AntimiR treatment corrects myotonic dystrophy primary cell defects across several CTG repeat expansions with a dual mechanism of action.

This study evaluated therapeutic antimiRs in primary myoblasts from patients with myotonic dystrophy type 1 (DM1). DM1 results from unstable CTG repeat expansions in the DMPK gene, leading to variable clinical manifestations by depleting muscleblind-like splicing regulator protein MBNL1. AntimiRs targeting natural repressors miR-23b and miR-218 boost MBNL1 expression but must be optimized for a better pharmacological profile in humans. In untreated cells, miR-23b and miR-218 were up-regulated, which correlated with CTG repeat size, supporting that active MBNL1 protein repression synergizes with the sequestration by CUG expansions in DMPK. AntimiR treatment improved RNA toxicity readouts and corrected regulated exon inclusions and myoblast defects such as fusion index and myotube area across CTG expansions. Unexpectedly, the treatment also reduced DMPK transcripts and ribonuclear foci. A leading antimiR reversed 68% of dysregulated genes. This study highlights the potential of antimiRs to treat various DM1 forms across a range of repeat sizes and genetic backgrounds by mitigating MBNL1 sequestration and enhancing protein synthesis.

Innovative Oral Nano/Gene Delivery System Based on Engineered Modified Saccharomyces cerevisiae for Colorectal Cancer Therapy.

The efficient delivery of RNA-based drugs to solid tumors remains a formidable obstacle. We aim to develop a safe and efficient oral drug delivery system compatible with RNA-based drugs that is urgently needed to overcome challenges such as enzymatic degradation and gastrointestinal barriers to facilitate effective treatment for treating colorectal cancer (CRC). To address these challenges, we utilized engineered modified Saccharomyces cerevisiae to evaluate the delivery efficacy of miR21-antagomir for treating CRC in preclinical mouse models, including adenomatosis polyposis coli mutant transgenic mice ApcMin/+ and in situ tumor-bearing mice. An orally deliverable gene delivery system, YS@NPs21, was designed. This gene delivery system demonstrated effectively suppressed tumor growth in both ApcMin/+ and in situ tumor-bearing mice models. This system exhibited tumor-targeting capability, effective inhibition of tumor growth, and low toxicity toward nontumor cells. Successful implementation of this innovative oral drug delivery system could offer a straightforward, safe, and RNA drug-compatible approach to CRC treatment, ultimately improving patient outcomes and reducing medical costs.

Suppression of miR-17 Alleviates Acute Respiratory Distress-associated Lung Fibrosis by Regulating Mfn2.

OBJECTIVE: Acute respiratory distress syndrome (ARDS) patients currently have relatively high mortality, which is associated with early lung fibrosis. This study aimed to investigate whether miR-17 suppression could alleviate ARDS-associated lung fibrosis by regulating Mfn2. METHODS: A mouse model of ARDS-related lung fibrosis was constructed via intratracheal instillation of bleomycin. The expression level of miR-17 in lung tissues was detected via quantitative real time polymerase chain reaction (qRT-PCR). In the ARDS mouse model of lung fibrosis, the mitigating effects of miR-17 interference were evaluated via tail vein injection of the miR negative control or the miR-17 antagomir. The pathological changes in the lung tissue were examined via HE staining and Masson's trichrome staining, and the underlying molecular mechanism was investigated via ELISA, qRT-PCR and Western blotting. RESULTS: Bleomycin-induced pulmonary fibrosis significantly increased collagen deposition and the levels of hydroxyproline (HYP) and miR-17. Interfering with miR-17 significantly reduced the levels of HYP and miR-17 and upregulated the expression of Mfn2. The intravenous injection of the miR-17 antagomir alleviated lung inflammation and reduced collagen deposition. In addition, interference with miR-17 could upregulate LC3B expression, downregulate p62 expression, and improve mitochondrial structure. CONCLUSION: Interfering with miR-17 can improve pulmonary fibrosis in mice by promoting mitochondrial autophagy via Mfn2.

Inhibition of miR-194-5p avoids DUSP9 downregulation thus limiting sepsis-induced cardiomyopathy.

