Target Of miR-125b Research Articles

MiR-29b inhibits non-small cell lung cancer progression by targeting STRN4.

Non-small cell lung cancer (NSCLC) is a malignant tumor with a high fatality, low overall cure, and survival rates worldwide. When only palliative therapy is available, the disease leads to malignant proliferation. Previous studies showed miR-29b serves as an NSCLC suppressor by inhibiting cells proliferation, migration, and invasion. However, the mechanism underlying NSCLC progression remains elusive. In this study, we identified Striatin 4 (STRN4), a target of miR-29b, which serves as a pro-oncogenic protein by promoting cells proliferation, migration, and invasion in NSCLC. Besides, the STRN4 was highly expressed in NSCLC and negatively regulated by miR-29b. Down-regulation of STRN4 inhibits NSCLC cells proliferation, migration, invasion, and promotes apoptosis in vitro, whereas overexpression-induced enhanced cell migration and invasion could be reverved by miR-29b. Notably, overexpression of miR-29b and down-regulation of STRN4 by shRNA suppressed cellular proliferation and delayed tumor progression in vivo. Together, these findings identify a miR-29b/STRN4 regulatory pathway in NSCLC progression, which may provide a new sight for the treatment of NSCLC.

Extracellular Vesicles-Encapsulated MicroRNA-125b Produced in Genetically Modified Mesenchymal Stromal Cells Inhibits Hepatocellular Carcinoma Cell Proliferation.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and one of the prominent causes of cancer mortality, leading to approximately 780,000 deaths per year worldwide. Down-regulation of microRNA-125b (miR-125b) is a prognostic indicator in HCC patients. Conversely, over-expression of miR-125b in HCC cells induces cell cycle arrest, inhibits proliferation, migration and invasion. Extracellular vesicles (EVs) function as intercellular messengers transferring proteins, RNAs, DNAs, carbohydrates, and lipids. Since EVs protect their cargo from degradation, delivery of therapeutic bioactive molecules, in particular miRNAs, through EVs represents an innovative avenue for cancer therapy. In this study, we evaluated a replacement strategy for the treatment of HCC via delivery of EVs secreted from human adipose tissue-derived mesenchymal stromal/medicinal signaling cells (ASCs) genetically modified with a lentiviral vector expressing miR-125b with a specific ExoMotif sequence tag to enhance the loading into extracellular vesicles. In particular, we determined that the delivery of miR-125b-loaded EVs produced in engineered ASCs specifically reduces HCC cell proliferation in vitro modulating a series of miR-125b targets, which belong to the p53 signaling pathway. This proof-of-concept study supports the development of innovative therapeutic strategies for HCC via EV-mediated miRNA delivery.

Novel miR-29b target regulation patterns are revealed in two different cell lines

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b, have been detected in various biological fluids and tissues from a large variety of disease models. However, how miRNAs “react” and function in different cellular environments is still largely unknown. In this study, the regulation patterns of miR-29b between human and mouse cell lines were compared for the first time. CRISPR/Cas9 gene editing was used to stably knockdown miR-29b in human cancer HeLa cells and mouse fibroblast NIH/3T3 cells with minimum off-targets. Genome editing revealed mir-29b-1, other than mir-29b-2, to be the main source of generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells.

