Inhibition Of miR-21 Research Articles

Knockdown of miR-182 changes the sensitivity of triple-negative breast cancer cells to cisplatin

Breast cancer is the most common malignancy that affects women. MicroRNAs (miRNAs) play an essential role in cancer therapy and regulate many biological processes such as cisplatin resistance. The study’s objective was to determine whether miR-182 dysregulation was the cause of cisplatin resistance in TNBC cell line MDA-MB-231. To determine the expression of miR-182, PCR was performed with primers specific to miR-182, and agarose gel electrophoresis was performed. To reduce the expression of miR-182 in MDA-MB-231 cells, anti-miR-182 oligonucleotides were used. RT-qPCR was used to confirm knockdown. The knockdown and control groups were treated with cisplatin at the same time. Propidium iodide (PI) and Annexin V staining were performed for apoptosis assay. Flow cytometric analysis was used to investigate the effect of miR-182 knockdown on cell cycle arrest. In comparison to untreated control MDA-MB-231 cells with MDA-MB-231 cells treated with anti-miR-182, there was a significant increase in the cisplatin-induced early apoptosis phase (p = 0.023). Also, inhibition of miR-182 significantly increased the cell cycle arrest at the G2/M phase in MDA-MB-231 cells (p = 0.031). Our results revealed that miR-182 inhibition may play a role in the overcoming of cisplatin resistance by inducing apoptosis and, cell cycle arrest in TNBC.

MicroRNA-155 Inhibition Activates Wnt/β-catenin Signaling to Restore Th17/Treg Balance and Protect against Acute Ischemic Stroke.

Acute ischemic stroke (AIS) is a dangerous neurological disease associated with an imbalance in Th17/Treg cells and abnormal activation of the Wnt/β-catenin signaling pathway. This study aims to investigate whether inhibition of miR-155 can activate the Wnt/β-catenin signaling pathway to improve Th17/Treg imbalance and provide neuroprotective effects against stroke. We employed a multi-level experimental design. Firstly, we analyzed the differential gene expression between the miR-155 antagomir-treated group and the control group using high-throughput sequencing to identify potential target genes. Subsequently, we conducted functional and pathway enrichment analysis of the differentially expressed genes using bioinformatics tools. Next, we performed in vivo animal experiments using a mouse model to validate the impact of miR-155 antagomir treatment on the Wnt/β-catenin signaling pathway and improvement of Th17/Treg cell ratios. Lastly, we conducted in vitro cell experiments to validate our findings further. High-throughput sequencing results showed significant differential expression between the miR-155 antagomir-treated group and the control group (BioProject: PRJNA1152758, SRA IDs: SRR30410532, SRR30410531, SRR30410530 for the disease group; SRR30410529, SRR30410528, SRR30410527 for the control group). Bioinformatics analysis revealed potential target genes associated with the Wnt/β-catenin signaling pathway and Th17/Treg cell imbalance. In vitro experiments demonstrated that miR-155 antagomir treatment significantly activated the Wnt/β-catenin signaling pathway and improved Th17/Treg cell ratios. In vivo, animal experiment results indicated that miR-155 antagomir treatment exhibited significant neuroprotective effects against AIS. This study demonstrates that miR-155 antagomir can improve Th17/Treg cell imbalance by activating the Wnt/β-catenin signaling pathway and exhibiting neuroprotective effects against AIS in a mouse model. These findings provide crucial support for miR-155 as a potential therapeutic strategy for stroke and lay the foundation for further research.Significance Statement This study identifies miR-155 as a pivotal regulator of the Th17/Treg cell balance and Wnt/β-catenin signaling pathway in AIS. By inhibiting miR-155, we demonstrate the potential to enhance neuroprotection and modulate immune responses, offering a promising therapeutic avenue for stroke management. These findings contribute to the growing understanding of molecular mechanisms in stroke and provide a foundation for developing miR-155-targeted therapies.

Loc646329 sponges miR-21 to reduce RAS/MAP kinase signaling pathway in oral squamous cell carcinoma (OSCC).