Sepsis-induced cardiomyopathy (SIC) is described as a reversible myocardial depression that occurs in patients with septic shock. Increasing evidence shows that microRNA-194-5p (miR-194-5p) participates in the regulation of oxidative stress, mitochondrial dysfunction, and apoptosis and its expression is associated with the occurrence and progression of cardiovascular disease; however, the effects of miR-194-5p in SIC are still unclear. This study explores whether miR-194-5p could modulate SIC by affecting oxidative stress, mitochondrial function, and apoptosis. Experimental septic mice were induced by intraperitoneal injection of lipopolysaccharide (LPS) in C57BL/6J mice. The biological role of miR-194-5p in SIC in vivo was investigated using cardiac echocardiography, ELISA, western blot, qRT-PCR, transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, bioinformatics analysis, and dual-luciferase reporter gene assay. Our major finding is that miR-194-5p antagomir mitigates sepsis-induced cardiac dysfunction, inflammation, oxidative stress, apoptosis and mitochondrial dysfunction in the hearts of septic mice, while miR-194-5p agomir triggers the opposite effects. Furthermore, dual-specificity phosphatase 9 (DUSP9) is a direct target of miR-194-5p and the cardioprotective effects of miR-194-5p antagomir on cardiac dysfunction, inflammation, apoptosis, mitochondrial dysfunction and oxidative stress are abolished through inhibiting DUSP9. Therefore, miR-194-5p inhibition could mitigate SIC via DUSP9 in vivo and the novel miR-194-5p/DUSP9 axis might be the potential treatment targets for SIC patients.

Inhibiting miR-618 Promotes Keratinocytes Proliferation and Migration to Enhance Wound Healing in Mice.

The delay in wound healing caused by chronic wounds or pathological scars is a pressing issue in clinical practice, imposing significant economic and psychological burdens on patients. In particular, with the aging of the population and the increasing incidence of diseases such as diabetes, impaired wound healing is one of the growing health problems. MicroRNA (miRNA) plays a crucial role in wound healing and regulates various biological processes. Our results show that miR-618 was significantly upregulated during the inflammatory phase of wound healing.Subsequently, miR-618 promotes the secretion of pro-inflammatory cytokines and regulates the proliferation and migration of keratinocytes. Mechanistically, miR-618 binds to the target gene-Atp11b and inhibits the PI3K-Akt signaling pathway, inhibiting the epithelial-mesenchymal transition (EMT) of keratinocytes. In addition, the PI3K-Akt signaling pathway induces the enrichment of nuclear miR-618, and miR-618 binds to the promoter of Lin7a to regulate gene transcription. Intradermal injection of miR-618 antagomir around full-thickness wounds in peridermal mice effectively accelerates wound closure compared to control. In conclusion, miR-618 antagomir can be a potential therapeutic agent for wound healing.

Microglia-targeted inhibition of miR-17 via mannose-coated lipid nanoparticles improves pathology and behavior in a mouse model of Alzheimer's disease.

Neuroinflammation and accumulation of Amyloid Beta (Aß) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aß accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aß burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.

Effect of miR-1297 on Kidney Injury in Rats with Diabetic Nephropathy through the PTEN/PI3K/AKT Pathway.

PURPOSE: This study aimed to determine the influence of miR-1297 on kidney injury in rats with diabetic nephropathy (DN) and its causal role. METHODS: A DN rat model was established through right kidney resection and intraperitoneal injection of streptozotocin (STZ). Sham rats did not undergo right kidney resection or STZ injection. The DN rats were divided into the DN model and antagomiR-1297 treatment groups. Kidney morphology was observed using hematoxylin and eosin staining. Renal function indices, including blood urea nitrogen (BUN), serum creatinine (SCr), and urinary protein, were measured using kits. Levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined through enzyme-linked immunosorbent assay (ELISA). Fibrin (FN), collagen type I (Col I), and α-smooth muscle actin (α-SMA) were assessed through western blotting and real-time reverse transcription-polymerase chain reaction. Apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. miR-1297 targets were predicted using bioinformatic software and verified through luciferase reporter assay. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway expression was analyzed through western blotting. RESULTS: AntagomiR-1297 reduced BUN (p = 0.005), SCr (p = 0.012), and urine protein (p < 0.001) levels and improved kidney tissue morphology. It prevented renal interstitial fibrosis by decreasing FN, Col I, and α-SMA protein levels (all p < 0.001). AntagomiR-1297 increased SOD (p = 0.001) and GSH-Px (p = 0.002) levels. Additionally, it reduced levels of cell inflammatory factors, including TNF-α, IL-6, and IL-1ß (all p < 0.001), and alleviated apoptosis (p < 0.001) in rat kidney tissue with DN. miR-1297 was pinpointed as a target for PTEN. AntagomiR-1297 increased PTEN expression and suppressed PI3K and AKT phosphorylation (all p < 0.001). CONCLUSIONS: AntagomiR-1297 can mitigate renal fibrosis, renal inflammation, apoptosis, and oxidative stress levels through the PTEN/PI3K/AKT pathway.

