miR-21 Inhibitor Research Articles

MicroRNA-124 negatively regulates STAT3 to alleviate hypoxic-ischemic brain damage by inhibiting oxidative stress.

MicroRNA-124 (miR-124) is implicated in various neurological diseases; however, its significance in hypoxic-ischaemic brain damage (HIBD) remains unclear. This study aimed to elucidate the underlying pathophysiological mechanisms of miR-124 in HIBD. In our study performed on oxygen-glucose deprivation followed by reperfusion (OGD)/R-induced primary cortical neurons, a substantial reduction in miR-124 was observed. Furthermore, the upregulation of miR-124 significantly mitigated oxidative stress, apoptosis, and mitochondrial impairment. We demonstrated that miR-124 interacts with the signal transducer and activator of transcription 3 (STAT3) to exert its biological function using the dual-luciferase reporter gene assay. As the duration of OGD increased, miR-124 exhibited a negative correlation with STAT3. STAT3 overexpression notably attenuated the protective effects of miR-124 mimics, while knockdown of STAT3 reversed the adverse effects of the miR-124 inhibitor. Subsequently, we conducted an HIBD model in rats. In vivo experiments, miR-124 overexpression attenuated cerebral infarction volume, cerebral edema, apoptosis, oxidative stress, and improved neurological function recovery in HIBD rats. In summary, the neuroprotective effects of the miR-124/STAT3 axis were confirmed in the HIBD model. MiR-124 may serve as a potential biomarker with significant therapeutic implications for HIBD.

MiR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

Hypoxic exercise is an effective intervention for obesity, because it promotes weight loss by regulating fatty acid (FA) metabolism. The regulation of peroxisome proliferator-activated receptor β (PPARβ) by miR-122 may be involved in this process, but the detailed mechanisms are unknown. In order to address this issue, we probed how miR-122 affected the expression of factors associated with FA metabolism in skeletal muscle of obese rats undergoing hypoxic training. By injecting adeno-associated virus 9 containing miR-122 overexpression vector or miR-122 inhibitor into skeletal muscles of rats with a 4-week hypoxic exercise regimen, the miR-122 expression level can be regulated. Body composition and blood lipid levels were analyzed, and PPARβ, carnitine palmitoyltransferase 1b (CPT1b), acetylCoA carboxylase 2 (ACC2), and FA synthase (FAS) mRNA and protein levels were evaluated using quantitative reverse transcription quantitative PCR(RT-qPCR) and Western blot analysis. We found that miR-122 overexpression increased low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels and decreased PPARβ, ACC2, and FAS expression. Conversely, miR-122 inhibition decreased TG level, increased high-density lipoprotein cholesterol (HDL-C) level, and upregulated PPARβ, ACC2, FAS, and CPT1b. These data indicated that the negative regulation of PPARβ by miR-122 promotes FA metabolism by altering the levels of the factors related to FA metabolism in skeletal muscle of obese rat under hypoxic training, thus providing molecular-level insight into the beneficial effects of this intervention.

Apigenin accelerates wound healing in diabetic mice by promoting macrophage M2-type polarization via increasing miR-21 expression.

The alteration of inflammatory phenotype by macrophage polarization plays an important role in diabetic wound repair. Apigenin has been reported to be anti-inflammatory and promote tissue repair; however, whether it regulates macrophage polarization to participate in diabetic wound repair remains to be investigated. We found that apigenin promoted miR-21 expression in LPS-stimulated RAW264.7 cells, inhibited cellular M1-type factor TNF-α and IL-1β secretion and increased M2-type factor IL-10 and TGF-β secretion, and accelerated macrophage conversion from M1 type to M2 type, whereas this protective effect of apigenin was counteracted by a miR-21 inhibitor. Moreover, we established a macrophage-HUVECs cell in vitro co-culture system and found that apigenin accelerated the migration, proliferation, and VEGF secretion of HUVECs by promoting macrophage miR-21 expression. Further, mechanistic studies revealed that this was mediated by the TLR4/Myd88/NF-κB axis. In in vivo study, diabetic mice had significantly delayed wound healing compared to non-diabetic mice, accelerated wound healing in apigenin-treated diabetic mice, and decreased M1-type macrophages and increased M2-type macrophages in wound tissues.