Hsa-miRNA-125b may induce apoptosis of HTR8/SVneo cells by targeting MCL1

MiR-125b regulates the kinds of cells that undergo apoptosis physiologically and pathologically. However, whether miR-125b affects the apoptotic behavior of trophoblasts and the underlying molecular regulatory mechanisms remains unclear. This study investigated the effect of miR-125b on apoptosis of HTR-8/SVneo cells in vitro. Constructed wild-type reporter vector (Wt-3′UTR) or mutated type reporter vector (Mut-3′UTR) reporter plasmids were transiently transfected into 293 T cells along with miR-125b mimics or a negative control. The luciferase reporter assay was used to validate whether the predicted MCL1 gene is a direct target of miR-125b. The HTR8/SVneo cells were transfected with miR-125 mimics, inhibitors, or a scramble control. Real-time polymerase chain reaction and western blotting were used to analyze mRNA and protein expression of the target gene MCL1. Flow cytometry was used to determine the effects on apoptosis. The luciferase activity assay validated the ability of miR-125b to specifically attenuate MCL1 transcription in the 293 T cell line, suggesting that MCL1 is a direct target of miR-125b. After transfection by miR-125b, relative expression and translation of the target gene MCL1 mRNA were repressed in HTR-8/SVneo cells. Trophoblast cells were induced to undergo apoptosis by overexpressing miR-125b in the HTR-8/SVneo cell line. In conclusion, MiR-125b may induced apoptosis of HTR8/SVneo cells by targeting MCL1. Our findings suggest that miR-125b may play a pivotal role in the pathophysiology of placentation.

Long Non-coding RNA H19 Suppression Protects the Endothelium Against Hyperglycemic-Induced Inflammation via Inhibiting Expression of miR-29b Target Gene Vascular Endothelial Growth Factor a Through Activation of the Protein Kinase B/Endothelial Nitric Oxide Synthase Pathway.

It has been shown that non-coding RNAs (ncRNAs) play an important regulatory role in pathophysiological processes involving inflammation. The vascular endothelial growth factor A (VEGFA) gene also participates in the inflammatory process. However, the relationships between ncRNAs and VEGFA are currently unclear. Here, this study was designed to determine the relationship between long non-coding RNA (lncRNA) H19, mircoRNA29b (miR-29b), and VEGFA in the development of diabetes mellitus (DM). We demonstrate that H19 is upregulated and miR-29b downregulated in individuals with DM and directly binds miR-29b. VEGFA is the target of miR-29b in the vascular endothelium of individuals with DM. We found that positive modulation of miR29b and inhibition of H19 and VEGFA significantly attenuates high glucose-induced endothelial inflammation and oxidative stress. We also found that the protein kinase B/endothelial nitric oxide synthase (AKT/eNOS) signal pathway in endothelial cells is activated through regulation of miR29b and H19 endogenous RNAs. We conclude that H19 suppression protects the endothelium against high glucose-induced inflammation and oxidative stress in endothelial cells by upregulation of miR-29b and downregulation of VEGFA through AKT/eNOS signal pathway activation. These results suggest a novel link between dysregulated ncRNA expression, inflammation, and the signaling pathway in the vascular endothelium of individuals with DM, indicating a promising strategy for preventing cardiovascular disease in such individuals.

Regulation of Fibroblast Apoptosis and Proliferation by MicroRNA-125b in Systemic Sclerosis.

To analyze the expression, regulation, and role of microRNA-125b (miR-125b) in systemic sclerosis (SSc). MiR-125b expression was assessed by quantitative polymerase chain reaction (qPCR) of RNA from dermal fibroblasts and whole skin biopsy specimens from healthy controls and SSc patients. To identify downstream effectors, RNA from healthy control fibroblasts was sequenced after miR-125b knockdown and further validated using qPCR and Western blotting. Fibrosis, apoptosis, and proliferation were assessed by Caspase-Glo 3/7 assay, Western blotting, immunofluorescence staining for cleaved caspase 3, and annexin V real-time assay in dermal fibroblasts. Expression of miR-125b was significantly down-regulated in SSc skin biopsy specimens by 53% (median fold change 0.47 [interquartile range 0.35-0.69]; P < 0.001) and in SSc dermal fibroblasts by 47% (median fold change 0.53 [interquartile range 0.36-0.58]; P < 0.001) compared to healthy control skin biopsy specimens and fibroblasts, respectively (n = 10 samples per group). Treatment with the histone deacetylase inhibitors trichostatin A and tubastatin A significantly decreased the expression of miR-125b in dermal fibroblasts. MiR-125b knockdown significantly reduced cell proliferation and α-smooth muscle actin (α-SMA) expression at the messenger RNA (mRNA) and protein levels. RNA-Seq identified BAK1, BMF, and BBC3 as potential targets of miR-125b. Quantitative PCR confirmed that knockdown of miR-125b up-regulated these genes (P < 0.01; n = 12). Bcl-2 homologous antagonist killer 1 showed the strongest induction confirmed at the protein level (P < 0.01; n = 10). Consequently, miR-125b knockdown increased apoptosis compared to scrambled control. Accordingly, miR-125b overexpression decreased apoptosis. Our findings indicate that miR-125b is down-regulated in SSc skin and primary dermal fibroblasts. MiR-125b down-regulation increases apoptosis and decreases proliferation and α-SMA expression in dermal fibroblasts, indicating that its compensatory, antifibrotic mechanism may be a potential novel therapeutic option.