Acacetin inhibited non-small-cell lung cancer (NSCLC) cell growth via upregulating miR-34a in vitro and in vivo.

Acacetin, one of the flavonoid compounds, is a natural product found in various plants, including Silver birch, and Damiana. Previous studies showed that acacetin has anti-cancer effects on many kinds of cancer cells, however, the role of and the mechanisms of actions of acacetin on non-small cell lung cancer (NSCLC) cells is still not fully understood. Herein, we found that, in vitro, acacetin inhibited the proliferation, invasion, and migration of NSCLC cells, A549 and H460, in a dose-dependent manner. Meanwhile, flow cytometry assay results showed that acacetin induced G2/M phase cell cycle arrest, and apoptosis of NSCLC cells. In vivo, acacetin suppressed tumor formation of A549-xenografted nude mice model with no obvious toxicities. Western blotting results showed that the protein levels of cell cycle-related proteins cyclin B1, cyclin D, and anti-apoptotic protein Bcl-2 had decreased, while the apoptosis-related protein Bak had increased both in NSCLC cells and in A549-xenografted tumor tissues. For investigating the molecular mechanism behind the biological effects of acacetin on NSCLC, we found that acacetin induced the expression levels of tumor suppressor p53 both in vitro and in vivo. MicroRNA, miR-34a, the direct target of p53, has been shown anti-NSCLC proliferation effects by suppressing the expression of its target gene programmed death ligand 1 (PD-L1). We found that acacetin upregulated the expression levels of miR-34a, and downregulated the expression levels of PD-L1 of NSCLC cells in vitro and of tumors in vivo. In vitro, knockdown p53 expression by siRNAs reversed the induction effects of acacetin on miR34a expression and abolished the inhibitory activity of acacetin on NSCLC cell proliferation. Furthermore, using agomir and antagomir to overexpress and suppress the expression miR-34a in NSCLC cells was also examined. We found that miR-34a agomir showed similar effects as acacetin on A549 cells, while miR-34a antagomir could partially or completely reverse acacetin's effects on A549 cells. In vivo, intratumor injection of miR-34a antagomir could drastically suppress the anti-tumor formation effects of acacetin in A549-xenografted nude mice. Overall, our results showed that acacetin inhibits cell proliferation and induces cell apoptosis of NSCLC cells by regulating miR-34a.

A Multifunctional Scaffold for Bone Infection Treatment by Delivery of microRNA Therapeutics Combined With Antimicrobial Nanoparticles.

Treating bone infections and ensuring bone repair is one of the greatest global challenges of modern orthopedics, made complex by antimicrobial resistance (AMR) risks due to long-term antibiotic treatment and debilitating large bone defects following infected tissue removal. An ideal multi-faceted solution would will eradicate bacterial infection without long-term antibiotic use, simultaneously stimulating osteogenesis and angiogenesis. Here, a multifunctional collagen-based scaffold that addresses these needs by leveraging the potential of antibiotic-free antimicrobial nanoparticles (copper-doped bioactive glass, CuBG) to combat infection without contributing to AMR in conjunction with microRNA-based gene therapy (utilizing an inhibitor of microRNA-138) to stimulate both osteogenesis and angiogenesis, is developed. CuBG scaffolds reduce the attachment of gram-positive bacteria by over 80%, showcasing antimicrobial functionality. The antagomiR-138 nanoparticles induce osteogenesis of human mesenchymal stem cells in vitro and heal a large load-bearing defect in a rat femur when delivered on the scaffold. Combining both promising technologies results in a multifunctional antagomiR-138-activated CuBG scaffold inducing hMSC-mediated osteogenesis and stimulating vasculogenesis in an in vivo chick chorioallantoic membrane model. Overall, this multifunctional scaffold catalyzes killing mechanisms in bacteria while inducing bone repair through osteogenic and angiogenic coupling, making this platform a promising multi-functional strategy for treating and repairing complex bone infections.