MicroRNA-124 plays an inhibitory role in cutaneous squamous cell carcinoma cells via targeting SNAI2, an immunotherapy determinant

PurposeMicroRNAs (miRs) play multiple roles during cutaneous squamous cell carcinoma (CSCC) progression. Previous studies suggest miR-124 could inhibit cancer development in CSCC. Methods: Obtained 63 pairs of CSCC and adjacent tissues for analysis. Cultured HaCaT and two CSCC cell lines (A431 and SCL-1) in DMEM (10 % FBS). Transfected cells using Lipofectamine 2000 with various miR-124 mimics, inhibitors, or Snail family transcriptional repressor 2 (SNAI2) expression plasmid. Performed a series of assays, including real-time quantitative PCR, Western blot, CCK8, wound healing, transwell, and luciferase reporter gene assay, to examine the effects of miR-124 on CSCC cells. Results: An evident downregulation of miR-124 in CSCC tissues, which was related to advanced disease stage and nodal metastasis. Overexpressing miR-124 could reduce the proliferation, migration, and invasion abilities of CSCC cells. It was verified that miR-124 targets the SNAI2 in CSCC cells. Moreover, ectopic expression of SNAI2 rescued the suppressive effects on CSCC cells induced by miR-124 overexpression. Furthermore, miR-124 increased cell sensitivity to cisplatin. Besides, SNAI2 is a critical factor in the immune-related aspects of CSCC and its modulation may influence the response to immunotherapy. Conclusion: We demonstrate that miR-124 inhibits CSCC progression through downregulating SNAI2, and thus it may be a molecular candidate for treating CSCC in the clinic.

MICRORNAS 125B, 199A-5P, AND 214 AS MODULATORS OF BONE HOMEOSTASIS

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.

Macrophage-derived exosomal miRNA-141 triggers endothelial cell pyroptosis by targeting NLRP3 to accelerate sepsis progression.

Sepsis, critical condition marked by severe organ dysfunction from uncontrolled infection, involves the endothelium significantly. Macrophages, through paracrine actions, play a vital role in sepsis, but their mechanisms in sepsis pathogenesis remain elusive. Objective: We aimed to explore how macrophage-derived exosomes with low miR-141 expression promote pyroptosis in endothelial cells (ECs). Exosomes from THP-1 cell supernatant were isolated and characterized. The effects of miR-141 mimic/inhibitor on apoptosis, proliferation, and invasion of Human Umbilical Vein Endothelial Cells (HUVECs) were assessed using flow cytometry, CCK-8, and transwell assays. Key pyroptosis-related proteins, including caspase-1, IL-18, IL-1β, NLR Family Pyrin Domain Containing 3 (NLRP3), ASC, and cleaved-GSDMD, were analyzed via Western blot. The interaction between miR-141 and NLRP3 was studied using RNAhybrid v2.2 and dual-Luciferase reporter assays. The mRNA and protein level of NLRP3 after exosomal miR-141 inhibitor treatment was detected by qPCR and Western blot, respectively. Exosomes were successfully isolated. miR-141 mimic reduced cell death and pyroptosis-related protein expression in HUVECs, while the inhibitor had opposite effects, increasing cell death, and enhancing pyroptosis protein expression. Additionally, macrophage-derived exosomal miR-141 inhibitor increased cell death and pyroptosis-related proteins in HUVECs. miR-141 inhibits NLRP3 transcription. Macrophages facilitate sepsis progression by secreting miR-141 decreased exosomes to activate NLRP3-mediated pyroptosis in ECs, which could be a potentially valuable target of sepsis therapy.

Oxidative stress promotes liver fibrosis by modulating the microRNA-144 and SIN3A-p38 pathways in hepatic stellate cells.

Background & Aims: Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro. Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A, and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis.

MiR-21 Expressed by Dermal Fibroblasts Enhances Skin Wound Healing Through the Regulation of Inflammatory Cytokine Expression.

The management of skin wound healing is still a challenge. MicroRNA-21 (miR-21) has been reported to play important roles in wound repair; however, the underlying mechanism needs to be further clarified. The present study aimed to study the direct role of miR-21 in skin wound healing in miR-21 KO mice and to investigate the role of miR-21 in controlling the migration and proliferation of primary human skin cells and its underlying mechanism(s). miR-21 KO and wild-type (WT) mice were used for in vivo wound healing assays, while mouse and human primary skin cells were used for in vitro assays. miR-21 inhibitors or mimics or negative control small RNAs were transfected to either inhibit or enhance miR-21 expression in the human primary dermal fibroblasts or epidermal cells. RNA sequencing analysis was performed to identify the potential molecular pathways involved. We found that the loss of miR-21 resulted in slower wound healing in miR-21 KO mouse skin and especially delayed the healing of dermal tissue. In vitro assays demonstrated that the reduced expression of miR-21 caused by its inhibitor inhibited the migration of human primary dermal fibroblasts, which could be enhanced by increased miR-21 expression caused by miR-21 mimics. RNA-sequence analysis revealed that the inhibition of miR-21 expression downregulated the inflammatory response pathways associated with the decreased expression of inflammatory cytokines, and the addition of IL-1β into the culture medium enhanced the migration and proliferation of dermal fibroblasts in vitro. In conclusion, miR-21 in dermal fibroblasts can promote the migration and growth of epidermal and dermal cells to enhance skin wound healing through controlling the expression of inflammatory cytokines.

