UTR Luciferase Reporter Assay Research Articles

MicroRNA-155-5p Targets JADE-1, Promoting Proliferation, Migration, and Invasion in Clear Cell Renal Cell Carcinoma Cells.

Clear cell renal cell carcinoma (ccRCC) incidence has been rising in recent years, with strong association between differential microRNA (miRNA) expression and neoplastic progression. Specifically, overexpression of miR-155-5p has been associated with promoting aggressive cancer in ccRCC and other cancers. In this study, we further investigate the role of this miRNA and one of its protein targets, Jade-1, to better understand the mechanism behind aggressive forms of ccRCC. Jade-1, a tumor suppressor, is stabilized by Von-Hippel Lindau (VHL), which is frequently mutated in ccRCC. Experiments featuring downregulation of miR-155-5p in two ccRCC cell lines (786-O and Caki-1) attenuated their oncogenic potential and led to increased levels of Jade-1. Conversely, knockdown experiments with an anti-Jade-1 shRNA in 786-O and Caki-1 cells showed increased metastatic potential through elevated proliferation, migration, and invasion rates. In a mouse xenograft model, downregulation of miR-155 decreased the rate of tumor implantation and proliferation. Direct interaction between miR-155-5p and Jade-1 was confirmed through a 3'UTR luciferase reporter assay. These findings further elucidate the mechanism of action of miR-155-5p in driving an aggressive phenotype in ccRCC through its role in regulating Jade-1.

Abstract 14672: Regulation of Succinate Signaling and Immunometabolism in Diabetes-Associated Atherosclerosis by Microrna-369-3p

Introduction: The risk of atherosclerotic cardiovascular disease (ASCVD) is increased in people with diabetes. Diabetes (D) is associated with impaired metabolism and functionality of macrophages (Mps), resulting in chronic inflammation. Identifying factors to combat metabolic alterations and promote resolution of inflammation remains an unmet goal. microRNAs (miRs) orchestrate multiple signaling events in Mps and regulate inflammation. Hypothesis: miR-369-3p regulates metabolic reprogramming in Mps during diabetes-associated atherosclerosis (D-AS). Methods and Results: miRNA-seq profiling from aortic intimal lesions of LDLR-/- mice on a high-fat sucrose containing (HFSC) diet revealed miR-369-3p downregulation with D-AS progression. miR-369-3p was also downregulated in PBMCs derived from D and D-associated ASCVD patients. Cell-type profiling showed miR-369-3p enrichment in mouse bone marrow derived Mps (BMDMs). oxLDL-stimulated BMDMs (foam cells, FCs) resulted in marked downregulation of miR-369-3p, increased secretion of IL1-β, and metabolic shift of oxidative phosphorylation (OXPHOS) to glycolysis, while miR-369-3p overexpression (miR-369-3p O/E) favored OXPHOS over glycolysis and reduced IL1-β secretion. Metabolomics revealed succinate, a key immunometabolite was profoundly elevated extracellularly from FCs, while miR-369-3p O/E reversed this phenotype. Extracellular succinate activates its receptor, GPR91. Indeed, GPR91 was upregulated in FCs while miR-369-3p O/E significantly repressed GPR91. 3’UTR luciferase reporter assay confirmed GPR91 as a miR-369-3p target. miR-369-3p O/E dampened GPR91-mediated inflammasome activation and mitochondrial dysfunction in FCs by decreasing p-ERK, p-DRP1, and NLRP3. Therapeutic delivery of miR-369-3p in LDLR-/- mice fed HFSC diet for 12 weeks reduced plaque size by ORO staining and plasma IL1-β. Flow cytometry of aortic Mps revealed that miR-369-3p decreased GPR91 expression by ~50%, along with a decrease in M1 and increase in M2-like-Mps. Conclusions: miR-369-3p confers a pro-resolving role in regulating lipid-driven inflammation and metabolic rewiring in Mps via targeting GPR91 and preventing D-AS plaque progression.