MiR-184-5p represses neuropathic pain by regulating CCL1/CCR8 signaling interplay in the spinal cord in diabetic mice.

BACKGROUND: Diabetic neuropathic pain (DNP) is a serious complication for diabetic patients involving nervous system. MicroRNAs (miRNAs) are small-noncoding RNAs which are dysregulated in neuropathic pain, and might be critical molecules for pain treatment. Our previous study has shown miR-184-5p was significantly downregulated in DNP. Therefore, the mechanism of miR-184-5p in DNP was investigated in this study. METHODS: A DNP model was established through streptozotocin (STZ). The pharmacological tools were injected intrathecally, and pain behavior was evaluated by paw withdrawal mechanical thresholds (PWMTs). Bioinformatics analysis, Dual-luciferase reporter assay and fluorescence-in-situ-hybridization (FISH) were used to seek and confirm the potential target genes of miR-184-5p. The expression of relative genes and proteins was analyzed by quantitative reverse transcriptase real-time PCR (qPCR) and western blotting. RESULTS: MiR-184-5p expression was down-regulated in spinal dorsal on days 7 and 14 after STZ, while intrathecal administration of miR-184-5p agomir attenuates neuropathic pain induced by DNP and intrathecal miR-184-5p antagomir induces pain behaviors in naïve mice. Chemokine CC motif ligand 1 (CCL1) was found to be a potential target of miR-184-5p and the protein expression of CCL1 and the mRNA expression of CCR8 were up-regulated in spinal dorsal on days 7 and 14 after STZ. The luciferase reporter assay and FISH demonstrated that CCL1 is a direct target of miR-184-5p. MiR-184-5p overexpression attenuated the expression of CCL1/CCR8 in DNP; intrathecal miR-184-5p antagomir increased the expression of CCL1/CCR8 in spinal dorsal of naïve mice. CONCLUSION: This research illustrates that miR-184-5p alleviates DNP through the inhibition of CCL1/CCR8 signaling expression.

[Effect of LncRNA GATA3-AS1 Targeting MiR-515-5p on Cell Proliferation and Apoptosis in Childhood Acute Lymphoblastic Leukemia Cells].

OBJECTIVE: To investigate the effects of long non-coding RNA (lncRNA) GATA3 antisense RNA 1 (GATA3-AS1) targeting miR-515-5p on the proliferation and apoptosis of childhood acute lymphoblastic leukemia (ALL) cells. METHODS: RT-qPCR was used to determine the expression of GATA3-AS1 and miR-515-5p in the plasma of controls and ALL children. Human ALL cells Jurkat were divided into si-GATA3-AS1, si-NC, miR-NC, miR-515-5p, si-GATA3-AS1+anti-miR-NC and si-GATA3-AS1+anti-miR-515-5p groups. CCK-8 assay was used to detect the cell proliferation, and flow cytometry was used to detect the cell apoptosis. The targeting relationship between GATA3-AS1 and miR-515-5p was determined by dual-luciferase reporter assay. RESULTS: The expression level of GATA3-AS1 in the plasma of ALL children was significantly higher than that of controls (P <0.001), while the expression level of miR-515-5p was significantly lower than that of controls (P <0.001). Compared with the si-NC group, the cell inhibition rate, apoptosis rate, and miR-515-5p expression level in si-GATA3-AS1 group were significantly increased (P <0.001). Compared with the miR-NC group, the cell inhibition rate and apoptosis rate in miR-515-5p group were significantly increased (P <0.001). GATA3-AS1 could directly and specifically bind to miR-515-5p. Compared with the si-GATA3-AS1+anti-miR-NC group, the cell inhibition rate and apoptosis rate in si-GATA3-AS1+anti-miR-515-5p group were significantly decreased (P <0.001). CONCLUSION: Down-regulation of GATA3-AS1 can inhibit proliferation and induce apoptosis of childhood ALL cells by targeting up-regulation of miR-515-5p expression.