Leonurine alleviates rheumatoid arthritis by regulating the Hippo signaling pathway

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease that can cause joint inflammation and damage. Leonurine (LE) is an alkaloid found in Leonurus heterophyllus. It has anti-inflammatory effects. Hypothesis/PurposeThe molecular mechanisms by which LE acts in RA are unclear and further investigation is required. MethodsMice with collagen-induced arthritis (CIA), and RA-fibroblast-like synoviocytes (FLSs) isolated from them were used as in vivo and in vitro models of RA, respectively. The therapeutic effects of LE on CIA-induced joint injury were investigated by micro-computed tomography, and staining with hematoxylin and eosin and Safranin-O/Fast Green. Cell Counting Kit-8, a Transwell® chamber, enzyme-linked immunosorbent assays, RT-qPCR, and western blotting were used to investigate the effects of LE on RA-FLS viability, migratory capacity, inflammation, microRNA-21 (miR-21) levels, the Hippo signaling pathway, and the effects and intrinsic mechanisms of related proteins. Dual luciferase was used to investigate the binding of miR-21 to YOD1 deubiquitinase (YOD1) and yes-associated protein (YAP). Immunofluorescence was used to investigate the localization of YAP within the nucleus and cytoplasm. ResultsTreatment with LE significantly inhibited joint swelling, bone damage, synovial inflammation, and proteoglycan loss in the CIA mice. It also reduced the proliferation, cell colonization, migration/invasion, and inflammation levels of RA-FLSs, and promoted miR-21 expression in vitro. The effects of LE on RA-FLSs were enhanced by an miR-21 mimic and reversed by an miR-21 inhibitor. The dual luciferase investigation confirmed that both YOD1 and YAP are direct targets of miR-21. Treatment with LE activated the Hippo signaling pathway, and promoted the downregulation and dephosphorylation of MST1 and LATS1 in RA, while inhibiting the activation of YOD1 and YAP. Regulation of the therapeutic effects of LE by miR-21 was counteracted by YOD1 overexpression, which caused the phosphorylation of YAP and prevented its nuclear ectopic position, thereby reducing LE effect on pro-proliferation-inhibiting apoptosis target genes. ConclusionLE regulates the Hippo signaling pathway through the miR-21/YOD1/YAP axis to reduce joint inflammation and bone destruction in CIA mice, thereby inhibiting the growth and inflammation of RA-FLSs. LE has potential for the treatment of RA.

Effect of miR-21 Inhibitor Coated with Ferric Oxide on Ovarian Cancer Cells Through Mitogen-Activated Protein Kinase Signaling Pathway

Ovarian cancer (OC) incidence has in recent years been on the rise among common gynecological cancers. Iron oxide nanoparticles (IONPs), as emerging nanomedicines, have been widely used in research on cancer therapy. However, with in-depth research on microRNA (miRNA), the effect of combining miRNA with nanoparticles on ovarian cancer is not yet clear. We deeply explored the mechanism of IONPs loading miRNA to regulate ovarian cancer cells. In this study, novelmiR-21 inhibitor-IONPs nanoparticles were prepared by loading miR-21 inhibitor into IONPs. Electron microscopy was used to observe nano-encapsulation and miR-21 expression was measured along with analysis of cell proliferation, apoptosis by flow cytometry, and phosphorylation of apoptotic proteins and mitogen-activated protein kinase (MAPK) signaling pathways by Western blot. Our results showed that miR-21 inhibitor-IONPs reduced miR-21 expression, thereby inhibiting ovarian cancer cells activities and promoting apoptosis. miR-21 inhibitor-IONPs also inhibited p-p38MAPK and p-ERK levels, which were increased after addition of MAPK agonist (U-46619). Moreover, ovarian cancer cell proliferation increased and apoptosis decreased. miR-21 inhibitor-IONPs can thus inhibit MAPK signaling, thereby reducing the activities of ovarian cancer cells. This study provides theoretical support for application of miR-21 inhibitor-IONPs as novel nanoparticles for the treatment of ovarian cancer.

