about the endocrine function of BAT controlling glucose homoeostasis under this thermoregulatory challenge. Here, we show that in male mice, activated BAT-derived extracellular vesicles (BDEVs) reprogram systemic glucose metabolism by promoting hepatic gluconeogenesis during cold stress. Cold exposure facilitates the selective packaging of miR-378a-3p—one of the BAT-enriched miRNAs—into EVs and delivery into the liver. BAT-derived miR-378a-3p enhances gluconeogenesis by targeting p110α. miR-378 KO mice display reduced hepatic gluconeogenesis during cold exposure, while restoration of miR-378a-3p in iBAT induces the expression of gluconeogenic genes in the liver. These findings provide a mechanistic understanding of BDEV-miRNA as stress-induced batokine to coordinate systemic glucose homoeostasis. This miR-378a-3p-mediated interorgan communication highlights a novel endocrine function of BAT in preventing hypoglycemia during cold stress.

Clinical and Translational MedicineVolume 12, Issue 1 e709 LETTER TO EDITOROpen Access Dysregulation of the miR-16-WWP1 signalling pathway leads to colorectal tumorigenesis Xiaorui Chen, Xiaorui Chen Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYi Zhao, Yi Zhao Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorQing Zhu, Qing Zhu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYanqing Liu, Yanqing Liu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYang Luo, Yang Luo Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorWei Cheng, Wei Cheng Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorBohan Zhang, Bohan Zhang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorKai Wang, Kai Wang Department of Gastrointestinal Surgery, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, ChinaSearch for more papers by this authorXiaohong Jiang, Xiaohong Jiang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorRui Liu, Rui Liu National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, ChinaSearch for more papers by this authorYanbo Wang, Corresponding Author Yanbo Wang ybwang@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorZhen Zhou, Corresponding Author Zhen Zhou zhenzhou@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorXi Chen, Corresponding Author Xi Chen xichen@nju.edu.cn orcid.org/0000-0002-5807-4219 Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China Research Unit of Extracellular RNA, Chinese Academy of Medical Sciences, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this author Xiaorui Chen, Xiaorui Chen Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYi Zhao, Yi Zhao Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorQing Zhu, Qing Zhu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYanqing Liu, Yanqing Liu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYang Luo, Yang Luo Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorWei Cheng, Wei Cheng Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorBohan Zhang, Bohan Zhang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorKai Wang, Kai Wang Department of Gastrointestinal Surgery, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, ChinaSearch for more papers by this authorXiaohong Jiang, Xiaohong Jiang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorRui Liu, Rui Liu National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, ChinaSearch for more papers by this authorYanbo Wang, Corresponding Author Yanbo Wang ybwang@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorZhen Zhou, Corresponding Author Zhen Zhou zhenzhou@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorXi Chen, Corresponding Author Xi Chen xichen@nju.edu.cn orcid.org/0000-0002-5807-4219 Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China Research Unit of Extracellular RNA, Chinese Academy of Medical Sciences, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this author First published: 26 January 2022 https://doi.org/10.1002/ctm2.709 Xiaorui Chen, Yi Zhao, and Qing Zhu contributed equally to this work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Dear Editor, Colorectal cancer (CRC) is one of the leading malignant tumour-related causes of death worldwide.1 WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a ubiquitin protein ligase2-4 and a potential oncogene in many cancer types.5-8 There is little work to explain the mechanisms by which WWP1 is regulated during tumorigenesis, particularly in CRC. We first determined the expression pattern of WWP1 by performing immunohistochemistry (IHC) in a commercial tissue microarray containing 90 pairs of CRC and adjacent normal tissue (ANT) specimens (Figure 1A; Table S1). The percentage of WWP1-positive cells was dramatically higher in CRC specimens compared with ANT specimens (Figure 1B,C). In addition, WWP1 protein levels were positively associated with clinical