Cardiac miR-19a/19b was induced and hijacked by CVB3 to facilitate virus replication via targeting viral genomic RdRp-encoding region.

Coxsackievirus B3 (CVB3) is one of the major pathogens of viral myocarditis, lacking specific anti-virus therapeutic options. Increasing evidence has shown an important involvement of the miR-17-92 cluster both in virus infection and cardiovascular development and diseases, while its role in CVB3-induced viral myocarditis remains unclear. In this study, we found that miR-19a and miR-19b were significantly up-regulated in heart tissues of CVB3-infected mice and exerted a significant facilitatory impact on CVB3 biosynthesis and replication, with a more pronounced effect observed in miR-19b, by targeting the encoding region of viral RNA-dependent RNA polymerase 3D (RdRp, 3Dpol) to increase viral genomic RNA stability. The virus-promoting effects were nullified by the synonymous mutations in the viral 3Dpol-encoding region, which corresponded to the seed sequence shared by miR-19a and miR-19b. In parallel, treatment with miR-19b antagomir not only resulted in a noteworthy suppression of CVB3 replication and infection in infected cells, but also demonstrated a significant reduction in the cardiac viral load of CVB3-infected mice, resulting in a considerable alleviation of myocarditis. Collectively, our study showed that CVB3-induced cardiac miR-19a/19b contributed to viral myocarditis via facilitating virus biosynthesis and replication, and targeting miR-19a/19b might represent a novel therapeutic target for CVB3-induced viral myocarditis.

Estradiol- and Progesterone-Associated Changes in microRNA-Induced Silencing and Reduced Antiseizure Efficacy of an Antagomir in Female Mice.

About one-third of individuals living with epilepsy have treatment-resistant seizures. Alternative therapeutic strategies are thus urgently needed. One potential novel treatment target is miRNA-induced silencing, which is differentially regulated in epilepsy. Inhibitors (antagomirs) of specific microRNAs (miRNAs) have shown therapeutic promise in preclinical epilepsy studies; however, these studies were mainly conducted in male rodent models, and research into miRNA regulation in females and by female hormones in epilepsy is scarce. This is problematic because female sex and the menstrual cycle can affect the disease course of epilepsy and may, therefore, also alter the efficacy of potential miRNA-targeted treatments. Here, we used the proconvulsant miRNA miR-324-5p and its target, the potassium channel Kv4.2, as an example to test how miRNA-induced silencing and the efficacy of antagomirs in epilepsy are altered in female mice. We showed that Kv4.2 protein is reduced after seizures in female mice similar to male mice; however, in contrast to male mice, miRNA-induced silencing of Kv4.2 is unchanged, and miR-324-5p activity, as measured by the association with the RNA-induced silencing complex, is reduced in females after seizure. Moreover, an miR-324-5p antagomir does not consistently reduce seizure frequency or increase Kv4.2 in female mice. As a possible underlying mechanism, we found that miR-324-5p activity and the silencing of Kv4.2 in the brain were differentially correlated with plasma levels of 17ß-estradiol and progesterone. Our results suggest that hormonal fluctuations in sexually mature female mice influence miRNA-induced silencing and could alter the efficacy of potential future miRNA-based treatments for epilepsy in females.

Osteoclast-targeted delivery of anti-miRNA oligonucleotides by red blood cell extracellular vesicles.