The reversion of DNA methylation-induced miRNA silence via biomimetic nanoparticles-mediated gene delivery for efficient lung adenocarcinoma therapy

BackgroundLung cancer is one of the fatal cancers worldwide, and over 60% of patients are lung adenocarcinoma (LUAD). Our clinical data demonstrated that DNA methylation of the promoter region of miR-126-3p was upregulated, which led to the decreased expression of miR-126-3p in 67 cases of lung cancer tissues, implying that miR-126-3p acted as a tumor suppressor. Transduction of miR-126-3p is a potential therapeutic strategy for treating LUAD, yet the physiological environment and properties of miRNA challenge current transduction approaches.MethodsWe evaluated the expression of miR-126-3p in 67 pairs of lung cancer tissues and the corresponding adjacent non-tumorous tissues by Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The relationship between the overall survival of lung cancer patients and miR-126-3p was analyzed by the Cancer Genome Atlas cohort database (Oncolnc, http://www.oncolnc.org). We analyzed DNA methylation Methylation-specific PCR (MSP) analysis. To determine whether ADAM9 is the direct target of miR-126-3p, we performed the 3′-UTR luciferase reporter assay. The protein levels in the cells or tissues were evaluated with western blotting (WB) analysis. The biodistribution of nanoparticles were monitored by in vivo tracking system.ResultsWe describe the development of novel stealth and matrix metalloproteinase 2 (MMP2)-activated biomimetic nanoparticles, which are constructed using MMP2-responsive peptides to bind the miR-126-3p (known as MAIN), and further camouflaged with red blood cell (RBC) membranes (hence named REMAIN). REMAIN was able to effectively transduce miRNA into lung cancer cells and release them via MMP2 responsiveness. Additionally, REMAIN possessed the advantages of the natural RBC membrane, including extended circulation time, lower toxicity, better biocompatibility, and immune escape. Moreover, in vitro and in vivo results demonstrated that REMAIN effectively induced apoptosis of lung cancer cells and inhibited LUAD development and progression by targeting ADAM9.ConclusionThe novel style of stealth and MMP2-activated biomimetic nanoparticles show great potential in miRNA delivery.

MiR-142-3p simultaneously targets HMGA1, HMGA2, HMGB1, and HMGB3 and inhibits tumorigenic properties and in-vivo metastatic potential of human cervical cancer cells

AimsHigh-mobility group (HMG) proteins are oncogenic in different cancers, including cervical cancer; silencing their individual expression using sh-RNAs, siRNAs, and miRNAs has had anti-tumorigenic effects, but the consequences of their collective downregulation are not known. Since multiple gene targeting is generally very effective in cancer therapy, the present study highlighted the consequences of silencing the expression of HMGA1, A2, B1, and B3 using sh-RNAs or miR-142-3p (that can potentially target HMGA1, A2, B1, and B3) in cervical cancer cell lines. Main methods3′ UTR luciferase reporter assays were performed to validate HMGA1, A2, B1, and B3 as targets of miR-142-3p in human cervical cancer cells. Annexin V/PI dual staining and flow cytometry analyses were used to detect apoptotic cells. miR-142-3p-mediated regulation of cell death, colony formation, migration, and invasion was investigated in human cervical cancer cells together with in vivo metastasis in zebrafish. Key findingsConcurrent knockdown of HMGA1, A2, B1, and B3 through their corresponding sh-RNAs inhibited cell viability and colony formation but induced apoptosis, and these effects were relatively reduced upon their individual knockdown. miR-142-3p targeted HMGA1, A2, B1, and B3 by binding to their 3′UTRs and induced apoptosis but inhibited proliferation, migration, and invasion of human cervical cancer cells. In addition, miR-142-3p expression decreased phospho-p65 and EMT-related proteins in cervical cancer cells and their in vivo metastatic potential upon implantation in zebrafish. SignificanceThese findings suggest that miR-142-3p acts as a tumor-suppressive miRNA by targeting HMGA1, A2, B1, and B3 and may serve as a potential therapeutic agent in human cervical cancer.