grades of CRC (Figure 1D). Kaplan–Meier survival analysis showed an increased risk of CRC-related deaths in patients with higher WWP1 protein levels (Figures 1E). We then confirmed WWP1 protein levels were consistently upregulated in 22 pairs of CRC specimens compared with ANT specimens (Figure 1F,G). FIGURE 1Open in figure viewerPowerPoint The expression pattern of WWP1 in colorectal cancer (CRC) tissues compared with adjacent normal tissue (ANT) specimens. (A) Image of immunohistochemistry (IHC) staining of WWP1 protein in human CRC tissue microarrays. (B) Representative images of IHC staining of WWP1 protein in CRC tissue microarrays. Scale bar, 100 μm. (C) IHC scores of WWP1 staining in 90 pairs of CRC and ANT specimens (n = 90 per group). (D) IHC scores of WWP1 staining in CRC tissue specimens with different grades (n = 10, 46 and 34, respectively). (E) Kaplan–Meier curves were generated to analyse the association of WWP1 protein levels in CRC tissue specimens with the overall survival of CRC patients. (F) Western blot analysis of the expression levels of WWP1 protein in 22 pairs of CRC and ANT specimens. (G) Densitometry analysis of the immunoblots from panel F (n = 22 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 To investigate whether WWP1 could regulate CRC cell proliferation and migration, we transfected a WWP1 overexpression plasmid into SW480 cells. As expected, a dose-dependent increase in WWP1 protein was observed following transfection with an increasing dose of the WWP1 plasmid; conversely, WWP1 protein expression was inhibited by the transfection of WWP1 siRNA (Figure 2A,B). Both the CCK-8 and EdU assays revealed that WWP1 siRNA could decrease the cell proliferation rate, while the WWP1 overexpression plasmid augmented it (Figure 2C–E). Likewise, transfection of WWP1 siRNA suppressed cell migration in transwell assay, while transfection of the WWP1 overexpression plasmid stimulated it (Figure 2F,G). FIGURE 2Open in figure viewerPowerPoint WWP1 functions as an oncogene and is conversely correlated with miR-16 in colorectal cancer (CRC). (A) Western blot analysis of WWP1 protein levels in SW480 cells transfected with control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid (0.5, 1.0 or 1.5 μg). (B) Densitometry analysis of the immunoblots from panel A (n = 3 per group). (C) The cell proliferation ability was analysed using the CCK-8 assay after the transfection of SW480 cells with equal doses of control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid (n = 3 per group). (D) The EdU proliferation assay was performed 24 h after the transfection of SW480 cells with equal doses of control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (E) Quantitative analysis of EdU-positive cells in panel D (n = 3 per group). (F) The cell migration ability was analysed using a transwell assay after the transfection of SW480 cells with equal doses of control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid. (G) Quantitative analysis of the cells that migrated to the bottom of the transwell membranes (n = 3 per group). (H) Quantitative RT-PCR analysis of the relative expression levels of WWP1 mRNA in CRC and ANT specimens (n = 22 per group). (I) Schematic description of the predicted duplexes formed by miR-16 and the 3′-UTR of WWP1 mRNA. The predicted free energy value of the hybrid and the seed recognition are indicated, and all nucleotides in this region are highly conserved across species. (J) Quantitative RT-PCR analysis of the relative expression levels of miR-16 in CRC and ANT specimens (n = 22 per group). (K) Pearson's correlation scatter plot of the fold changes of WWP1 protein and miR-16 in CRC samples. (L) Western blot analysis of WWP1 protein levels in SW480, HT29 and HCT116 cells transfected with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor. (M) The relative luciferase activity in 293T transfected with wild type or mutant WWP1 3′-UTR (n = 3 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 We explored how the WWP1 protein was regulated in CRC. No significant change in WWP1 mRNA levels and low correlation between WWP1 protein and mRNA levels were detected in the above-mentioned 22 pairs of CRC samples (Figure 2H; Figure S1), suggesting a posttranscriptional mechanism for the regulation of WWP1. Because miRNAs serve as vital posttranscriptional regulators in various cancers, we speculated that some miRNAs might target WWP1 in CRC. miR-16 was predicted to be an upstream regulator of WWP1 by two bioinformatic algorithms TargetScan and miRcode. The 3′-UTR of WWP1 possesses a conserved miR-16 binding site with a suitable value of minimum free energy of typical miRNA-target pairs (Figure 2I). Consistently, miR-16 levels were downregulated in the above-mentioned 22 pairs of CRC samples (Figure 2J). Pearson's correlation scatter