Oral squamous cell carcinoma (OSCC) is a prevalent and aggressive form of head and neck cancer. Emerging evidence suggests that microRNAs (miRNAs) play a crucial role in the development and progression of OSCC. In particular, miR-21 has been implicated in various cellular processes, including proliferation, apoptosis, and invasion; it could be a potential therapeutic target for OSCC. In this study, The Cancer Genome Atlas (TCGA) for OSCC (TCGA-OSCC) clinical information was used. Patient outcomes were analyzed through survival analysis using Kaplan-Meier curves and log-rank tests. The HSC-3cell line was used as the OSCC cell line, which is known for its aggressive characteristics. The expression of Loc646329, miR-21, PTEN, PDCD4, and SPRY2 was evaluated by RT-PCR, flow cytometry, and scratch assay. Also, the effect of Loc646329 expression on OSCC was evaluated using the in vivo Xenograft Mouse Model. The results showed that overexpression of Loc646329 leads to the downregulation of miR-21, PDCD4, and SPRY2 genes while the PTEN gene is upregulated. In other words, the overexpression of Loc646329 by miR-21 inhibition (thorough targeting RAS/MAPK pathway) induces apoptosis and stops the cell cycle in OSCC cells. In vivo tests showed that elevated Loc646329 expression was linked to positive pathological outcomes, such as smaller tumor size, reduced lymph node metastasis, and enhanced overall survival in OSCC patients. In conclusion, the identification of Loc646329 as a sponge for miR-21 and its impact on the RAS/MAPK signaling pathway offers new opportunities for the development of innovative therapeutic strategies for OSCC. Further investigations into this regulatory axis may uncover additional targets for precision medicine approaches in the treatment of OSCC.

MiR-21 suppression in macrophages promotes M2-like polarization and attenuates kidney ischemia-reperfusion injury.

MicroRNA-21 (miR-21) is one of the most frequently upregulated miRNAs in response to kidney ischemia-reperfusion (IR) injury, exhibiting both protective and pathogenic effects depending on the cell type, disease state, and target signaling. In this study, we analyzed the function of miR-21 in various cell types to elucidate its role in ischemia-induced inflammation and acute kidney injury (AKI). Utilizing a mouse model of IR injury, we observed significant upregulation of miR-21 in renal tubular epithelial cells and macrophages following IR. Deletion of miR-21 in macrophages mitigated IR-induced pro-inflammatory cytokine production and AKI. However, conditional deletion of miR-21 in proximal tubules or nonproximal tubules did not protect the kidneys against these effects. Mechanistically, miR-21 inhibition promoted M2-like polarization in macrophages and suppressed M1-like polarization and proinflammatory cytokine production in kidneys. Invitro, miR-21 knockdown in the mouse macrophage cell line Raw246.7 or genetic deletion of miR-21 in bone marrow-derived macrophages (BMDMs) increased the percentage of CD206+ cells (M2 phenotype) while decreasing the percentage of CD86+ cells (M1 phenotype) and the expressions of proinflammatory cytokines. Overexpression of miR-21 in Raw264.7 cells reduced the percentage of CD206+ cells. Furthermore, we demonstrated that signal transducer and activator of transcription 3 (STAT3) was a target gene of miR-21 in macrophages, and miR-21 deletion promoted M2 macrophage polarization via STAT3 activation. In conclusion, miR-21 plays a role in regulating macrophage polarization, and the blockade of miR-21/STAT3 signaling may represent a novel therapeutic strategy for the prevention or treatment of AKI.