Osteoporosis (OP) affects millions worldwide but currently cannot be cured. Suppressing the level of miR-214 in osteoclasts by the anti-miRNA oligonucleotide (AMO) anti-miR-214 reverses bone absorption and provides a potential treatment. Here we report a peptide-guided delivery strategy using red blood cell extracellular vesicles (RBCEVs) as the vehicle to realize osteoclast-targeted delivery of anti-miR-214. A bi-functional peptide, TBP-CP05, which binds to both the CD63 on RBCEVs and receptors on osteoclasts, acts as the guide. TBP-CP05 binds with RBCEVs through CP05, displays the TRAP-binding peptide (TBP) on the surface of EVs, and endows RBCEVs with osteoclast-targeting capability both in vitro and in vivo. Intravenous injection of the osteoclast-targeting RBCEVs (OT-RBCEVs) led to the enrichment of EVs in the bone skeleton, significant inhibition of the osteoclast activity, elevated osteoblast activity, and improved bone density in osteoporotic mice. Altogether, this work demonstrates efficient guidance of drug-loaded EVs to the targeted cells in vivo using bi-functional fusion peptides, and showcases that targeted delivery of anti-miR-214 by OT-RBCEVs may be a viable method for OP treatment. SIGNIFICANCE STATEMENT. Surface functionalization of EVs endows these nanovesicles cell-specific targeting property which guides the drug cargos to specific tissues and cells with higher accuracy, longer retention, and minimal off-target effects. Methods to functionalize EVs with minimal procedures are highly desired for clinical applications. Here we present a facile method using a bifunctional fusion peptide to guide RBCEVs to osteoclasts. A simple incubation of the bifunctional peptide and RBCEVs results in osteoclast-targeting RBCEVs (OT-RBCEVs) that effectively deliver anti-miR-214 to osteoclasts in vivo in a mouse model of osteoporosis, bringing a potential therapy to osteoporotic patients. This is, to our knowledge, the first report on peptide functionalization of RBCEVs and osteoclast-targeted delivery using RBCEVs.

[The effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells by targeting miR-200b-5p].

Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p.

Intestinal recruitment of CCR6-expressing Th17 cells by suppressing miR-681 alleviates endotoxemia-induced intestinal injury and reduces mortality.

INTRODUCTION: Sepsis or endotoxemia can induce intestinal dysfunction in the epithelial and immune barrier. Th17 cells, a distinct subset of CD4+ T-helper cells, act as "border patrol" in the intestine under pathological condition and in the previous studies, Th17 cells exhibited an ambiguous function in intestinal inflammation. Our study will explore a specific role of Th17 cells and its relevant mechanism in endotoxemia-induced intestinal injury. MATERIALS AND METHODS: Lipopolysaccharide was used to establish mouse model of endotoxemia. miR-681 was analyzed by RT-PCR and northern blot analysis and its regulation by HIF-1α was determined by chromatin immunoprecipitation and luciferase reporter assay. Intestinal Th17 cells isolated from endotoxemic mice were quantitatively evaluated by flow cytometry and its recruitment to the intestine controlled by miR-681/CCR6 pathway was assessed by using anti-miRNA treatment and CCR6 knockout mice. Intestinal histopathology, villus length, intestinal inflammation, intestinal permeability, bacterial translocation and survival were investigated, by histology and TUNEL analysis, ELISA, measurement of diamine oxidase, bacterial culture, with or without anti-miR-681 treatment in endotoxemic wild-type and (or) CCR6 knockout mice. RESULTS: In this study, we found that miR-681 was significantly promoted in intestinal Th17 cells during endotoxemia, which was dependent on hypoxia-inducible factor-1α (HIF-1α). Interestingly, miR-681 could directly suppress CCR6, which was a critical modulator for Th17 cell recruitment to the intestines. In vivo, anti-miR-681 enhanced survival, increased number of intestinal Th17 cells, reduced crypt and villi apoptosis, decreased intestinal inflammation and bacterial translocation, resulting in protection against endotoxemia-induced intestinal injury in mice. However, CCR6 deficiency could neutralize the beneficial effect of anti-miR-681 on the intestine during endotoxemia, suggesting that the increment of intestinal Th17 cells caused by anti-miR-681 relies on CCR6 expression. CONCLUSION: The results of the study indicate that control of intestinal Th17 cells by regulating novel miR-681/CCR6 signaling attenuates endotoxemia-induced intestinal injury.

Anti-miR-96 and Hh pathway inhibitor MDB5 synergistically ameliorate alcohol-associated liver injury in mice.