Long noncoding RNA IRAIN acts as tumor suppressor via miR-125b in multiple myeloma.

Long noncoding RNAs (lncRNAs) are aberrantly expressed in a variety of cancer types. The lncRNA IGF1R antisense imprinted non-protein coding RNA (IRAIN) is associated with various cancer types, yet the role of IRAIN in multiple myeloma (MM) progression remains unclear. In the present study it was identified that IRAIN may act as a tumor suppressor in MM, whilst microRNA (miR)-125b may promote tumorigenesis. Downregulation of IRAIN significantly increased the expression of miR-125b. Furthermore, by using dual-luciferase reporter assays, IRAIN was identified as a target of miR-125b. Knockdown of IRAIN promoted MM cell proliferation in vitro. Thus, expression levels of IRAIN may be used to predict the clinical prognosis of patients with MM and may be a novel therapeutic target for treating MM.

MicroRNA-29b inhibits cell growth and promotes sensitivity to oxaliplatin in colon cancer by targeting FOLR1.

The present study was aimed to explore the functional role of microRNA (miR)-29b in colon cancer, as well as underlying mechanisms. Expressions of miR-29b and folate receptor 1 (FOLR1) were measured in both human colon tumor samples and cell lines. Colon cancer cell lines SW480 and SW620 were transfected with miR-29b mimic, antisense oligonucleotides (ASO)-miR-29b, small interfering (siRNA) against FOLR1 (si-FOLR1), or corresponding negative controls (NCs), and then were incubated with or without oxaliplatin (L-OHP). Thereafter, cell viability, cytotoxicity, cell apoptosis, and expression of FOLR1, ATP Binding Cassette Subfamily G Member 2 (ABCG2) and p-glycoprotein (p-gp) were analyzed. We found that miR-29b was significantly decreased, while FOLR1 was statistically elevated in colon cancer samples and cell lines compared to the nontumor samples and nontumourigenic immortalized human colon epithelial cell line FHC. Overexpression of miR-29b markedly inhibited cell viability, promoted sensitivity to L-OHP, stimulated cell apoptosis (all p < .05), and decreased the levels of ABCG2 and p-gp in cancer cells, whereas suppression of miR-29b showed contrary results. Moreover, we observed that FOLR1 was a direct target of miR-29b and was negatively regulated by miR-29b. In addition, the findings revealed that the effects of FOLR1 inhibition on cell viability, sensitivity to L-OHP, cell apoptosis, and the levels of ABCG2 and p-gp were similar to overexpression of miR-29b. Taken together, our study suggests that miR-29b inhibits cell growth and promotes sensitivity to L-OHP in colon cancer by targeting FOLR1.

Copper Transporter ATP7A (Copper-Transporting P-Type ATPase/Menkes ATPase) Limits Vascular Inflammation and Aortic Aneurysm Development: Role of MicroRNA-125b.

Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

MicroRNA-mediated downregulation of K+ channels in pulmonary arterial hypertension.

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel β1 subunit (BKCaβ1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaβ1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

TNF-α-elicited miR-29b potentiates resistance to apoptosis in peripheral blood monocytes from patients with rheumatoid arthritis.