Effect of Long Non-Coding RNA LINC01268 on the Malignant Biological Behaviors of Acute Myeloid Leukemia Cells

To investigate the effect of long non-coding RNA LINC01268 on apoptosis of acute myeloid leukemia (AML) cells and related mechanisms. The expression levels of LINC01268 and miR-217 in peripheral blood samples from AML patients and AML cell lines HL-60 and KG-1 were detected by qRT-PCR. HL-60 cells were divided into pcDNA3.1-NC, pcDNA3.1-LINC01268, si-NC, si-LINC01268, miR-NC, miR-217 mimics, si-LINC01268 + inhibitor-NC and si-LINC01268+ miR-217 inhibitor groups. The mRNA expressions of LINC01268 and miR-217 were detected by qRT-PCR. The targeting relationship between LINC01268 and miR-217 was detected by dual-luciferase reporter assay. Cell viability was detected by CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The expression of cell cycle and apoptosis-related proteins p21, Bcl-2, Bax, caspase-3 and PI3K/AKT signaling pathway-related proteins were detected by Western blot. The expression of LINC01268 in peripheral blood samples of AML patients and AML cell lines HL-60 and KG-1 was increased (P < 0.05), and the expression of miR-217 was decreased (P < 0.05). Compared with si-NC group and miR-NC group, the viability of HL-60 cells was decreased in si-LINC01268 group and miR-217 mimics group (P < 0.05), the proportion of cells in G1 phase and apoptosis rate were increased (P < 0.05), the protein expression levels of p21, Bax and caspase-3 were increased (P < 0.05), while the protein expression level of Bcl-2 was decreased (P < 0.05). LINC01268 targeted and negatively regulated the expression of miR-217, and inhibiting the expression of miR-217 partially reversed the effects of LINC01268 interference on the viability, cell cycle and apoptosis of HL-60 cells. Interference with LINC01268 could inhibit the activity of PI3K/AKT signaling pathway. Inhibiting the expression of miR-217 could partially reverse the inhibition of LINC01268 interference on PI3K/AKT signaling pathway. LINC01268 is highly expressed and miR-217 is lowly expressed in AML cells. LINC01268 can promote the activity of PI3K/AKT signaling pathway, increase the survival rate and inhibit the apoptosis of AML cells by targeting miR-217 expression.

MiR-153-3p via PIK3R1 Is Involved in Cigarette Smoke-Induced Neurotoxicity in the Brain.

Cigarettes contain various chemicals that cause damage to nerve cells. Exposure to cigarette smoke (CS) causes insulin resistance (IR) in nerve cells. However, the mechanisms for a disorder in the cigarette-induced insulin signaling pathway and in neurotoxicity remain unclear. Therefore, we evaluated, by a series of pathology analyses and behavioral tests, the neurotoxic effects of chronic exposure to CS on C57BL/6 mice. Mice exposed to CS with more than 200 mg/m3 total particulate matter (TPM) exhibited memory deficits and cognitive impairment. Pathological staining of paraffin sections of mouse brain tissue revealed that CS-exposed mice had, in the brain, neuronal damage characterized by thinner pyramidal and granular cell layers and fewer neurons. Further, the exposure of SH-SY5Y cells to cigarette smoke extract (CSE) resulted in diminished insulin sensitivity and reduced glucose uptake in a dose-dependent fashion. The PI3K/GSK3 insulin signaling pathway is particularly relevant to neurotoxicity. microRNAs are involved in the PI3K/GSK3β/p-Tau pathway, and we found that cigarette exposure activates miR-153-3p, decreases PI3K regulatory subunits PIK3R1, and induces Tau hyperphosphorylation. Exposure to an miR-153 inhibitor or to a PI3K inhibitor alleviated the reduced insulin sensitivity caused by CS. Therefore, our results indicate that miR-153-3p, via PIK3R1, causes insulin resistance in the brain, and is involved in CS-induced neurotoxicity.

