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T cells utilized in adoptive T cell immunotherapy are typically activated in vitro. Although these cells demonstrate proliferation and anti-tumor activity following activation, they often face difficulties in sustaining long-term survival post-reinfusion. This issue is attributed to the induction of T cells into a terminal differentiation state upon activation, whereas early-stage differentiated T cells exhibit enhanced proliferation potential and survival capabilities. In previous study, we delineated four T cell subsets at varying stages of differentiation: T N , T SCM , T CM , and T EM , and acquired their miRNA expression profiles via high-throughput sequencing. In the current study, we performed a differential analysis of miRNA across these subsets, identifying a distinct miRNA, hsa-miR-744-5p, characterized by progressively increasing expression levels upon T cell activation. This miRNA is not expressed in T SCM but is notably present in T EM . Target genes of miR-744-5p were predicted, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, revealing that these genes predominantly associate with pathways related to the 'Wnt signaling pathway'. We established that miR-744-5p directly targets STK11, influencing its expression. Further, we investigated the implications of miR-744-5p on T cell differentiation and functionality. Overexpression of miR-744-5p in T cells resulted in heightened apoptosis, reduced proliferation, an increased proportion of late-stage differentiated T cells, and elevated secretion of the cytokine TNF-α. Moreover, post-overexpression of miR-744-5p led to a marked decline in the expression of early-stage differentiation-associated genes in T cells (CCR7, CD62L, LEF1, BCL2) and a significant rise in late-stage differentiation-associated genes (KLRG1, PDCD1, GZMB). In conclusion, our findings affirm that miR-744-5p contributes to the progressive differentiation of T cells by downregulating the STK11 gene expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

With the rapid development of next-generation sequencing technology, several studies have shown that ncRNAs can act as competitive endogenous RNAs (ceRNAs) and are involved in various biological processes, such as proliferation, differentiation, apoptosis, and migration of breast cancer (BC) cells, and plays an important role in BC progression as a molecular target for its diagnosis, treatment, prognosis, and differentiation of subtypes and age groups of BC patients. Based on the description of ceRNA-related biological functions, this study screened and sorted the sequencing analysis and experimental verification conclusions of BC-related ceRNAs and found that the ncRNAs mediated ceRNA networks can promote the development of BC by promoting the expression of genes related to BC proliferation, drug resistance, and apoptosis, inducing the production of epithelial-mesenchymal transition (EMT) to promote metastasis and activating cancer-related signaling pathways., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The capacity of the liver to restore its architecture and function assures good prognoses of patients who suffer serious hepatic injury, cancer resection, or living donor liver transplantation. Only a few studies have shed light on the mechanisms involved in the termination stage of LR. Here, we attempt to further verify the role of the p53/miR-34a/SIRT1 positive feedback loop in the termination of liver regeneration and its possible relationship with liver cancer., Method: We performed partial hepatectomy (PH) in mice transfected with adenovirus (Ade) overexpressing P53 and adenovirus-associated virus (AAV) overexpressing miR-34a. LR was analyzed by liver weight/body weight, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and cell proliferation, and the related cellular signals were investigated. Bile acid (BA) levels during LR were analyzed by metabolomics of bile acids., Results: We found that the P53/miR-34a/SIRT1 positive feedback loop was activated in the late phase of LR. Overexpression of P53 or miR-34a terminated LR early and enhanced P53/miR-34a/SIRT1 positive feedback loop expression and its proapoptotic effect. T-β-MCA increased gradually during LR and peaked at 7 days after PH. T-β-MCA inhibited cell proliferation and promoted cell apoptosis via facilitating the P53/miR-34a/SIRT1 positive feedback loop during LR by suppressing FXR/SHP. The P53/miR-34a/SIRT1 positive feedback loop was abolished in HCC patients with P53 mutations., Conclusions: The P53/miR-34a/SIRT1 positive feedback loop plays an important role in the termination of LR. Our findings showed the molecular and metabolic mechanisms of LR termination and provide a potential therapeutic alternative for treating P53-wild-type HCC patients.