plots confirmed the negative correlation between WWP1 protein and miR-16 (Figure 2K). We assessed the alteration of WWP1 protein in three CRC cell lines (SW480, HT29, and HCT116) by transfecting miR-16 mimic or inhibitor. As expected, the cellular level of miR-16 altered dramatically after transfection (Figure S2A). Consequently, WWP1 protein expression was markedly reduced by miR-16 transfection, while miR-16 inhibitor promoted WWP1 protein expression in CRC cells (Figure 2L; Figure S2B). However, the WWP1 mRNA level was not altered by miR-16 (Figure S2C). Furthermore, we determined whether miR-16 directly binds to the WWP1 3′-UTR via luciferase reporter assay. Luciferase reporter activity was dramatically altered when miR-16 level was changed while the mutated luciferase reporter was no longer affected by miR-16 (Figure 2M). We investigated the cellular phenotypes mediated by the miR-16-WWP1 axis. Cell proliferation rate was significantly reduced by miR-16 mimic, whereas miR-16 inhibitor markedly boosted proliferation (Figure 3A,C,F). The WWP1 overexpression plasmid was sufficient to rescue the suppression of cell proliferation by miR-16 (Figure 3B,D,F). Furthermore, miR-16 mimic markedly decreased cell migration ability, whereas miR-16 inhibitor increased the number of migrated cells (Figure 3E,G). WWP1 overexpression dramatically attenuated miR-16-mediated suppression on cell migration (Figure 3E,G). Likewise, miR-16 mimic decreased cell invasion; in contrast, miR-16 inhibition had an opposite effect on invasion (Figure S3). Thus, miR-16 may regulate proliferation, migration, and invasion in a WWP1-dependent manner. FIGURE 3Open in figure viewerPowerPoint The effects of miR-16 on WWP1 expression and function in vitro and in vivo. (A) Cell proliferation ability was analysed using the CCK-8 assay after the transfection of SW480 cells with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor (n = 3 per group). (B) Cell proliferation ability was analysed using the CCK-8 assay after the cotransfection of SW480 cells with control mimic + control plasmid, miR-16 mimic + control plasmid, control mimic + WWP1 overexpression plasmid, or miR-16 mimic + WWP1 overexpression plasmid (n = 3 per group). (C) The EdU proliferation assay was performed after the transfection of SW480 cells with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (D) The EdU proliferation assay was performed after the cotransfection with control mimic + control plasmid, miR-16 mimic + control plasmid, control mimic + WWP1 overexpression plasmid, or miR-16 mimic + WWP1 overexpression plasmid. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (E) Cell migration ability was analysed using a transwell assay after the transfection with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or were cotransfected with control mimic + control plasmid, miR-16 mimic + control plasmid, control mimic + WWP1 overexpression plasmid, or miR-16 mimic + WWP1 overexpression plasmid. (F) Quantitative analysis of EdU-positive cells in panels C and D (n = 3 per group). (G) Quantitative analysis of the cells that migrated to the bottom of the transwell membranes (n = 3 per group). (H) Representative images of the excised tumours. SW480 cells were infected with control LV or a lentivirus overexpressing miR-16 (LV-miR-16) or were cotransfected with LV-miR-16 and WWP1 overexpression plasmid. Then, the cells were implanted subcutaneously into four-week-old SCID male mice. Tumour growth was evaluated at day 24 after cell implantation. (I) Representative images of H&E-stained sections of xenografted tumours and representative images of IHC staining for WWP1 and Ki-67 in xenografted tumours. (J) Quantitative analysis of IHC staining for WWP1 and Ki-67 (n = 5 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 We investigated the effects of miR-16-WWP1 axis on CRC tumour growth in vivo. An SW480 cell line with stably knockdown of WWP1 by lentivirus (LV-shWWP1) was implanted into SCID mice. Additionally, SW480 cells infected with control lentivirus or miR-16 overexpression lentivirus (LV-miR-16) or co-infected with LV-miR-16 and the WWP1 overexpression vector were also implanted into SCID mice. Tumour growth was markedly decreased in the LV-shWWP1 group compared to the control lentivirus group (Figure S4A,B). Slower mitosis, reduced malignancy, lower level of WWP1, and the proliferation marker gene Ki-67 were observed in the LV-shWWP1 group by H&E staining and immunohistochemical staining (Figure S4C,D). LV-miR-16 clearly reduced tumour growth in vivo, whereas the WWP1 overexpression plasmid attenuated this effect (Figure 3H; Figure S5). Western blotting and immunohistochemical staining showed decreased WWP1 protein level in the LV-miR-16 group and rescued expression of WWP1 in the co-infection group (Figure 3I,J; Figure