Red ginger confers antioxidant activity, inhibits lipid and sugar metabolic enzymes, and downregulates miR-21/132 expression

Ginger is a spice and medicinal plant with several varieties. This study aimed to understand the antioxidant, antidiabetic, and antiobesity properties of red ginger (RG) (Zingiber officinale var. rubrum), through pharmacoinformatics coupled with in vitro studies. Additionally, the suppression of miR-21/132 expression by RG was studied. Two RG extracts were sequentially produced using hexane (RGH) and ethanol (RGE) and characterized using UHPLC-Q-Orbitrap HRMS-based untargeted metabolomics analysis. Seven compounds identified in RGE and six in RGH were subjected to molecular docking tests on iNOS, lipase, α-glucosidase, α-amylase, and FTO protein receptors. Overall, 5,7-dihydroxy-2-(4-hydroxyphenyl)-6,8-bis(3,4,5-trihydroxyoxan-2-yl)-4H-chromen-4-one and pheophorbide A from RGE, and nictoflorin and rutin from RGH showed superior binding to most receptors. In vitro studies confirmed the ability of both RGE and RGH extracts to scavenge DPPH and ABTS radicals; inhibit activities of three metabolic enzymes, lipase (EC50 85.58 and 105.50 μg/mL), α-glucosidase (EC50 of 92.56 and 106.20 μg/mL), and α-amylase (EC50 of 96.60 and 111.80 μg/mL). Ex vivo RGE and RGH considerably suppressed protein expression associated with obesity, diabetes, and oxidative stress, including miR-21/132. This presents new insights into the molecular mechanism of RG in combating metabolic syndrome; however, further in vivo and clinical trials are needed to validate these findings.

MiR-155 promotes m6A modification of SOX2 mRNA through targeted regulation of HIF-1α and delays wound healing in diabetic foot ulcer in vitro models.

Diabetic foot ulcers (DFU) are one of the most destructive complications of diabetes mellitus. The aim of this study was to link miR-155 and SOX2 with DFU to explore the regulation of wound healing by DFU and its potential mechanism. Human keratinocytes (HaCaT) were induced with advanced glycation end products (AGEs) to construct DFU models in vitro. AGE-induced HaCaTcells were subjected to CCK-8 assays, flow cytometry, and wound healing assays to evaluate cell proliferation, apoptosis, and migration capacity, respectively. RT-qPCR and Western blotting were used to determine gene and protein expression levels, respectively. N6-methyladenosine (M6A) levels in total RNA were assessed using an M6A methylation quantification kit. Our results suggested that the inhibition of miR-155 promoted wound healing in an in vitro DFU model, while the knockdown of HIF-1α reversed this process, and that HIF-1α was a target protein of miR-155. In addition, knockdown of HIF-1α promoted the m6A level of SOX2 mRNA, inhibited the expression of SOX2, and inhibited the activation of the EGFR/MEK/ERK signaling pathway, thus inhibiting the proliferation and migration of HaCaTcells and promoting the apoptosis of HaCaTcells, while overexpression of SOX2 reversed this effect. We also found that METTL3 knockdown had the opposite effect of HIF-1α knockdown. Inhibition of miR-155 promoted the expression of HIF-1α and attenuated the m6A modification of SOX2 mRNA, thereby promoting the expression of SOX2 and activating the downstream EGFR/MEK/ERK signaling pathway to promote wound healing in an in vitro DFU model.

Circ-ADAM9 Knockdown Reduces Insulin Resistance and Placental Injury in Diabetic Mice via MAPK Pathway Inactivation.