Exploiting the Metabolic Vulnerability of Leukemic Stem Cells (LSCs) in Inv(16) Acute Myeloid Leukemia (AML) with the Combination of Miristen, a Mir-126 Inhibitor, and Venetoclax

AML with inv(16)(p13q22) expresses the leukemogenic fusion gene CBFB-MYH11 and upregulates microRNA (miR)-126 that supports homeostasis, quiescence, and activity of LSCs. Genetic or pharmacologic depletion of miR-126 via miR-126 knock out (KO) or miRisten (a CpG-anti-miR-126 inhibitor) reduces LSCs in a Cbfb-MYH11 ( CM) knock-in (KI) AML model and inv(16) AML patient-derived xenografts (PDXs), respectively [PMID: 34686664]. However, the antileukemic underpinnings of miR-126 inhibition in inv(16) AML remain to be elucidated. LSCs reportedly rely on fatty acid metabolism/oxidative phosphorylation (OXPHOS) for their bioenergetic metabolic needs. To assess the role of miR-126 in metabolism, we performed metabolomic profiling for Lin-/c-Kit+ cells isolated from CM KI AML (miR-126 high) mice and CM with miR-126 KO mice ( CM/miR-126 Δ /Δ). Our data revealed that CM/miR-126 Δ /Δ had a significant decrease of short, medium as well as long-chain acyl carnitines and fatty acid (2 to 11 fold, p&amp;lt;0.05) compared to CM/miR-126 WT/ WT mice, suggesting that miR-126 deficit decreased fatty acid oxidation (FAO). Moreover, electron microscope imaging demonstrated mitochondrial fusion (i.e., larger and networked mitochondria) in CM Lin-/c-Kit+Sca1+ (LSK) cells vs syngeneic controls. In contrast, mitochondria fragmentation (fission) and decreased FAO/OXPHOS were observed in murine CM with miR-126 KO LSK. Accordingly, FAO and Seahorse assays demonstrated miRisten treatment led to mitochondria fragmentation and reduced FAO/OXPHOS and in human CD34 + CD38 ‒ inv(16) blasts (enriched for LSCs) vs normal CD34 + CD38 ‒ cells. Sprouty Related EVH1 Domain Containing 1 (SPRED1), a downstream target of miR-126, inhibits ERK/CREB activation and in turn increases antiapoptotic protein BCL-2 via phosphorylation and stabilization. In inv(16) AML cells, upregulated miR-126 downregulated SPRED1 and in turn increased ERK/CREB signaling and BCL-2 levels. This increase augments the ubiquitination of DRP1, a protein otherwise involved in mitochondrial fission, via NFE2-related factor 2 (NRF2), and increases Carnitine Palmitoyltransferase 1B (CPT1B), a rate-controlling protein for FAO and OXPHOS. In contrast, miR-126 KO or miR-126 inhibition by miRisten led to SPRED1 upregulation and decreased ERK/CREB signaling and BCL-2 levels, thereby resulting in increased Dynamin-related protein 1 (DRP1) and mitochondrial fission and a decrease in CPT1B, FAO, and OXPHOS. Thus, given the interplay of miR-126 with BCL-2 and its implication in mitochondrial fusion and increase of OXPHOS in inv(16) blasts and LSCs, we hypothesized a synergism between miRisten and the BCL-2 inhibitor venetoclax (VEN). In fact, combination of miRisten/VEN synergistically decreased FAO/OXPHOS, enhanced mitochondria fission and increased apoptosis of murine and human inv(16) blasts and LSCs [combination indices (CIs)&amp;lt;1, (ED50-ED95)]. Thus, we treated CM mice with scrambled oligonucleotides (sODN) (20 mg/kg i.v., daily for 3 weeks), miRisten (20 mg/kg i.v., daily for 3 weeks), VEN (100 mg/kg oral gavage, daily for 2 weeks), or miRisten/VEN (same dosages as single agents). Compared with sODN or single agents, miRisten/VEN significantly reduced bone marrow (BM) leukemic blast infiltration [miRisten/VEN 33.6±3.4% vs miRisten 49.8±2.6% (p=0.001), VEN 56.2±4.7% (p=0.001), or sODN 60.0±2.6% (p&amp;lt;0.0001)] and spleen weights [miRisten/VEN 0.228±0.038g vs miRisten 0.495±0.058g (p=0.001), VEN 0.479±0.06g (p=0.002), or sODN 0.853±0.039g (p&amp;lt;0.0001)] and prolonged survival of CM mice [median survival miRisten/VEN 125d vs miRisten 108d (p=0.005), VEN 106.5d (p=0.039), or sODN 91d (p&amp;lt;0.0001), respectively; secondary transplant median survival: miRisten/VEN 92d vs miRisten 70.5d (p=0.048), VEN 54d (p=0.006), or sODN 45d (p=0.0002)]. Similar results were also observed in inv(16) PDXs with significant reduction of BM huCD45+ leukemic blasts by miRisten/VEN compared to single agents or sODN [miRisten/VEN 13.7±1.9% vs miRisten 27.9±3.4% (p=0.003), VEN 25.3±4.6%(p=0.032), or sODN 68.6±5.3% (p&amp;lt;0.0001)]; survival analyses for treated PDX mice and secondary transplant recipients are in progress. In summary, our study provides the mechanism for combining miRisten with VEN in the treatment of inv(16) AML. While VEN is FDA approved for AML, miRisten has just undergone a successful toxicology evaluation and is underway to the clinic.