S6). H&E staining showed that LV-miR-16 caused slower mitosis and less malignancy, but the WWP1 overexpression plasmid neutralized this effect (Figure 3I). The Ki-67 level was decreased in tumours from the LV-miR-16 group but was restored in the co-infection group (Figure 3I,J). These results demonstrated that CRC tumour growth could be inhibited in vivo by miR-16 via targeting WWP1. Moreover, we investigated the relationship between miR-16 and another important regulatory miRNA of WWP1, miR-452, in CRC cells. miR-452 could target and repress WWP1 translation in two CRC cell lines (Figure 4A–C). However, miR-452 and miR-16 did not display synergism in vitro (Figure 4D–I). FIGURE 4Open in figure viewerPowerPoint The effects of miR-452 on WWP1 expression and function in colorectal cancer (CRC) cells. (A) Quantitative RT-PCR analysis of the relative expression levels of miR-452 in SW480 and HT29 cells transfected with control mimic, miR-452 mimic, control inhibitor or miR-452 inhibitor (n = 3 per group). (B) Western blot analysis of WWP1 protein levels in SW480 and HT29 cells transfected with control mimic, miR-452 mimic, control inhibitor or miR-452 inhibitor. (C) Densitometry analysis of the immunoblots of WWP1 protein from panel B (n = 3 per group). (D) Western blot analysis of WWP1 protein levels in SW480 and HT29 cells transfected with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or co-transfected with miR-16 mimic + miR-452 mimic or miR-16 inhibitor + miR-452 inhibitor. (E) Densitometry analysis of the immunoblots of WWP1 protein from panel D (n = 3 per group). (F) The Edu proliferation assay was performed after transfection with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or co-transfection with miR-16 mimic + miR-452 mimic or miR-16 inhibitor + miR-452 inhibitor. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (G) Cell migration ability was analysed using a transwell assay after transfected with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or co-transfected with miR-16 mimic + miR-452 mimic or miR-16 inhibitor + miR-452 inhibitor. (H) Quantitative analysis of EdU-positive cells in panel F (n = 3 per group). (I) Quantitative analysis of the cells that migrated to the bottom of the transwell membranes in panel G (n = 3 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 Overall, we showed that miR-16 can target WWP1 to suppress CRC tumorigenesis and that downregulation of miR-16 in CRC abolishes this repression of WWP1, resulting in more malignant tumour features. Our findings also have clinical relevance because targeting WWP1 protein with miR-16 has the potential to inhibit cellular proliferation and migration for CRC treatment. Indeed, the ubiquitin-proteasome system has become a popular target for developing therapeutics against tumors.9 Our results strongly support this promising strategy because overexpression of miR-16 or silencing of WWP1 strongly inhibits tumour growth in vivo. Future studies are needed to evaluate the therapeutic efficacy of WWP1 inhibition (e.g., with miR-16 or siRNA) in disrupting target protein ubiquitination and inducing antitumor activity. ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 32022015 and 31871295), the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (No. SN-ZJU-SIAS-008), the CAMS Innovation Fund for Medical Sciences (No. CIFMS-2021-I2M-5-015), and the Fundamental Research Funds for the Central Universities (No. 020814380146 and 020814380162). CONFLICT OF INTEREST The authors declare no conflict of interest. Supporting Information Filename Description ctm2709-sup-0001-SuppMat.docx35.7 KB SUPPORTING INFORMATION ctm2709-sup-0002-tablesS1.xlsx28.7 KB SUPPORTING INFORMATION ctm2709-sup-0003-FigureS1-S7.pdf1.1 MB SUPPORTING INFORMATION Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Siegel RL, Miller KD, Jemal A. 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CrossrefCASWeb of Science®Google Scholar Volume12, Issue1January 2022e709 FiguresReferencesRelatedInformation

Extracellular vesicles (EVs) play an important role in the communication between tissues and cells. However, it is difficult to screen and trace EVs secreted by specific tissues in vivo, which affects the functional study of EVs in certain tissues under pathophysiological conditions. In this study, a Cre-dependent CD63(flag)-EGFP co-expressed with mCherry protein system expressing mice was constructed, which can be used for the secretion, movement, and sorting of EVs from specific tissues in vivo. This mouse model is an ideal research tool for studying the secretion amount, target tissue, and functional molecule screening of EVs in specific tissues under different pathophysiological conditions. Moreover, it provides a new research method to clarify the mechanism of secreted EVs in the pathogenesis of the disease.