Gestational diabetes mellitus (GDM) significantly risks maternal and neonatal health. Circular RNAs (circRNAs) regulate various diseases but their role in GDM is unclear. We investigated the involvement of circ-ADAM9 in GDM. We analyzed circ-ADAM9 expression in GDM-related microarray data (GSE182737) and measured its levels in the blood of GDM patients. In a high-fat diet-induced GDM mouse model, we inhibited circ-ADAM9 expression and tracked blood glucose levels, serum insulin, lipid levels, placental apoptosis, and reactive oxygen species (ROS) levels. Pathological changes in pancreatic tissues and fetal outcomes were also examined. Molecular interactions were explored using bioinformatics tools and validated through luciferase assays, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting in high glucose (HG)-induced human trophoblast cells (HTR-8/SVneo). We further investigated the involvement of circ-ADAM9/miR-375/FPR2 axis in HG-induced injury in HTR-8/SVneo cells by assessing cell viability, apoptosis, ROS production, and antioxidant levels. Both GDM patients and GDM-induced mice exhibited a substantial upregulation of circ-ADAM9. Knockdown of circ-ADAM9 lowered blood glucose, alleviated insulin resistance, improved lipid metabolism, decreased placental apoptosis and ROS levels, and reduced pancreatic lesions in GDM mice. Circ-ADAM9 downregulation also improved fetal viability, weight, and crown-rump length. In HG-induced HTR-8/SVneo cells, circ-ADAM9 overexpression and miR-375 downregulation were evident. Overexpression of miR-375 inhibited circ-ADAM9 activity, substantiating their binding interaction. In GDM mice, circ-ADAM9 deficiency restored miR-375 expression. TargetScanHuman predicted and luciferase assays confirmed the miR-375-FPR2 interaction and elevated FPR2 levels in GDM mice were reduced by circ-ADAM9 silencing. In HG-induced HTR-8/SVneo cells, circ-ADAM9 knockdown restored cell viability, suppressed apoptosis and ROS levels, and enhanced antioxidant enzyme levels. These effects were reversed by miR-375 inhibition or FPR2 overexpression, suggesting circ-ADAM9 upregulates FPR2 expression by sponging miR-375 and modulating the MAPK pathway. This study is the first to demonstrate the expression and function of circ-ADAM9 in the progression of GDM. Circ-ADAM9 downregulation ameliorates insulin resistance and placental injury in GDM by modulating the miR-375/FPR2 axis and inactivating the MAPK pathway, which may offer a novel therapeutic target for the treatment of GDM.

Tectorigenin Inhibits Glycolysis-induced Cell Growth and Proliferation by Modulating LncRNA CCAT2/miR-145 Pathway in Colorectal Cancer.

Colorectal cancer (CRC) places a heavy burden on global health. Tectorigenin (Tec) is a type of flavonoid-based compound obtained from the Chinese medical herb Leopard Lily Rhizome. It was found to exhibit remarkable anti-tumor properties in previous studies. However, the effect and molecular mechanisms of Tec in colorectal cancer have not been reported. The objective of this study was to explore the action of Tec in proliferation and glycolysis in CRC and the potential mechanism with regard to the long non-coding RNA (lncRNA) CCAT2/micro RNA-145(miR-145) pathway in vitro and in vivo . The anti-tumor effect of Tec in CRC was examined in cell and animal studies, applying Cell Counting Kit-8 (CCK-8) assay as well as xenograft model experiments. Assay kits were utilized to detect glucose consumption and lactate production in the supernatant of cells and animal serum. The expression of the glycolysis-related proteins was assessed by Western Blotting, and levels of lncRNA CCAT2 and miR-145 in CRC tissue specimens and cells were assessed by realtime quantitative PCR (RT-qPCR). Tec significantly suppressed cell glycolysis and proliferative rate in CRC cells. It could decrease lncRNA CCAT2 in CRC cells but increase the expression of miR-145. LncRNA CCAT2 overexpression or inhibition of miR-145 could abolish the inhibitive effects of Tec on the proliferation and glycolysis of CRC cells. The miR-145 mimic rescued the increased cell viability and glycolysis levels caused by lncRNA CCAT2 overexpression. Tec significantly inhibited the growth and glycolysis of CRC xenograft tumor. The expression of lncRNA CCAT2 decreased while the expression of miR-145 increased after Tec treatment in vivo. Tec can inhibit the proliferation and glycolysis of CRC cells through the lncRNA CCAT2/miR-145 axis. Altogether, the potential targets discovered in this research are of great significance for CRC treatment and new drug development.

RGLS8429-Mediated miR-17 Inhibition Leads to Acute PKD1/2 De-repression and Ameliorates Preclinical ADPKD

RGLS8429-Mediated miR-17 Inhibition Leads to Acute PKD1/2 De-repression and Ameliorates Preclinical ADPKD

DJ-1 regulates astrocyte activation through miR-155/SHP-1 signaling in cerebral ischemia/reperfusion injury.