MicroRNA-21 promotes head and neck squamous cell carcinoma (HNSCC) induced transition of bone marrow mesenchymal stem cells to cancer-associated fibroblasts

BackgroundMost patients diagnosed with head and neck tumor will present with locally advanced disease, requiring multimodality therapy. Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. We aimed to determine whether microRNA-21(miR-21) would play a vital role in HNSCC induced transition of human bone marrow mesenchymal stem cells (hBMSCs) to cancer-associated fibroblasts (CAFs).MethodsIn this study, we used electron microscope to observed exosomes collected from human tissue and two cell lines. We co-cultured hBMSCs with exosomes from FaDu and Cal-27 cells with miR-21 inhibited or not, then assessed cell cycle changes of hBMSCs with flow cytometry and determined expression level of α-SMA and FAP through qRT-PCR and Western blot.ResultsWe observed an up-regulation of miR-21 expression in HNSCC tissue and FaDu and Cal-27 cells. Importantly, the exosomes derived from both cells induced CAFs-like characteristics in hBMSCs. while treatment with a miR-21 inhibitor effectively suppressed the transition of hBMSCs to CAFs and reversed the changes in the cell cycle distribution. This suggests that miR-21 plays a crucial role in facilitating the transition of hBMSCs to CAFs and modulating the cell cycle dynamics.ConclusionOur findings highlight the significance of miR-21 in mediating the communication between HNSCC cells and hBMSCs through exosomes, leading to the promotion of CAFs-like features and alterations in the cell cycle of hBMSCs.

Modulation of miR-29 influences myocardial compliance likely through coordinated regulation of calcium handling and extracellular matrix

MicroRNAs (miRNAs) control the expression of diverse subsets of target mRNAs, and studies have found miRNA dysregulation in failing hearts. Expression of miR-29 is abundant in heart, increases with aging, and is altered in cardiomyopathies. Prior studies demonstrate that miR-29 reduction via genetic knockout or pharmacologic blockade can blunt cardiac hypertrophy and fibrosis in mice. Surprisingly, this depended on specifically blunting miR-29 actions in cardiomyocytes versus fibroblasts. To begin developing more translationally relevant vectors, we generated a novel transgene-encoded miR-29 inhibitor (TuD-29) that can be incorporated into a viral-mediated gene therapy for cardioprotection. Here, we corroborate that miR-29 expression and activity is higher in cardiomyocytes versus fibroblasts and demonstrate that TuD-29 effectively blunts hypertrophic responses in cultured cardiomyocytes and mouse hearts. Furthermore, we found that adeno-associatedvirus (AAV)-mediated miR-29 overexpression in mouse hearts induces early diastolic dysfunction, whereas AAV:TuD-29 treatment improves cardiac output by increasing end-diastolic and stroke volumes. The integration of RNA sequencing and miRNA-target interactomes reveals that miR-29 regulates genes involved in calcium handling, cell stress and hypertrophy, metabolism, ion transport, and extracellular matrix remodeling. These investigations support a likely versatile role for miR-29 in influencing myocardial compliance and relaxation, potentially providing a unique therapeutic avenue to improve diastolic function in heart failure patients.

High-Precision Single-Cell microRNA Therapy by a Functional Nanopipette with Sensitive Photoelectrochemical Feedback.