Alcohol-associated liver disease (ALD) and its complications are significant health problems worldwide. Several pathways in ALD are influenced by alcohol that drives inflammation, fatty acid metabolism, and fibrosis. Although miR-96 has become a key regulator in several liver diseases, its function in ALD remains unclear. In contrast, sonic hedgehog (SHH) signaling has a well-defined role in liver disease through influencing the activation of hepatic stellate cells (HSCs) and the inducement of liver fibrosis. In this study, we investigated the expression patterns of miR-96 and Hh molecules in mouse and human liver samples. We showed that miR-96 and Shh were upregulated in ethanol-fed mice. Furthermore, alcoholic hepatitis (AH) patient specimens also showed upregulated FOXO3a, TGF-ß1, SHH, and GLI2 proteins. We then examined the effects of Hh inhibitor MDB5 and anti-miR-96 on inflammatory and extracellular matrix (ECM)-related genes. We identified FOXO3 and SMAD7 as direct target genes of miR-96. Inhibition of miR-96 decreased the expression of these genes in vitro in AML12 cells, HSC-T6 cells, and in vivo in ALD mice. Furthermore, MDB5 decreased HSCs activation and the expression of ECM-related genes, such as Gli1, Tgf-ß1, and collagen. Lipid nanoparticles (LNPs) loaded with the combination of MDB5, and anti-miR-96 ameliorated ALD in mice. Our study demonstrated that this combination therapy could serve as a new therapeutic target for ALD.

Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair.

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

miR-200b-3p antagomir inhibits neuronal apoptosis in oxygen-glucose deprivation (OGD) model through regulating ß-TrCP.

BACKGROUND: Hypoxia-ischemic brain damage (HIBD) is a primary cause of morbidity and disability in survivors of preterm infants. We previously discovered that miR-200b-3p plays an important role in HIBD via targeting Slit2. This study was designed to identify novel targets of miR-200b-3p and investigate the relationship between miR-200b-3p and its downstream effectors. METHODS AND RESULTS: Cultured primary rat hippocampal neurons were used in the model of oxygen-glucose deprivation (OGD) and RT-qPCR was utilized to detect the alterations of miR-200b-3p in these cells following the OGD. Our study found that the expression of miR-200b-3p was up-regulated in neurons post OGD. Bioinformatics analysis identified that ß transducin repeat-containing protein (ß-TrCP) is a target gene of miR-200b-3p, and our luciferase reporter gene assay confirmed that miR-200b-3p can interact with ß-TrCP mRNA. Hypoxia-ischemic brain damage was induced in three-day-old SD rats and inhibition of miR-200b-3p by injection of antagomir into bilateral lateral ventricles enhanced ß-TrCP expression at both the mRNA and protein levels in rats' brains. TUNEL staining and CCK-8 assays found that the survival of hippocampal neurons in the miR-200b-3p antagomir group was improved significantly (p＜0.05), whereas apoptosis of neurons in the miR-200b-3p antagomir group was significantly decreased (p＜0.05), as compared with the OGD group. However, silencing of ß-TrCP by ß-TrCP siRNA impaired the neuroprotective effect of miR-200b-3p antagomir. H&E staining showed that miR-200b-3p attenuated the pathological changes in the hippocampal region of rats with HIBD. CONCLUSION: Our study has demonstrated that ß-TrCP is a target gene of miR-200b-3p and that inhibition of miR-200b-3p by antagomir attenuates hypoxia-ischemic brain damage via ß-TrCP.

MicroRNA-9a-5p-NOX4 inhibits intestinal inflammatory injury by regulating the M1 polarization of intestinal macrophages.

We found that the expression of microRNA (miRNA)-9a-5p decreased in inflammatory bowel diseases (IBD; ulcerative colitis and Crohn's disease). Further, we revealed the effects and mechanisms of miRNA-9a-5p for regulating IBD progression. In C57BL/6N mice, IBD was induced with dextran sodium sulfate (DSS), and the effects of endogenous miRNA-9a-5p were mimicked/antagonized through intraperitoneal injection of miRNA-9a-5p agomir and antagomir. In animal experimentation, agomir could inhibit intestinal inflammation and tissue damage, and reduce the mucosal barrier permeability. Antagomir, on the other hand, could promote barrier damage, whose effect was associated with the M1 macrophage polarization. This study finds that miRNA-9a-5p targets NOX4 to suppress ROS production, which plays an important role in mucosal barrier damage in IBD.