CD14-positive monocytes from patients with rheumatoid arthritis (RA) are more resistant to apoptosis, which promotes their persistence at the inflammatory site and thereby contributes crucially to immunopathology. We sought to elucidate one mechanism underlying this unique pathogenesis: resistance to apoptosis and the potential involvement of miR-29b in this process. CD14-positive peripheral blood monocytes (PBMs) from RA patients were observed to be resistant to spontaneous apoptosis compared to PBMs from healthy volunteers. Intriguingly, expression of miR-29b was significantly upregulated in PBMs from RA patients than those from healthy volunteers, and this upregulation was correlated with RA disease activity. Functionally, forced expression of the exogenous miR-29b in CD14-positive Ctrl PBMs conferred resistance to spontaneous apoptosis and Fas-induced death, thereafter enhancing the production of major proinflammatory cytokines in there cells. Following identification of the potential miR-29b target transcripts using bioinformatic algorithms, we showed that miR-29b could directly bind to the 3'-UTR of the high-mobility group box-containing protein 1 (HBP1) and inhibited its transcription in PBMs. Importantly, stable expression of the exogenous HBP1 in differentiated THP-1 monocytes effectively abolished miR-29b-elicited resistance to Fas-induced apoptosis. Finally, among patients with RA and good clinical responses to immunotherapy, expression levels of miR-29b were significantly compromised in those treated with infliximab (a TNF-α inhibitor) but not in those treated with tocilizumab (a humanized mAb against the IL-6 receptor), pointing to a potential association between miR-29b activation and TNF-α induction. The available data collectively suggest that TNF-α-elicited miR-29b potentiates resistance to apoptosis in PBMs from RA patients via inhibition of HBP1 signaling, and testing patients for miR-29b/HBP1 expression ratios may provide more accurate prognostic information and could influence the recommended course of immunotherapy.

MiR-29b Reverses T helper 1 cells/T helper 2 cells Imbalance and Alleviates Airway Eosinophils Recruitment in OVA-Induced Murine Asthma by Targeting Inducible Co-Stimulator

Asthma is a complex chronic disease and the pathogenesis is still not entirely clear. In this study, we aimed to clarify the role and mechanism of miR-29b in the development of asthma. We observed that miR-29b levels were decreased in the lung and spleen of OVA-induced asthmatic mice. Reverse transcription-quantitative polymerase chain reaction and flow cytometry demonstrated that the inducible co-stimulator (ICOS) expression at mRNA and protein levels was elevated in the lung of asthmatic mice, and miR-29b expression in the lung of asthmatic mice was negatively associated with ICOS mRNA levels by Pearson Correlation analysis. Additional, flow cytometry showed that the percentage of CD4⁺ICOS⁺ T cells in the lung and spleen was regulated by miR-29b, and dual luciferase reporter assay confirmed ICOS was a target gene of miR-29b. Furthermore, miR-29b overexpression in asthmatic mice was induced with miR-29b agomir by intranasal administration; miR-29b alleviated total inflammatory cell infiltration and CCL24 levels, decreased IL-5 levels in bronchoalveolar lavage fluid and serum, and upregulated IFN-γ expression in serum. This study demonstrates that miR-29b targets ICOS, thereby reverses the imbalance of T helper 1 cells (Th1)/Th2 responses and decreases eosinophils recruitment in the airway, which are key features of allergic airway inflammation. Therefore, miR-29b might be an attractive candidate target for asthma treatment.

MiR-125b Suppression Inhibits Apoptosis and Negatively Regulates Sema4D in Avian Leukosis Virus-Transformed Cells.

Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3′-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.

Mir-29b mediates the regulation of Nrf2 on airway epithelial remodeling and Th1/Th2 differentiation in COPD rats.