Reactive astrocyte activation in the context of cerebral ischemia/reperfusion (I/R) injury gives rise to two distinct subtypes: the neurotoxic A1 type and the neuroprotective A2 type. DJ-1 (Parkinson disease protein 7, PARK7), originally identified as a Parkinson's disease-associated protein, is a multifunctional anti-oxidative stress protein with molecular chaperone and signaling functions. SHP-1 (Src homology 2 domain-containing phosphatase-1) is a protein tyrosine phosphatase closely associated with cellular signal transduction. miR-155 is a microRNA that participates in cellular functions by regulating gene expression. Recent studies have uncovered the relationship between DJ-1 and astrocyte-mediated neuroprotection, which may be related to its antioxidant properties and regulation of signaling molecules such as SHP-1. Furthermore, miR-155 may exert its effects by influencing SHP-1, providing a potential perspective for understanding the molecular mechanisms of stroke. A middle cerebral artery occlusion/reperfusion (MCAO/R) model and an oxygen-glucose deprivation/reperfusion (OGD/R) model were established to simulate focal cerebral I/R injury invivo and invitro, respectively. The invivo interaction between DJ-1 and SHP-1 has been experimentally validated through immunoprecipitation. Overexpression of DJ-1 attenuates I/R injury and suppresses miR-155 expression. In addition, inhibition of miR-155 upregulates SHP-1 expression and modulates astrocyte activation phenotype. These findings suggest that DJ-1 mediates astrocyte activation via the miR-155/SHP-1 pathway, playing a pivotal role in the pathogenesis of cerebral ischemia-reperfusion injury. Our results provide a potential way for exploring the pathogenesis of ischemic stroke and present promising targets for pharmacological intervention.

Inhibition of MiR-155 Using Exosomal Delivery of Antagomir Can Up-Regulate PTEN in Triple Negative Breast Cancer.

The most aggressive form of breast cancer (BC) is Triple-Negative BC (TNBC), with the poorest prognosis, accounting for nearly 15% of all cases. Since there is no effective treatment, novel strategies, especially targeted therapies, are essential to treat TNBC. Exosomes are nano-sized microvesicles derived from cells and transport various intracellular cargoes, including microRNAs (miRNAs). MiRNAs, small non-coding RNA, are an influential factor in the development of cancerous transformations in cells. Bioinformatics analysis of genes related to TNBC revealed that PTEN plays a crucial role in the disease. Relative expression of this gene was analyzed with RT-qPCR in 14 TNBC clinical samples. Electroporation was used to load miRNA antagomir into exosomes extracted from the conditioned medium. Then, the expression of miR-155 and PTEN was evaluated in MDA-MB-231 cells treated with antagomir-loaded exosomes. Based on the bioinformatics analysis, miR-155 is a potent inhibitor of PTEN. Following treatment with antagomir-loaded exosomes, RT-qPCR showed significantly reduced miR- 155 and increased PTEN levels in MDA-MB-231 cells. Based on the results of this study, exosomes can be effectively used as a cargo of oligonucleotides like miRNA mimics and antagomirs in targeted therapies.

MiR-206 Suppresses Triacylglycerol Accumulation via Fatty Acid Elongase 6 in Dairy Cow Mammary Epithelial Cells.

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA-mRNA networks.

Fecal let-7b and miR-21 directly modulate the intestinal microbiota, driving chronic inflammation