T cells used in immune cell therapy, represented by T cell receptor therapy (TCR-T), are usually activated and proliferated in vitro and are induced to a terminally differentiated phenotype, with limited viability after transfusion back into the body. T cells exhibited a robust proliferative potential and in vivo viability in the early stages of progressive differentiation. In this study, we identified microRNAs that regulate T cell differentiation. After microRNA sequencing of the four subsets: Naïve T cells (T N ), stem cell-like memory T cells (T SCM ), central memory T cells (T CM ）, and effector memory T cells (T EM ), miR-150 was identified as the most highly expressed miRNA among the four subsets and was lowly expressed in the T SCM cells. We predicted the target genes of miR-150 miRNA and performed Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes analyses. We observed that the target genes of miR-150 were enriched in pathways associated with T-cell differentiation. FOXP1 and RC3H1 were identified as key target genes of miR-150 in the regulation of T-cell function. We examined the effects of miR-150 on the differentiation and function of healthy donor T-cells. We observed that miR-150 overexpression promoted T-cell differentiation to effector T-cells and effector memory T-cells, enhanced apoptosis, inhibited cell proliferation and increased secretion of pro-inflammatory cytokines such as IFN-γ and TNF-α. In addition, the expressions of early differentiation-related genes (ACTN1, CERS6, BCL2, and EOMES), advanced differentiation-related genes (KLRG1), and effector-function-related genes (PRF1 and GZMB) were significantly decreased after overexpression of miR-150. Collectively, our results suggested that miR-150 can promote progressive differentiation of T cells and the downmodulation of miR-150 expression while performing adoptive immunotherapy may inhibit T-cell differentiation and increase the proliferative potential of T cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Licorice has previously been shown to affect gene expression in cells; however, the underlying mechanisms remain to be clarified. We analyzed the microRNA expression profile of serum from mice treated by gavage with licorice decoction, and obtained 11 differentially expressed microRNAs (DEmiRNAs). We also screened differentially expressed genes (DEgenes) based on RNA-Seq data, and 271 common genes were identified by intersection analysis of the predicted target genes of 11 DEmiRNAs and the DEgenes. The miRNA-gene network showed that most of the hub genes were immune-related. KEGG enrichment analysis of the 271 genes identified three significant pathways, and the 21 genes involved in these three pathways, and the 11 DEmiRNAs, were constructed into a miRNA pathway-target gene network, in which mmu-miR-27a-3p stood out. Compared to ImmPort, there were 13 immune genes within the above group of 21 genes, and three intersected with the mmu-miR-27a-3p predicted target genes, Cd28 , Grap2 and Cxcl12 , of which the expression of Cd28 changed most significantly. We confirmed the regulation of Cd28 by mmu-miR-27a-3p using a dual-luciferase assay, and further confirmed that overexpression of mmu-miR-27a-3p could significantly downregulate the expression of Cd28 in lymphocytes. These results indicate that mmu-miR-27a-3p could be involved in the licorice-mediated regulation of the expression of Cd28 in mice.

Aims: We previously showed that the protective effects of endothelial progenitor cells (EPCs)-released exosomes (EPC-EXs) on endothelium in diabetes. However, whether EPC-EXs are protective in diabetic ischemic stroke is unknown. Here, we investigated the effects of EPC-EXs on diabetic stroke mice and tested whether miR-126 enriched EPC-EXs (EPC-EXs miR126 ) have enhanced efficacy., Methods: The db/db mice subjected to ischemic stroke were intravenously administrated with EPC-EXs 2 hours after ischemic stroke. The infarct volume, cerebral microvascular density (MVD), cerebral blood flow (CBF), neurological function, angiogenesis and neurogenesis, and levels of cleaved caspase-3, miR-126, and VEGFR2 were measured on day 2 and 14., Results: We found that (a) injected EPC-EXs merged with brain endothelial cells, neurons, astrocytes, and microglia in the peri-infarct area; (b) EPC-EXs miR126 were more effective than EPC-EXs in decreasing infarct size and increasing CBF and MVD, and in promoting angiogenesis and neurogenesis as well as neurological functional recovery; (c) These effects were accompanied with downregulated cleaved caspase-3 on day 2 and vascular endothelial growth factor receptor 2 (VEGFR2) upregulation till day 14., Conclusion: Our results indicate that enrichment of miR126 enhanced the therapeutic