Aims Overconsumption of sugar-sweetened beverages (SSBs) is associated with an increased risk of metabolic disorders, including obesity and diabetes. However, accumulating evidence also suggests the potential negative impact of consuming nonnutritive sweeteners (NNSs) on weight and glycaemic control. The metabolic effects of sucralose, the most widely used NNS, remain controversial. This study aimed to compare the impact of intake of dietary sucralose (acceptable daily intake dose, ADI dose) and sucrose-sweetened water (at the same sweetness level) on lipid and glucose metabolism in male mice. Materials and methods Sucralose (0.1 mg/mL) or sucrose (60 mg/mL) was added to the drinking water of 8-week-old male C57BL/6 mice for 16 weeks, followed by oral glucose and intraperitoneal insulin tolerance tests, and measurements of bone mineral density, plasma lipids, and hormones. After the mice were sacrificed, the duodenum and ileum were used for examination of sweet taste receptors (STRs) and glucose transporters. Results A significant increase in fat mass was observed in the sucrose group of mice after 16 weeks of sweetened water drinking. Sucrose consumption also led to increased levels of plasma LDL, insulin, lipid deposition in the liver, and increased glucose intolerance in mice. Compared with the sucrose group, mice consuming sucralose showed much lower fat accumulation, hyperlipidaemia, liver steatosis, and glucose intolerance. In addition, the daily dose of sucralose only had a moderate effect on T1R2/3 in the intestine, without affecting glucose transporters and plasma insulin levels. Conclusion Compared with mice consuming sucrose-sweetened water, daily drinking of sucralose within the ADI dose had a much lower impact on glucose and lipid homeostasis.

Rationale: Targeting vascular smooth muscle cell (VSMC) phenotypic switching is a promising therapeutic approach for atherosclerosis. Dysregulation of PGC1 alpha (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), a key regulator of cellular energy metabolism, has been implicated in the pathogenesis of atherosclerosis, yet its role in atherosclerosis remains controversial. Objective: The current study aimed to determine whether and how PGC1 alpha in VSMCs regulates atherosclerosis progression. Methods and Results: We generated transgenic rabbits with SMC-specific PGC1 alpha overexpression and showed that these rabbits developed significantly less aortic atherosclerosis than their nontransgenic littermates after high-cholesterol diet feeding, while total plasma cholesterol levels were similar. As indicated by the restored expression of VSMC differentiation marker genes, the high-cholesterol diet-induced phenotypic switching in the aortic media was largely reversed in transgenic rabbits, accompanied by decreased levels of synthetic phenotype genes, proinflammatory cytokines, adhesion molecules, macrophage infiltration, MMPs (matrix metalloproteinases), reactive oxygen species production and senescence. Ex vivo studies further showed that VSMC-specific PGC1 alpha overexpression markedly suppressed the promotive effect of high-cholesterol diet feeding on the association of SRF (serum response factor) with ELK1 (ETS transcription factor ELK1), a TCF (ternary complex factor) that acts as a myogenic repressor in VSMCs, thereby preserving the VSMC contractile phenotype. Furthermore, knockdown of PGC1 alpha remarkably increased ERK (extracellular signal-regulated kinase)1/2-ELK-1 signaling, which promoted phenotypic switching and proliferation of cultured rabbit VSMCs. In addition, we showed that PGC1 alpha can regulate EGFR (epidermal growth factor receptor)-ERK1/2 MAPK (mitogen-activated protein kinase) signaling via modulating PPAR gamma (peroxisome proliferator-activated receptor gamma) activity in RVSMCs (rabbit vascular smooth muscle cells). Finally, we showed that these beneficial results of SMC-specific PGC1 alpha overexpression can be extrapolated from rabbits to human VSMCs and clinical settings. Conclusions: We demonstrated a critical role of PGC1 alpha in maintaining the contractile phenotype of VSMCs and highlighted the therapeutic potential of PGC1 alpha for atherosclerosis.