MiR-223-3p targets FBXW7 to promote epithelial-mesenchymal transition and metastasis in breast cancer.

BackgroundBreast cancer is the most common malignant tumor diagnosed in women. It is the second leading cause of cancer‐related death among women in the world. Aberrant expression of microRNAs (miRNAs) have been identified to be involved in the development and progression of breast cancer. The aim of this study was to investigate the function of miR‐223‐3p in breast cancer progression and metastasis.MethodsqRT‐PCR was used to analyze the expression levels of miR‐223‐3p in breast cancer tissues and cell lines. Wound healing and Matrigel assays were used to examine cell motility and invasiveness. FBXW7 3′‐UTR construct and luciferase reporter assays were performed for the target gene.ResultsmiR‐223‐3p was overexpressed in breast cancer tissue and cell lines. A high level of miR‐223‐3p was associated with poor prognosis in breast cancer patients. In addition, overexpressed miR‐223‐3p promoted the migration and invasion of breast cancer cells in vitro and in vivo. Mechanistically, we found that tumor suppressor gene FBXW7 is a target of miR‐223‐3p. Luciferase activity reporter assay indicated miR‐223‐3p could directly bind with the 3′‐UTR of FBXW7. miR‐223‐3p exhibited its oncogenic role partly by decreasing FBXW7 expression, and consequently promoted the invasion and metastasis of breast cancer cells.ConclusionsOur study revealed a physical and functional relationship among miR‐223‐3p and FBXW7. By negatively regulating FBXW7 expression, miR‐223‐3p exerts a tumor promotion role promoting cell invasion and metastasis in breast cancer.

Abstract 9561: Loss of Full Length Pumilio 1 Abrogates Gene P27 Silencing-Mediated Cell Proliferation and Secures Mirna-221 Mimics Therapeutics in the Heart

Introduction: MiR-221 mimics have been demonstrated to be cardioprotective and anti-fibrotic in the treatment of myocardium infarction and heart failure. However, miR-221 is a known oncomiR as the upregulation of miR-221 and the downregulation of its target p27 are associated with tumor progression and poor prognosis. Therefore the treatment of miR-221 mimics raise safety concerns. Hypothesis: miR-221 fails to trigger P27 mediated neoplastic cellular proliferation due to loss of full length pumilio-1 (FL PUM-1) expression in vivo . Methods: H9c2, Hela and primary cardiac fibroblasts were used to study the regulations of miR-221 mimics on P27 by RT-qPCR and Western blot, cell proliferation by cell number, CCk-8 and Ki67 staining. The interaction with PUM1 was assessed by siRNA transfection, p27 3’UTR cloning and luciferase reporter assay. In vivo 2 days after miR-221 mimic (1mg/kg and 4mg/kg) i.v. injection in the rats, the liver, heart, kidney and muscle were collected to measure p27 and PUM1 by RT-qPCR and WB. Pum1 cDNA was cloned using primers flanking its start and stop codons, and inserted into pcDNA3.1 vector. 10 colonies from each tissue were sequenced to verify mRNA splicing variants. PUM1 cleavage were verified by the incubations of cathepsin K (CatK) inhibitor odanacatib (ODN). Results: in vitro miR-221 mimics significantly downregulated the expression of P27 and increased cell proliferation. FL PUM1 (140kDa) and isoforms of 105 and 90 kDa were detected. The transfections of siRNAs to knockdown PUM1 but not PMU2 abolished miR-221-mediated effects and direct binding of miR-221 on p27 3’UTR. In vivo following two dosages miR-221 mimic treatments, the expressions of miR-221 increased in the heart, liver, kidney and muscle for 6.8 and 17.3 fold, 33.4 and 506.5 fold, 2.2 and 3.5 fold, and no significant increases respectively. P27 was not affected. FL-PUM1 was not detected in all tissues, but only isoforms. 105 kDa was generated through alternative RNA slicing and 90 kDa by CatK cleavage. Conclusions: This is the first study to address the safety concerns with respect to the possible pro-proliferative effects of miR-221 mimic therapeutics in cardiovascular applications. It provides essential insights of the context-dependent nature of miRNA functionality.