This work proposes the concept of single-cell microRNA (miR) therapy and proof-of-concept by engineering a nanopipette for high-precision miR-21-targeted therapy in a single HeLa cell with sensitive photoelectrochemical (PEC) feedback. Targeting the representative oncogenic miR-21, the as-functionalized nanopipette permits direct intracellular drug administration with precisely controllable dosages, and the corresponding therapeutic effects can be sensitively transduced by a PEC sensing interface that selectively responds to the indicator level of cytosolic caspase-3. The experimental results reveal that injection of ca. 4.4×10-20mol miR-21 inhibitor, i.e., 26488 copies, can cause the obvious therapeutic action in the targeted cell. This work features a solution to obtain the accurate knowledge of how a certain miR-drug with specific dosages treats the cells and thus provides an insight into futuristic high-precision clinical miR therapy using personalized medicine, provided that the prerequisite single-cell experiments are courses of personalized customization.

Splicing factor ESRP1 derived circ_0068162 promotes the progression of oral squamous cell carcinoma via the miR-186/JAG axis.

Oral squamous cell carcinoma (OSCC) is a common malignancy in the oral and maxillofacial regions with an increasing incidence rate. Circular RNA (circRNA) is a recently discovered long-chain non-coding RNA family member. The objective of this study was to analyze the role of circ_0068162 in OSCC development. We downloaded sample data GSE145608 from the Gene Expression Omnibus database. Online databases Starbase, TargetScan and miRDB were used to predict the target microRNAs (miRNAs) and genes. Cell viability and proliferation were assessed using the CCK-8 and EdU assays, respectively. Cell migration and invasion abilities were detected using transwell assay. The double luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the interaction relationship between the identified target molecules. RNase R and actinomycin D treatment were performed to analyze the stability of circ_0068162. We found that circ_0068162 was overexpressed in the cytoplasm of OSCC cells and clinical OSCC tissues. Knockdown of circ_0068162 inhibited the growth, migration and invasion of OSCC cells. We also identified miR-186 as the target miRNA of circ_0068162, and JAG1 and JAG2 as the target genes of miR-186. The miR-186 inhibitor rescued the effects of sh-circ_0068162 and JAG1/JAG2 overexpression rescued the effects of miR-186 mimic in OSCC cells. Furthermore, ESRP1 promoted the biosynthesis of circ_0068162. The circ_0068162/miR-186/JAGs/ESRP1 feedback loop is closely related to OSCC development.

Abstract 18622: The Role of MicroRNA-98 in the Context of Myocardial Ischemia-Reperfusion Injury During Late Pregnancy

BACKGROUND: We have shown that the late-pregnant (LP) rodent exhibits a higher susceptibility to myocardial ischemia-reperfusion injury (IRI) compared to non-pregnant (NP). The molecular mechanisms remain unclear. The dysregulation of multiple microRNAs (miRs) in IRI suggests their potential as promising targets for therapeutic interventions. Methods: Female Sprague-Dawley rats in both NP and LP states underwent occlusion of the left anterior descending coronary artery (LAD) for a duration of 45 minutes, followed by reperfusion for 3 hours or 24 hours. Expression of miR was assessed via fluorescent in situ hybridization. MicroRNA-microarray profiling was conducted on hearts of NP and LP rats subjected to IRI. Female H9c2 rat cardiomyoblast cells were transfected with miR mimics and inhibitors, and subjected to hypoxia/reoxygenation. Blood samples were obtained from NP, LP, and ischemic heart disease (IHD) patients. MiR98 inhibitor was administered at the initiation of reperfusion. Results: MicroRNA-microarray analysis revealed upregulation of microRNA-98-5p (miR-98) in LV of LP at baseline, and a further upregulation after IRI. The expression of Stat3 and Pgc-1α were decreased in LV of LP rats compared to NP rats upon IRI. Fluorescent in situ hybridization revealed expression of miR-98 in cardiomyocytes in NP rats, which was increased in LP rats. Overexpression of miR-98 in vitro in female H9c2 cells decreased Stat3 and Pgc-1α levels, and promoted apoptosis, oxidative stress, and inflammatory markers. MiR-98 inhibitor in LP rats at the onset of reperfusion reduced infarct size, apoptosis, oxidative stress, and inflammatory markers and was associated with upregulation of Stat3 and Pgc-1α. In humans, plasma miR-98 levels were significantly higher in healthy LP compared to healthy NP individuals and even higher in LP patients with IHD patients. CONCLUSIONS: We show the detrimental effects of miR-98 by promoting cardiomyocyte oxidative stress, inflammation, and apoptosis via its targets Stat3 and Pgc-1α in the context of late pregnancy. MiR-98 could be a novel cardio-protective strategy or biomarker in late pregnancy.