Abstract Secreted microRNAs (miRNAs) are novel endocrine factors that play essential pathological and physiological roles. Here, we report that pancreatic β cell‐released exosomal miR‐29 family members (miR‐29s) regulate hepatic insulin sensitivity and control glucose homeostasis. Cultured pancreatic islets were shown to secrete miR‐29s in response to high levels of free fatty acids (FFAs) in vitro. In vivo, high levels of FFAs, promoted by either high‐fat diet (HFD) feeding (physiopathological) or fasting (physiological), increased the secretion of miR‐29s into plasma. Intravenous administration of exosomal miR‐29s attenuated insulin sensitivity. The overexpression of miR‐29s in the β cells of transgenic (TG) mice promoted the secretion of miR‐29s and inhibited the insulin‐mediated suppression of glucose output in the liver. We used selective overexpression of traceable heterogenous mutant miR‐29s in β cells to confirm that islet‐derived exosomal miR‐29s target insulin signalling in the liver and blunt hepatic insulin sensitivity. Moreover, in vivo disruption of miR‐29s expression in β cells reversed HFD‐induced insulin resistance. In vitro experiments demonstrated that isolated exosomes enriched in miR‐29s inhibited insulin signalling in the liver and increased hepatic glucose production. These results unveil a novel β cell‐derived secretory signal—exosomal miR‐29s—and provide insight into the roles of miR‐29s in manipulating glucose homeostasis.

Oxaliplatin resistance is a major challenge in the clinical treatment for advanced colorectal cancer (CRC). Long non-coding RNAs (lncRNAs) are involved in tumorigenesis and progression as critical regulators, while their potential roles in chemoresistance are poorly understood. In this study, we report that the LINC00460-miR-149-5p/miR-150-5p-mutant p53 feedback loop is responsible for oxaliplatin resistance in CRC. First, LINC00460 was found to exhibit higher expression in oxaliplatin-resistant CRC (CRC/OxR) cells compared with parental oxaliplatin-sensitive ones, and this expression pattern depends on mutant p53 (SW480/OxR), not wild-type p53 (HCT116/OxR). Oxaliplatin-induced LINC00460 in SW480/OxR cells was mainly located in the cytoplasm and was associated with AGO2 protein. LINC00460 functions as a competing endogenous RNA (ceRNA) to promote oxaliplatin resistance through sequestering miR-149-5p/miR-150-5p and upregulating the expression of the microRNA (miRNA) target p53. Knockdown of LINC00460 sensitized SW480/OxR cells to oxaliplatin by modulating p53 in vitro and in vivo. In turn, mutant p53 positively regulated the expression of LINC00460, thus forming a feedback loop. Clinical data showed that LINC00460 was upregulated in CRC tissues compared with paired normal tissues and was significantly correlated with clinical stage and node (N) status. Our findings uncover a mechanism for the LINC00460-miR-149-5p/miR-150-5p-mutant p53 feedback loop in oxaliplatin resistance of CRC, and they provide potential therapeutic targets for tumor chemoresistance.