ABSTRACT Inflammatory bowel diseases (IBD) etiology is multifactorial. Luminal microRNAs (miRNAs) have been suspected to play a role in the promotion of chronic inflammation, but the extent to which fecal miRNAs are interacting with the intestinal ecosystem in a way that contribute to diseases, including IBD, remains unknown. Here, fecal let-7b and miR-21 were found elevated, associated with inflammation, and correlating with multiple bacteria in IBD patients and IL-10–/– mice, model of spontaneous colitis. Using an in vitro microbiota modeling system, we revealed that these two miRNAs can directly modify the composition and function of complex human microbiota, increasing their proinflammatory potential. In vivo investigations revealed that luminal increase of let-7b drastically alters the intestinal microbiota and enhances macrophages’ associated proinflammatory cytokines (TNF, IL-6, and IL-1β). Such proinflammatory effects are resilient and dependent on the bacterial presence. Moreover, we identified that besides impairing the intestinal barrier function, miR-21 increases myeloperoxidase and antimicrobial peptides secretion, causing intestinal dysbiosis. More importantly, in vivo inhibition of let-7b and miR-21 with anti-miRNAs significantly improved the intestinal mucosal barrier function and promoted a healthier host-microbiota interaction in the intestinal lining, which altogether conferred protection against colitis. In summary, we provide evidence of the functional significance of fecal miRNAs in host-microbiota communication, highlighting their therapeutic potential in intestinal inflammation and dysbiosis-related conditions, such as IBD.

MiR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including β-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

Silencing miR-155–5p expression improves intestinal damage through inhibiting inflammation and ferroptosis in necrotizing enterocolitis

BackgroundNecrotizing enterocolitis (NEC) is a condition characterized by acquired damage to the mucosal lining, predominantly affecting premature infants. Bioinformatics assessments uncovered a notable rise in miR-155–5p expression in the intestinal tissues of infants suffering from NEC. Nevertheless, the development of NEC's underlying mechanisms and the role of miR-155–5p are still not well understood. This research aimed to explore the role of miR-155–5p in NEC and to elucidate its underlying mechanisms. MethodsTo replicate NEC in vitro, lipopolysaccharide (LPS) was employed, whereas an in vivo rat model of NEC was established using formula feeding and exposure to hypoxia. Subsequently, levels of inflammatory cytokines, cell survival, and apoptosis rates were assessed. Various biochemical indicators such as glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured utilizing a purchased diagnostic kit. For the assessment of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) within FHC cells, analysis by flow cytometry was conducted. Additionally, the technique of Western blotting was utilized to analyze the levels of ferroptosis-associated proteins. Moreover, hematoxylin and eosin (H&E) staining was carried out to observe the histopathological alterations in the intestinal tissue samples from rats with necrotizing enterocolitis (NEC). ResultsReducing miR-155–5p improved the survival of FHC cells exposed to LPS, decreased cell apoptosis, inflammation, and ferroptosis, and mitigated intestinal damage in NEC rats. Furthermore, SLC7A11 was found to be a direct target of miR-155–5p. The inhibition of miR-155–5p decreased LPS-induced inflammation and ferroptosis in both FHC cells and NEC rats by promoting SLC7A11 expression. This effect was evidenced by increased levels of ferroptosis-related proteins FTH1 and GPX4, decreased COX-2 and ACSL4 levels, lower lipid peroxidation marker MDA, reduced antioxidant markers GSH, SOD, and CAT, fewer IL-6 and TNF-α, and suppression of the IκBα/NF-κB p65 signaling pathway. ConclusionsIn conclusion, reducing miR-155–5p could improve intestinal damage in NEC by inhibiting inflammation and ferroptosis. These findings may provide theoretical insights for the development of new therapies for NEC.

Inflammatory factor-mediated miR-155/SOCS1 signaling axis leads to Treg impairment in systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disorder associated with the decrease and functional impairment of regulatory T cells (Tregs). In the current study, we explored the interplay of miR-155 and suppressor of cytokine signaling 1 (SOCS1) in regulating Treg function and stability in SLE. MethodsClinical samples from healthy subjects and SLE patients were collected, and a mouse model of SLE was established to profile the expression pattern of miR-155 and SCOS1 in Tregs. Tregs isolated from mouse spleen were stimulated by inflammatory cytokines to confirm involvement of miR-155/SOCS1 axis in dictating Treg stability and function. We also administrated synthetic miR-155 inhibitor in SLE animal model to evaluate the potential effect on rescuing Treg function and alleviating SLE progression. ResultsTregs from SLE patients and SLE-induced mice exhibited a downregulation of SOCS1 and an upregulation of miR-155. In Tregs stimulated by inflammatory cytokines, Nuclear factor kappa B (NF-κB) signaling activation was required for the change of SOCS1 and miR-155 expression. miR-155 served as a negative regulator to dampen SOCS1 expression in inflammation-stimulated Tregs. The transfection of miR-155 mimic impaired the suppressive function and differentiation of Tregs through targeting SOCS1. In contrast, miR-155 inhibition improved Treg function under inflammatory stimulation and alleviated SLE conditions in the mouse model. ConclusionInflammation-induced miR-155 impairs Treg stability and function in SLE through decreasing SOCS1 expression. Targeting miR-155 might be developed as an intervention to mitigate SLE conditions.