efficacy of EPC-EXs on diabetic ischemic stroke by attenuating acute injury and promoting neurological function recovery., (© 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Introduction: Inhibiting apoptosis of trophoblasts in women with gestational diabetes mellitus (GDM) is expected to guarantee adequate nutrition for the fetus and avoid abortion. MiR-193b is one of the most downregulated miRNAs in GDM patients. However, less is known about the role of miR-193b in autophagy and apoptosis in GDM patients., Methods: We detected the expression of miR-193b in GDM patients. Then, we cultured human trophoblasts (HTR8 cells) with high glucose (HG) to simulate a diabetic environment in vitro, and further explored the effects of miR-193b on apoptosis and autophagy of HG-treated HTR8 cells., Results: The expression of miR-193b was significantly downregulated in the peripheral blood of GDM patients compared with healthy controls, and decreased miR-193b caused apparent autophagy and a substantially high apoptosis rate in HG-treated HTR8 cells. These effects were reversed by enhancing miR-193b expression or using the autophagy inhibitor 3-MA. Inhibiting miR-193b induced the pro-autophagic, cytostatic, and pro-apoptotic effects reduced by 3-MA in HTR8 cells upon HG treatment. Moreover, the expression of insulin-like growth factor-binding protein 5 (IGFBP5) was upregulated notably in the peripheral blood of GDM patients, and IGFBP5 appears to represent a direct miR-193b target. Note that silencing IGFBP5 blocked autophagy and apoptosis in HG-treated HTR8 cells, an effect that was diminished by inhibiting miR-193b., Conclusion: Our data indicate that aberrantly low expression of miR-193b in HG-induced trophoblasts results in massive apoptosis events by upregulating IGFBP5-induced autophagy, which may trigger GDM. Therefore, miR-193b may became a potential target for GDM therapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The ischemia/reperfusion (I/R) process in patients with ST-segment elevation myocardial infarction (STEMI) triggers an immune response, resulting in myocyte death. Krüppel-Like Factor 2 (KLF2), which is highly expressed in endothelial cells (ECs) under laminar flow, exerts anti-inflammatory effects. In this study, we explored the role of small extracellular vesicles (EVs) from KLF2-overexpressing ECs (KLF2-EVs) in the immunomodulation and its implications in myocardial I/R injury. Methods and Results: The small EVs were isolated from KLF2-overexpressing ECs' supernatant using gradient centrifugation. Mice were subjected to 45 min of ischemia followed by reperfusion, and KLF2-EVs were administrated through intravenous injection. KLF2-EVs ameliorated I/R injury and alleviated inflammation level in the serum and heart. We employed the macrophage depletion model and splenectomy and showed that Ly6C high monocyte recruitment from bone marrow was the main target of KLF2-EVs. miRNA-sequencing of KLF2-EVs and bioinformatics analysis implicated miRNA-24-3p (miR-24-3p) as a potent candidate mediator of monocyte recruitment and CCR2 as a downstream target. miR-24-3p mimic inhibited the migration of Ly6C high monocytes, and miR-24-3p antagomir reversed the effect of KLF2-EVs in myocardial I/R. Conclusion: Our data demonstrated that KLF2-EVs attenuated myocardial I/R injury in mice via shuttling miR-24-3p that restrained the Ly6C high monocyte recruitment. Thus, KLF2-EVs could be a potential therapeutic agent for myocardial I/R injury., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: To explore the mechanism of levonorgestrel (LNG)-ameliorating adenomyosis through long non-coding RNA H19 (lncRNA H19)/miR-17/Toll-like receptor 4 (TLR4) pathway., Patients and Methods: A total of 71 cases of adenomyosis and 54 cases of normal endometrium were sampled. Quantitative polymerase chain reaction (qPCR) was employed to quantify lncRNA H19, miR-17, and TLR4 mRNA, while Western blot (WB) was used to quantify TLR4 protein. Effects of LNG on normal endometrial stromal cells (ESCs) were evaluated. Suppression/over-expression vectors of lncRNA H19, miR-17, and TLR4 were constructed to observe their effects on ESCs., Results: MiR-17 and TLR4 mRNA were up-regulated and lncRNA H19 was down-regulated in adenomyosis. After LNG treatment, lncRNA H19 was up-regulated while miR-17 and TLR4 were down-regulated. LNG, up-regulation of lncRNA H19, and down-regulation of miR-17 and TLR4 portend increased apoptosis, G1-arrested cells, as well as inhibited inflammation. Dual-luciferase reporter (DLR) assay conformed the targeting relation of lncRNA H19/miR-17/TLR4 pathway., Conclusion: LNG ameliorates adenomyosis via lncRNA H19/miR-17/TLR4 pathway., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Liang et al.)