MicroRNA-20a suppresses RANKL-modulated osteoclastogenesis and prevents bone erosion in mice with rheumatoid arthritis through the TLR4/p38 pathway.

Abnormal osteoclast formation plays a significant part in rheumatoid arthritis (RA). As potent therapeutic biomarkers, microRNAs (miRNAs) have obtained increasing attention. Recently, treatment regimens regarding miRNAs have been implicated in skeletal diseases. The aim of this study is to assess the expression and function of miR-20a during osteoclast proliferation and differentiation and its correlation with bone erosion in RA mice. The expression of miR-20a was observed to be diminished in the ankle tissues of RA mice relative to that in normal controls evaluated by RT-qPCR. Hematoxylin and eosin staining, Safranin O-fast green staining, and tartrateresistant acid phosphatase staining were used to evaluate the effects of miR-20a on RA symptoms. The proliferation and differentiation of osteoclasts, and bone erosion were repressed by agomiR-20a injection. 3'UTR luciferase reporter assays were conducted to validate the putative binding between miR-20a and receptor activation of nuclear factor-κB ligand (RANKL). The protein expression and phosphorylation level of toll-like receptor4 (TLR4)/p38 pathway-related factors were detected by Western blot. miR-20a inhibited proliferation and differentiation potentials to osteoclasts partly through the TLR4/p38 pathway. The current work provides evidence that miR-20a hinders proliferation and differentiation of osteoclasts by targeting RANKL through the TLR4/p38 pathway.

Abstract 16176: Newly Identified MiR-6770-3p Dramatically Induces Angiogenesis in Human Endothelial Cells While Uncommonly Decreasing Cell Proliferation

Micro-RNAs (miRs) are small non-coding RNAs that alter the expression of multiple mRNA targets. Although they participate in physiological processes, dysregulation of their expression is implicated in various diseases. We investigated whether miRs could be involved in the regulation of FXYD1 expression, a sub-unit of the Na+/K+-ATPase pump, that we previously described as both cardio- and vasculoprotective, under oxidative stress conditions. Using in silico analysis, we identified 3 potential miRs that could target FXYD1 mRNA: miR-3178, miR-3960 and miR-6770-3p. Using a FXYD1-3’UTR-luciferase reporter assay, we found that the overexpression of miR-6770-3p in HEK293T cells resulted in the largest reduction in luciferase activity, showing a strong targeting of FXYD1 mRNA. A mutation of the binding site in the 3’UTR of FXYD1 restored luciferase activity, confirming the binding site of miR-6770-3p. Mir-6770-3p is a novel miR that currently has no known function. MiR-6770-3p is the minor strand of its 5p/3p miR duplex in both the human endothelial cell (EC) line, EA.hy.926, and human dermal microvascular (HMVECs). Its overexpression dramatically increased endothelial tube formation in a Matrigel angiogenesis assay (>4 fold change vs. control transfection, p<0.05) and was also associated with a 2-3-fold increase of VEGFR2 and Endoglin mRNA levels, respectively, in both cell types. Conversely, it was also uncommonly associated with a decrease in cell proliferation, suggesting that the pro-angiogenic effect of miR-6770-3p was not due to increased proliferation. Interestingly and contrary to our previous findings, miR-6770-3p was the major strand in Human Umbilical Vein ECs (HUVECs). In this cell type, overexpression of the 3p strand in fact decreased tube formation, while retaining its negative effect on proliferation. The difference in miR major and minor strand may partly explain the differences in functional behaviours in HUVECs, as compared to HMVECs. In conclusion, we have identified a novel miR which has the potential to modulate vessel formation in vitro, and induce critical regulators of angiogenesis; however, further work is required to better understand how this miR performs these functions.