COPD, or Chronic obstructive pulmonary disease, is an inflammation-related disease and lead to cachexia and muscle wasting. Altered nuclear factor erythroid 2-related factor 2 (Nrf2) expression is found in patients of COPD because it is involved in pulmonary protective effects. MiR-29b could be activated by Nrf2. We hypothesized that miR-29b might mediate the regulation of Nrf2 on Th1/Th2 differentiation and airway epithelial remodeling in COPD rats. SD rats were exposed to smoke for COPD induction. Expression of Nrf2 mRNA and miR-29b in lung tissues was quantified. Expression of Nrf2 and matrix metalloproteinase 2 (MMP2) were also detected by immunohistochemistry and western blot. Th1 markers and Th2 markers were measured by ELISA in peripheral blood. Flow cytometry was used to detect the Th1/Th2 ratio. miR-29b and Nrf2 was manipulated at mRNA level in A549 cells using transfection. Cellular growth and migration were measured in transfectants. In lung tissues of COPD rats, expression of Nrf2 and miR-29b decreased. MMP2, a target of miR-29b, had an opposite expression to miR-29b in peripheral blood. Levels of inflammatory factors and Th1/Th2 ratio increased. MiR-29b mediated the regulation of Nrf2 on remodeling of lung epithelial cells. Blocking Nrf2 expression in A549 cells led to the opposite expression of miR-29b and further decreased MMP2 production; meanwhile, cell growth and motility were improved. Different miR-29b levels affected MMP2 expression and cellular characteristics. The findings suggested that miR-29b was a regulator the pathological progress of COPD. It mediates the effect of Nrf2 on Th1/Th2 differentiation and on remodeling process of airway epithelial cells.

Abstract 3558: Interrogation of onco-microRNA 125b reveals androgen receptor as a key upstream transcriptional factor that is targetable in female gastric cancer

Abstract Background &amp; Aims: Our prior study revealed a miRNA-regulatory network by an integrated analysis of microRNA and mRNA profiling of gastric cancer (GC). It defined a GC microRNA subtype that was associated with poor survival in GC cases. In this study, we further demonstrate that onco-microRNA miR-125b, a key node in this microRNA regulatory network, is upregulated in GC and associated with poor overall survival by interfering in the apoptosis pathway and this pathway can be regulated by androgen receptor (AR). Our study also clarifies the potential clinical utility of the AR antagonist Bicalutamide on GC treatment. Methods: To further investigate the role of AR-miR-125b axis in GC, we conducted both in vitro experiment and clinical study with a cohort of 373 GC samples. The expression of miR-125b and AR was analyzed and compared with patient survival, and its implications were evaluated between genders of GC cases. We explored the tumor suppressive effect of bicalutamide using in vitro assays and in vivo subcutaneous xenotransplanted tumor model of human GC cell lines in nude mice. Results: The markedly up regulated expression of AR-miR-125b axis was validated in 373 GC samples. Univariate and multivariate analyses find that the miR-125b and/or AR were associated with poor prognosis and this trend was more pronounced among female than male cases. Pathway analysis shows that the predicted targets of miR-125b are highly involved in apoptosis/program death pathway. The robust apoptosis genes, BIK and CASP6 are validated as the directed targets of miR-125b. miR-125b can suppress apoptosis and increase cell growth, migration and invasion in GC cell lines. Furthermore, low expression of BIK and CASP6 in GC were associated with poor disease-free survival. There was a notable positive correlation between miR-125b and AR level in GC samples. Chip, EMSA and luciferase-binding assay confirmed that AR can direct regulated miR-125b expression as a transcriptional factor. We examined AR antagonist bicalutamide to inhibit proliferation and increased apoptosis in AR positive gastric cancer cells. The effect of bicalutamide is more apparent in female than in male nude mice. Conclusions: Our findings suggest that miR-125b regulated apoptosis pathway is important to GC progression and that the AR/miR-125 may be an important clinical biomarker for GC survival and possible therapeutic target for GC treatment. Citation Format: Ben Liu, Meng Zhou, Da Yang, Qinghua Wang, Qiang Zhang, Xiangchun Li, Meng Yang, Qiong Wang, Lian Li, Luyang Liu, Hongji Dai, Fengju Song, Hong Zheng, Wei Zhang, Kexin Chen. Interrogation of onco-microRNA 125b reveals androgen receptor as a key upstream transcriptional factor that is targetable in female gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3558.