Exosomes (EXs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. We have recently revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived EXs (cEPC-EXs) on protecting endothelial cells against hypoxia injury. However, the relationship between the changes of cEPC-EXs and the effects of exercise on ischemic stroke (IS) is unknown. Here, we investigated whether exercise-regulated EPC-EXs contribute to the beneficial effects of exercise on IS. C57BL/6 mice received moderate treadmill exercise (10 m/min) for 4-wks and then were subjected to middle cerebral artery occlusion (MCAO) stroke. The neurologic deficit score (NDS), infarct volume, microvessel density, cell apoptosis, angiogenesis/neurogenesis, sensorimotor functions were determined on day 2 (acute stage) and/or day 28 (chronic stage) post-stroke. The miR-126 and EPC-EX levels were analyzed by RT-PCR or nanoparticle tracking analysis combined with microbeads and used for correlation analyses. The function of EPC-EXs from exercised mice was detected in a hypoxia neuron model. Cell apoptosis, axon growth ability and gene expressions (cas-3 and Akt) were measured. Our data showed that: i) On day 2, exercised mice had decreased NDS and infarct volume, reduced cell apoptosis rate and cleaved cas-3 level, and a higher microvessel density than those in control (no-exercise) mice. The levels of EPC-EXs in plasma and brain tissue were raised and positively correlated in exercised mice. Meanwhile, the miR-126 level in cEPC-EXs and in ischemic tissue were upregulated in exercised mice. The EPC-EXs and their carried miR-126 levels negatively correlated with the infarct volume and cell apoptosis, whereas positively correlated with microvessel density. In addition, cEPC-EXs from exercised mice elicited protective effects on neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by miR-126 and PI3k inhibitors in vitro. ii) On day 28, exercised mice had less infarct volume, higher microvessel density, angiogenesis/neurogenesis and better sensorimotor functions. The levels of BDNF, p-TrkB/TrkB and p-Akt/Akt were upregulated in the brain of exercised mice. These recovery indexes correlated with the levels of cEPC-EXs and their miR-126. In conclusion, our data suggest that moderate exercise intervention has protective effects on the brain against MCAO-induced ischemic injury in both acute and chronic stages which might via the release of miR-126 enriched EPC-EXs., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Breast cancer (BC) remains a serious health threat for women due to its high incidence and the trend of rejuvenation. Accumulating evidence has highlighted that microRNAs (miRNAs) and messenger RNAs (mRNAs) could play important roles in various biological processes involved in the pathogenesis of BC. The present study aimed to identify potential prognostic biomarkers associated with BC., Methods: Here, original gene expression profiles of patients with BC was downloaded from The Cancer Genome Atlas (TCGA) database. TargetScan, miRDB, and miRTarBase databases were used to predict the target genes of prognostic-related differentially expressed miRNAs (DEMs). Subsequently, functional enrichment analysis and topological analysis were performed on the overlaps of target genes and differentially expressed mRNAs (DEGs), and Kaplan-Meier analysis was used to predict prognosis-related target genes to identify prognostic biomarkers., Results: A total of 218 DEMs and 2222 DEGs were extracted in which eight miRNAs were associated with prognosis, and 278 target DEGs were screened out incorporated into functional enrichment analysis and protein-protein interaction network visualization studies. Additionally, five hub genes (CXCL12, IGF1, LEF1, MMP1, and RACGAP1) were observed as potential biomarkers for BC prognosis through survival analysis., Conclusion: We performed a distinctive correlation analysis of miRNA-mRNA in BC patients, and identified eight miRNAs and five hub genes may be effective biomarkers for the prognosis of BC patients., (© 2019 Wiley Periodicals, Inc.)

Efficient and targeted delivery approach to transfer exogenous genes into macrophages is still a great challenge. Current gene delivery methods often result in low cellular uptake efficiency in vivo in some types of cells, especially for the Kupffer cells (KCs). In this article, we demonstrate that amphiphilic core-shell nanoparticles (NPs) consisting of well-defined hydrophobic poly(methyl methacrylate) (PMMA) cores and branched polyethyleneimine (PEI) shells (denoted as PEI@PMMA NPs) are efficient nanocarriers to deliver microRNA (miRNA)-loaded plasmid to the KCs. Average hydrodynamic diameter of PEI@ PMMA NPs was 279 nm with a narrow size distribution. The NPs also possessed positive surface charges up to +30 mV in water, thus enabling effective condensation of negatively charged plasmid DNA. Gel electrophoresis assay showed that the resultant PEI@PMMA NPs were able to completely condense miRNA plasmid at a weight ratio of 25:1 (N/P ratio equal to 45:1). The Cell Counting Kit-8 assay and flow cytometry results showed that the PEI@PMMA/miRNA NPs displayed low cytotoxicity and cell apoptosis activity against the KCs. The maximum cell transfection efficiency reached 34.7% after 48 hours, which is much higher than that obtained by using the commercial Lipofectamine™ 2000 (1.7%). Bio-transmission electron microscope observation revealed that the PEI@PMMA NPs were mainly distributed in the cytoplasm of the KCs. Furthermore, when compared to the control groups, the protein expression of target nuclear factor κB P65 was considerably inhibited (P<0.05) both in vitro and in vivo. These results demonstrate that the PEI@PMMA NPs with a unique amphiphilic core-shell nanostructure are promising nanocarriers for delivering miRNA plasmid to KCs.