Danggui-Shaoyao-San (DSS) ameliorating cognitive impairment in ischemia–reperfusion vascular dementia mice through miR-124 regulating PI3K/Akt signaling pathway

Vascular dementia (VD) is a disease characterized by cognitive impairment and memory loss due to brain cell damage caused by cerebral vascular ischemia. Danggui-Shaoyao-San (DSS) has been used clinically to treat diseases for centuries. The VD model was established by bilateral common carotid artery (BCCA) repeated ischemia–reperfusion (I/R) and caudal bleeding. Target prediction of DSS and miR-124 in PI3K/Akt signaling pathway by network pharmacology. The effect of DSS on cognitive dysfunction were evaluated through methods such as behavioral experiments, cerebral blood flow monitoring, HE and Nissl staining, western blot, and q-PCR. Prediction result showed that both DSS and miR-124 could target Akt1. DSS treatment significantly reduced hippocampal cell damage, improved learning and memory ability. Mechanically, DSS treatment up-regulated the expression levels of PI3K and Akt protein, and its gene. Bcl-2/Bax index is up-regulated and cell apoptosis reduced. LC3II/LC3I index decreased and autophagy of brain cells increased. Moreover, DSS down-regulated the expression level of miR-124. And inhibition of miR-124 up-regulate the expression of PI3K, Akt. These results suggested that DSS can reduce the content of miR-124 in the hippocampus of VD mice, thus regulating the PI3K/Akt signaling pathway and improving the learning and memory ability of VD mice.

MiR-21 regulates skeletal muscle atrophy and fibrosis by targeting TGF-beta/SMAD7-SMAD2/3 signaling pathway

Long-term denervation-induced atrophy and fibrosis of skeletal muscle due to denervation leads to poor recovery of muscle function. Studies have shown that the transforming growth factor-β1 (TGF-β1)-Smad signaling pathway plays a central role in muscle atrophy and fibrosis. Recent studies demonstrate the role of microRNAs (miRs) in various pathological conditions, including muscle regeneration. miR-21 has been shown to play a dynamic role in inflammatory responses and in accelerating injury responses to fibrosis. We used both RNA sequencing and quantitative RT-PCR strategies to examine the alternations of miRNAs during denervation-induced gastrocnemius muscle atrophy and fibrosis. Our data showed that MiR-21 was upregulated in denervated gastrocnemius muscle tissue, and TGF-β1treatment increased miR-21 expression. Inhibition of miR-21 reduced gastrocnemius muscle fibrosis and significantly downregulated the expression of p-SMAD2/3 and the fibrosis-associated markers TGF-β1, connective tissue growth factor, alpha smooth muscle actin. Masson's trichrome staining revealed that atrophy and fibrosis in gastrocnemius muscle tissue were reduced in the miR-21 inhibition group compared to the control group. We confirmed that SMAD7 is a direct target of miR-21 using a dual luciferase assay. Furthermore, Immunofluorescence and Western blot analyses revealed that miR-21 inhibition reduced SMAD2/3 phosphorylation and nuclear translocation. While SMAD7-siRNA abolished the effect. Consequently, the discovery that miR-21 regulates the atrophy and fibrosis of the gastrocnemius muscle offers a possible therapeutic approach for their management.