Abstract 15057: miR-132 Inhibitor CDR 132L Attenuates Cardiac Remodeling by Exhibiting Anti-Fibrotic Effects

Introduction: Increased cardiac expression levels of miRNA miR-132 have been associated with pathological cardiac remodeling including cardiac fibrosis. As a selective inhibitor for miR-132, the synthetic antisense oligonucleotide CDR132L, is capable to reverse cellular pathology and restore normal heart function. Pharmacological inhibition of miR-132 as well as genetic knockout has been shown to protect mice from transverse aortic constriction induced cardiac remodeling including interstitial fibrosis. Methods and Results: Treatment effects of CDR132L were investigated in several different pig models of ischemic and non-ischemic heart failure (HF) with the aim to further elucidate CDR132L’s mode of action and confirm species-independent effects on fibrotic remodeling. In a pig model of subacute post myocardial infarction (MI) induced HF, CDR132L was administered on day 3 and day 28 by intravenous (IV) or intracoronary (IC) infusion at doses of 1, 5 and 10 mg/kg and followed until day 56. In a chronic post-MI HF large animal model, beneficial effects were confirmed in a 6-month study comparing 5 monthly and 3 monthly repeated IV administrations of 5 mg/kg CDR132L starting one month post-MI. Further, CDR132L was tested in a porcine model of percutaneous aortic constriction by implantation of a reduction stent in the thoracic aorta and two IC injections of CDR132L on day 0 and day 28 followed for 56 days. As part of the pharmacodynamic analyses in these models, paraffin sections of cardiac tissue from the left ventricular remote region were stained with picrosirius red (PSR) and fibrosis was assessed based on collagen quantity. Analysis of fibrosis in PSR-staining revealed that the degree of interstitial fibrosis was significantly reduced in CDR132L treatment groups compared to placebo between 22% and 61%. Moreover, results of the first-in-human safety and tolerability study of CDR132L in patients with stable HF of ischemic origin (NCT04045405) showed encouraging positive trends for relevant cardiac fibrosis biomarkers. Conclusion: By blocking aberrant miR-132 expression, CDR132L is capable to halt and reverse pathological cardiac remodeling partly due to its anti-fibrotic effects.

Abstract 15115: Efficacy and Safety of miR-132 Inhibitor CDR132L in Patients With Reduced Left Ventricular Ejection Fraction After Myocardial Infarction: Rationale for and Design of the HF-REVERT Trial

Background: Inhibition of miR-132 effectively prevents and reverses heart failure (HF) in relevant models, making it an attractive HF target. CDR132L is a synthetic antisense oligonucleotide (ASO) selectively blocking miR-132. Translational in vitro and in vivo studies have demonstrated meaningful efficacy of CDR132L on left ventricular (LV) structure and function. CDR132L was safe and well tolerated in a Phase 1b study in chronic HF patients. The indicative effects of the miR-132 inhibitor CDR132L may be particularly beneficial in patients with active adverse remodelling, e.g. after acute myocardial infarction with early decline in ejection fraction. These patients have been relatively understudied and therapeutic options are limited. Method: The HF-REVERT (P h ase 2, Multicenter, Randomized, Parallel, 3-arm, Placebo-controlled Study to Assess E f ficacy and Safety of CD R 132L in Pati e nts with Reduced Left V entricular E jection F r ac t ion (≤ 45%) after Myocardial Infarction; NCT05350969) is a proof-of-concept study to evaluate the efficacy of CDR132L in improving cardiac function and safety in patients with reduced LV ejection fraction (LVEF). HF-REVERT aims to assess the efficacy and safety of 5 mg/kg and 10 mg/kg CDR132L, given as 3 single intravenous doses administered 28 days apart as an add-on to standard-of-care treatment, in a larger cohort of patients (N = 280) with HF and reduced LVEF (≤ 45%) after MI with a follow-up of 10 months. Key inclusion criteria are the diagnosis of acute STEMI/NSTEMI and the development of systolic dysfunction (LVEF ≤ 45%) after MI index event and elevated N-terminal pro B-type natriuretic peptide (NT-proBNP). The study consists of a 6-month double-blind treatment period with a read out of the primary endpoint LVESVI (left ventricular end systolic volume index) followed by a 6-month open label observation period. Discussion: The HF-REVERT trial currently tests CDR132L in patients with MI and systolic dysfunction. This trial may therefore underpin the concept of miR-132 inhibition to prevent/reverse cardiac remodeling in HF patients. The results will inform the design of a subsequent outcome trial to test the effect of CDR132L in HF patients.