MicroRNAs (miRNAs) are classes of small, non-coding RNAs that regulate the translation of target mRNA transcripts. In this study, we demonstrated that miR-592 was downregulated in human hepatocellular carcinoma (HCC) and could suppress growth of the human HCC cell line HepG2. A tumor oncogene, DEK, was identified as a direct target of miR-592. Luciferase report assay indicated miR-592 regulates DEK expression though bind to its 3'UTR. Furthermore, knockdown of DEK also suppressed cell proliferation of HepG2 cells, which was consist with miR-592. At last, we suggested that DEK was upregulated in HCC tissues inversely with miR-592. These results demonstrated that miR-592 targets DEK transcript and suppresses HCC cell growth, and may provide potential therapeutic target in human HCC.

Objective: To test a novel gene carrier, named polyethyleneimine nanogel particles (PEI-NP), for delivering the mircroRNA (miRNA) into Kupffer cells (KCs) for silencing the expression of nuclear transcription factor κB (NF-κB) P65., Methods: The capacity of PEI-NPs to carry miRNA was tested by gel electrophoresis. RAW264.7 cell line was transfected with the cationic polymers which mixed together PEI-NP and miRNA. Then, the cytotoxicity of the RAW264.7 cells was detected by cell counting kit-8 (CCK-8); the cell apoptosis and transfection efficiency were analyzed by flow cytometry combined with annexin V-FITC/PI staining; the gene and protein levels of NF-κB P65 were determined respectively by real-time quantitative PCR (qRT-PCR) and Western blotting. The distribution of the cationic polymers in RAW264.7 cells and liver KCs was observed by transmission electron microscope (TEM). The protein level of NF-κB P65 in KCs was detected by immunohistochemistry., Results: PEI-NPs were able to completely combine with miRNA at the least mass ratio of 20:1. The highest transfection efficiency of 48 hours was (33.63±1.94)% in vitro. The protein level of NF-κB P65 at 48 hours of PEI-NP/miRNA transfection into RAW264.7 cells was significantly inhibited. TEM showed the cationic polymers in RAW264.7 cytoplasm and only in KCs of liver tissue after transfection. Immunohistochemistry indicated that the protein expression of NF-κB P65 in KCs was significantly lower than that in control group., Conclusion: Macrophages and KCs were successfully transfected with the cationic polymers in vitro and in vivo, respectively. The expression of NF-κB P65 was knocked down significantly after transfection.

Coronary artery disease (CAD) is the most common cardiovascular disease worldwide. In this study, we investigated the pathogenesis of CAD. We downloaded the GSE98583 dataset, including 12 CAD samples and 6 normal samples, from the Gene Expression Omnibus (GEO) database and screened differentially expressed genes (DEGs) in CAD versus normal samples. Next, we performed functional enrichment analysis, protein-protein interaction (PPI) network, and functional module analyses to explore potential functions and regulatory functions of identified DEGs. Next, transcription factors (TFs) and microRNAs (miRNAs) targeting DEGs were predicted. In total, 456 DEGs were identified in CAD and normal samples, including 175 upregulated and 281 downregulated genes. These genes were enriched in the intestinal immune network for immunoglobulin A production and the mitogen-activated protein kinase signaling pathway (e.g., TGFBR2 and EGF). The PPI network contained 212 genes, and HIST1H2BJ, HIST1H2AC, EGF, and EP300 were hub genes with degrees higher than 10. Four significant modules were identified from the PPI network, with genes in the modules mainly enriched in the inflammatory response, protein ubiquitination involved in ubiquitin-dependent protein catabolic processes, protein transport, and mitochondrial translational elongation, respectively. Two TFs (E2F1 and FOXK1) and five miRNAs (miR-122A, miR-516-5P, miR-507, miR-342, and miR-520F) were predicted to target 112 DEGs. miR-122A reportedly targets both LRP10 and IQGAP1 in the TF-miRNA target regulatory network. The abnormal expression of TGFBR2, EGF, LRP10, and IQGAP1 may be implicated in CAD pathogenesis. Our study provides targets and potential regulators for investigating CAD pathogenesis. [ABSTRACT FROM AUTHOR]