Abstract 763: The regulation of miR-125b1-peroxiredoxin like 2A anti-oxidative activity in oral squamous cell carcinoma

Abstract Head and neck cancer, including oral squamous cell carcinoma (OSCC), is the sixth most common cancer worldwide. MicroRNAs are small non-coding RNAs that are involved in the modulation of biological/pathological properties. Peroxiredoxin like 2A (PRXL2A) has been reported to be an antioxidant protein that protects cells from oxidative stress. Our previous study identified an association between PRXL2A up-regulation in OSCC and a worse patient prognosis. The miR-125 family of genes drive pluripotent regulation across a wide variety of cancers. In this study, we identify the oncogene eligibility of PRXL2A and clarify miR-125b as its upstream regulator. Down-regulation of miR-125b can be observed in OSCC tumors. Lower miR-125b expression in tumors results in a worse patient prognosis at the relatively early stage. Reporter assays were able to validate that PRXL2A is a direct target of miR-125b. Exogenous miR-125b expression in OSCC cells results in increased oxidative stress and drug sensitivity, and suppressor activity that is paralleled by the knockout of PRXL2A gene. The suppressor activity of miR-125b is able to be rescued by PRXL2A, which suggests the existence of a miR-125b-PRXL2A regulatory axis that is part of OSCC pathogenesis. Nuclear factor erythroid-2-related factor was found to be a downstream effector of the miR-125b-PRXL2A cascade. As a whole, this study has pinpointed novel clues demonstrating that down-regulation of miR-125b suppressor underlies up-regulation of PRXL2A in OSCC, and this then protects the affected tumor cells from oxidative stress resulting in a worse prognosis. Citation Format: Yi-Fen Chen, Yun-Yen Wei, Cheng-Chieh Yang, Kuo-Wei Chang, Shu-Chun Lin. The regulation of miR-125b1-peroxiredoxin like 2A anti-oxidative activity in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 763.

Abstract 2034: Activation of cancer-associated fibroblasts via the transfer of miR-125b containing extracellular vesicles from tumor cells

Abstract Tumor cells secrete elevated amounts of extracellular vesicles (EVs) as a mean of communication between them and other cells in the tumor microenvironment. Here, we established a series of breast cancer cell lines releasing fluorescent EVs to identify the common cell types that receive tumor EVs within the tumor microenvironment. Among the different cell types, cancer-associated fibroblasts consistently take up the most EVs from cancer cells. Using next generation sequencing, we find that miR-125b is among the top enriched miRNAs in EVs released by mouse isogenic triple-negative breast cancer 4T1 and 4TO7 cell lines. Co-injection of 4TO7 cells with 4T1 EVs results in an upregulation of fibroblast activation in vivo while blockage of miR-125b in 4T1 EVs rescues the effect indicating that EV-delivery of miR-125b is crucial for the fibroblast activation in mouse breast cancer models. Moreover, EVs from human breast cancer cells also contain abundant miR-125b and fibroblasts within the xenografted tumor that take up these EVs gain higher levels of miR-125b and upregulation of cancer-associated fibroblast markers. Both mouse and human fibroblasts that are transfected with miR-125b mimics show an activated phenotype similar to the knockdown of established miR-125b targets. In summary, normal fibroblasts within the tumor microenvironment actively take up the miR-125b-containing EVs from breast cancer cells and develop into cancer-associated fibroblasts. Citation Format: Luyen Tien Vu, Boya Peng, Daniel Xin Zhang, Victor Ma, Andrew Grimson, William C. Cho, Judy Lieberman, Minh TN Le. Activation of cancer-associated fibroblasts via the transfer of miR-125b containing extracellular vesicles from tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2034.

MicroRNA-125b controls growth of ovarian granulosa cells in polycystic ovarian syndrome by modulating cyclin B1 expression.