MiR-421 up-regulation by the oleanolic acid derivative K73-03 regulates epigenetically SPINK1 transcription in pancreatic cancer cells leading to metabolic changes and enhanced apoptosis

SPINK1 overexpression promotes cancer cell aggressiveness and confers chemo-resistance to multiple drugs in pancreatic cancer. Oleanolic acid (OA) derivatives possess active effects against different cancers. Here we report the effect of K73-03, a new novel OA derivative, against pancreatic cancer through mitochondrial dysfunction via miR-421/SPINK1 regulation. We examined the binding ability of miR-421 with SPINK1-3’UTR Luciferase reporter assays. Moreover, miR-421/SPINK1 expressions in pancreatic cancer, with or without K73-03 treatment, were evaluated. Cells viability, migration, autophagy, mitochondrial function and apoptosis were examined with or without K73-03 treatment. We established that the K73-03 effect on the miR-421 that plays a crucial role in the regulation of SPINK1 in pancreatic cancer. Our findings indicated that K73-03 inhibited the mitochondrial function that led to inducing autophagy and apoptosis through epigenetic SPINK1 down-regulation via miR-421 up-regulation in pancreatic cancer. Furthermore, the inhibition of miR-421 expression in pancreatic cancer cells abolished the efficacy of K73-03 against SPINK1 oncogenic properties. We found an interesting finding that the interaction between miR-421 and SPINK1 is related to mitochondrial function through the effect of K73-03. Further, SPINK1 appear to be the molecular targets of K73-03 especially more than gemcitabine.

Role of miR-96/EVI1/miR-449a Axis in the Nasopharyngeal Carcinoma Cell Migration and Tumor Sphere Formation.

The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3′UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.

MicroRNA-125b as a tumor suppressor by targeting MMP11 in breast cancer.

BackgroundBreast cancer is a common type of tumor in women worldwide. MicroRNAs have been identified as regulators in many human cancers. The aim of this study was therefore to investigate the functional role of miR‐125b in regulating breast cancer progression.MethodsWe used the StarBase database to investigate the expression of miRNA‐125b in breast cancer and adjacent normal tissues. MMP11 3′‐UTR construct and luciferase reporter assays was performed for target genes. Cell proliferation was evaluated by CCK‐8 and colony formation assay. The migration and invasion were assessed by transwell assay.ResultsLuciferase reporter assays showed miRNA‐125b directly targeted MMP11. miRNA‐125b by transfection with its mimic in breast cancer cells significantly suppressed breast cancer cell proliferation and migration. Western blot revealed that overexpression of miRNA‐125b substantially reduced MMP11 protein expression. We used the UALCAN database to investigate the expression of MMP11 in human breast cancer and adjacent normal tissues. In addition, we found that miRNA‐125b spoiled MMP11 induced breast cancer cell proliferation and migration promotion effect.ConclusionsmiRNA‐125b mimic inhibited proliferation, migration, and invasion of breast cancer cells through targeting MMP11 protein.

MiRNA-96-5p impacts the progression of breast cancer through targeting FOXO3.