Exosome-delivered microRNAs confer adriamycin-resistance through modulating the immune and metabolism-related gene PTEN in HER2-negative breast cancer.

e12500 Background: Anthracycline is widely used in breast cancer (BCa) chemotherapy, but acquired drug resistance remains a challenge. Increasing evidence suggests that changes in the tumour immune microenvironment (TIME) consistently contribute to the development of chemoresistance. Methods: TIME scores and tumor-infiltrating immune cells (TICs) scores were used to investigate the prognosis, characteristics and gene transcriptomic profiling of HER2-negative BCa patients who received anthracycline chemotherapy. Exosomes from MCF/7-ADR (ADR-exos) and MCF/7-S cell lines were characterized. Dysregulated miRNAs were obtained by using miRNA microarray analysis on BCa cells and their corresponding exosomes. Protein profiling was performed to identify differentially expressed proteins in up- and down-regulated miR-222 cells. Ability of ADR-exos to transfer drug-resistance mediated by miR-222 was assessed by immunofluorescence assay and flow cytometry. Conditions to load miR-222 mimic or inhibitor into exosomes were optimized. Influence of miR-222 inhibitor-containing exosomes on the downstream pathway of miR-222 and the potential therapeutic effects were evaluated. Relationship between miR-222/PTEN and drug resistance in circulating exosomes from mice after doxorubicin chemotherapy was investigated. Results: Firstly, we discovered the complicated and heterogeneous TICs subtypes and their unique biological behaviors in HER2-negative BCa. Then, we identified 85 differentially expressed immunologic signature gene sets and 7202 corresponding immune-related genes in three subtypes quantified by gene set variation analysis. We identified 12 up-regulated and 13 down-regulated genes using miRNA microarray analysis in doxorubicin-resistant cells and their exosomes. Mass spectrometry found 45 differentially expressed proteins affected by miR-222. Moreover, we successfully established miRNA mimic/inhibitor-containing exosomes to explore the solo function of exosomal miR-222 in doxorubicin-resistance both in vitro and in vivo. For the first time, we detected the levels of miR-222 and its target gene PTEN in circulating exosomes from mice after doxorubicin-based chemotherapy and explored the potential clinical implications of miR-222/PTEN-containing exosomes in chemotherapy and immune suppression of BCa. Conclusions: Doxorubicin-resistant cells can transmit drug-resistance to sensitive cells via delivering miR-222 by modulating the immune- and metabolism-related gene PTEN without interference from other relevant drug-resistance factors in exosomes. Therefore, miR-222-containing exosomes and their target gene PTEN may be a promising biomarker for predicting chemotherapy efficacy and tumor immunity regulation in BCa patients receiving anthracycline chemotherapy.

Hyperoxia-Induced miR-195 Causes Bronchopulmonary Dysplasia in Neonatal Mice.

Background: Exposure to hyperoxia is an important factor in the development of bronchopulmonary dysplasia (BPD) in preterm newborns. MicroRNAs (miRs) have been implicated in the pathogenesis of BPD and provide a potential therapeutic target. Methods: This study was conducted utilizing a postnatal animal model of experimental hyperoxia-induced murine BPD to investigate the expression and function of miR-195 as well as its molecular signaling targets within developing mouse lung tissue. Results: miR-195 expression levels increased in response to hyperoxia in male and female lungs, with the most significant elevation occurring in 40% O2 (mild) and 60% O2 (moderate) BPD. The inhibition of miR-195 improved pulmonary morphology in the hyperoxia-induced BPD model in male and female mice with females showing more resistance to injury and better recovery of alveolar chord length, septal thickness, and radial alveolar count. Additionally, we reveal miR-195-dependent signaling pathways involved in BPD and identify PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) as a novel specific target protein of miR-195. Conclusions: Our data demonstrate that high levels of miR-195 in neonatal lungs cause the exacerbation of hyperoxia-induced experimental BPD while its inhibition results in amelioration. This finding suggests a therapeutic potential of miR-195 inhibition in preventing BPD.