IntroductionThere is a lot of evidence that suggests that microRNAs (miRs) play an imperative role in the pathogenesis of polycystic ovary syndrome (PCOS). This study was designed to decipher the role of miR-125b in PCOS pathogenesis.Material and methodsExpression analysis of miR-125b was determined by real-time quantitative polymerase chain reaction and the KGN ovarian granulosa cell viability was examined by CCK-8 assay. DAPI assay and flow cytometry were carried out for the detection of apoptosis and cell cycle distribution respectively. Protein levels were checked by immunoblotting.ResultsThe miR-125b transcript levels were considerably high in polycystic ovaries and ovarian granulosa KGN cells. The inhibition of miR-125b expression decreased the viability of the KGN cells by arresting the cells at the G2/M check point. Target Scan analysis revealed cyclin B1 as the target of miR-125b and suppression of miR-125b caused considerable up-regulation of cyclin B1 expression. Like miR-125b inhibition, cyclin B1 silencing also inhibited the KGN cell viability via G2/M arrest. Ectopic expression of miR-125b was unable to nullify the effects of cyclin-B silencing on KGN cell viability but the overexpression of cyclin B1 nullified the effects of the miR-125b suppression on KGN cell proliferation.ConclusionsSince miR-125b controls the proliferation rate of granulosa cells in polycystic ovaries, it might be addressed as a potential therapeutic target for PCOS patients

2183-P: miR-125b Is Regulated by Glucose via AMPK and Impairs ß-Cell Function

miRNAs are tiny RNAs that repress gene expression post-transcriptionally, necessary for normal endocrine function. AMPK is essential for glucose homeostasis and a suggested target of metformin. β-cell-specific inactivation of AMPK in mice (AMPKdKO) impairs glucose homeostasis and β-cell function. AMPKdKO mice present dysregulation of several miRNAs that may contribute to these defects, including miR-125b (∼2.5 fold upregulated). High levels of miR-125b are associated with hyperglycaemia (HbA1c) in prediabetic, T2D and T1D subjects, suggesting miR-125b as a biomarker or contributor to the development of diabetes. The role of miR-125b in β-cells remains elusive although in other cell types regulates apoptosis, growth and differentiation. We aim to determine miR-125b function in β-cells and the role of glucose and AMPK in the regulation of its expression. miR-125b level in human islets strongly correlates with BMI and is induced by glucose in both mouse and human islets, while feeding mice a ketogenic (low sugar) diet reduced islet miR-125b. These effects disappeared in islets with β-cell specific deletion of AMPK whereas treatment of human islets with highly specific AMPK activators significantly decreased miR-125b, indicating that glucose regulates miR-125b via AMPK in both mice and humans. miR-125b overexpression in MIN6 cells limited cytokine-induced apoptosis whilst strongly reducing insulin content. RNA-Seq on these cells suggests a role for miR-125b in metabolism, respiration and cytokine receptor interactions and RNA-IP of the miRNA-induced silencing complex revealed novel miR-125b targets such as mannose-6-phosphate receptor (MPR), important for enzyme sorting within secretory granules. Preliminary data show that β-cell-specific inhibition of miR-125b using a molecular “sponge” improves glucose tolerance in mice fed a high-fat diet. In summary, we reveal miR-125b as an important regulator of β-cell function that might contribute to the deleterious effects of hyperglycaemia on β-cells. Disclosure R. Cheung: None. G. Pizza: None. D.M. Rolando: None. P.L. Chabosseau: None. M. Nguyen-Tu: None. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma. A. Martinez-Sanchez: None. Funding Medical Research Council UK

Propranolol suppresses infantile hemangioma cell proliferation and promotes apoptosis by upregulating miR-125b expression.

Propranolol could repress infantile hemangioma cell growth and induce apoptosis. miR-125b could inhibit cell proliferation in some tumors. However, whether propranolol exerts its proliferation inhibition and apoptosis-promoting effect by regulating the expression of miR-125b needs to be further investigated. In tumor tissue and endothelial cells isolated from infantile hemangioma patients, we found that the expression levels of miR-125b were significantly decreased. In-vitro analysis revealed that propranolol increased the expression of miR-125b in hemangioma cells in a dose-dependent and time-dependent manner. Interestingly, it was observed that regression of miR-125b expression by its inhibitor could abrogate the effect of propranolol on hemangioma cell growth and apoptosis. In addition, our data further identified TFAP4 as a direct target of miR-125b. Collectively, our data provided evidence that propranolol may repress infantile hemangioma cell growth and promote apoptosis through upregulating the miR-125b expression, which exerted its suppression of tumor development by targeting TFAP4.