BackgroundBreast cancer is the most common malignant tumor in women worldwide, with a high mortality rate. MicroRNAs are small non‐coding RNAs that negatively regulate the expression of target genes by interacting with the target gene 3'‐UTR, and participate in cell differentiation, proliferation, apoptosis and metabolism. The function of miRNA‐96‐5p in the progression of breast cancer has not been reported.MethodsWe used the StarBase database to investigate the expression of miRNA‐96‐5p in breast cancer and adjacent normal tissues. FOXO3 3'‐UTR construct and luciferase reporter assays was performed for the target gene. Expression levels of miRNAs including its target were analyzed by qRT‐PCR and western blot. Cell proliferation was detected by CCK8 and colony formation, EdU assay.ResultsLuciferase reporter assays showed miRNA‐96‐5p directly targeted FOXO3. Abrogation of miRNA‐96‐5p by transfection with its inhibitors in breast cancer cells significantly suppressed miRNA‐96‐5p expression and breast cancer cells proliferation. Western blot revealed that overexpression of miRNA‐96‐5p substantially reduced FOXO3 protein expression. We used the GEPIA, UALCAN and KM‐plotter databases to investigate the expression of FOXO3 in human breast cancer and adjacent normal tissues, and its correlation with survival. In addition, we found that FOXO3 spoiled miR‐96‐5p induced breast cancer cell proliferation block effecting.ConclusionsmiRNA‐96‐5p may exert a tumor promotion role through negatively regulating tumor suppressor gene FOXO3 and promoting cell proliferation.

Abstract WP127: Intravenous Administration of Microrna-122 Mimic for Treatment of Ischemic and Hemorrhagic Strokes in Rats

MicroRNAs(miRs)are very promising next generation drug targets due to their unique miR-target binding that is different from the traditional ligand-receptor. A single miR binds to complementary bases in the 3’ untranslated regions(3’UTR) of hundreds of target genes and down-regulates these genes. MiR therapeutics have been developed for treatment of various diseases, with several miR drugs(e.g., miR-122/miR-155 inhibitors, miR-16/29/34 mimics) being advanced into human trials in the last decade. Our previous studies showed that intravenous (i.v.) miR-122 mimic (2.4mg/kg, wrapped in PEG-liposomes) improves outcomes after suture middle cerebral artery occlusion(MCAO)-induced ischemic stroke (IS) with a 6 hour time window. In pilot whole genome miR expression studies, we demonstrated that microRNA-122 (miR-122) is the most significantly decreased miR in blood after intraventricularautologousfresh blood-induced intracerebral hemorrhage (ICH) in rats. As compared to sham controls, miR-122 decreased 28 fold at 3 hrs, 34 fold at 24 hrs, 31 fold at 7 days and 19 fold at 14 days in blood after ICH in rats. These miRNA expression data suggest that elevating miR-122 in blood has great potential to treat ICH in addition to IS. Therefore, we hypothesized that i.v. miR-122 mimic improves outcomes after ICH, in addition to IS. Our miR-122 targetome studies show that a set of miR-122 target genes (Pla2g2a, Vcam1, Nos2, Rhbdf1, Olig1, Nrep) are responsible for the therapeutic efficacy of miR-122 mimic on ischemic stroke, while another set of genes (Ywhaq, Klrk1, Tpst1, Vars2)account for efficacy in ICH. Using3’UTR luciferase reporter assays and anti-sense Morpholino Oligos (MOs), we show that miR-122 binds to 3’UTR of its target genes Pla2g2a and Vcam1, rather than Nos2. In addition, in vivo MO-miR-122-Pla2g2a blocks miR-122 mimic treatment-induced decrease of Pla2g2a in blood cells after ischemic stroke. In summary, our data suggest miR-122 mimic can treat IS and ICH. Therefore, the miR-122 mimic treatment for IS and ICH could likely be performed without brain imaging (e.g. in the ambulance, and/or emergency room) since it is broadly efficacious for both IS and ICH.

MiR-146a Promotes Tolerogenic Properties of Dendritic Cells and through Targeting Notch1 Signaling

ABSTRACT Background MiR-146a has been shown to negatively regulate innate immune, inflammatory response and antiviral pathway, however, its role in the tolerogenic responses remains largely unknown. This study aimed to investigate the role of miR-146a in the OVA-induced allergic inflammation of dendritic cells (DCs). Methods Bone marrow-derived DCs (BMDCs) were treated with OVA (100 µg/ml) for 24 h. MiR-146a expressions were assessed by quantitative RT-PCR. BMDCs were transfected with miR-146a mimics or inhibitor. Cell surface markers were analyzed by flow cytometry. Cytokine levels were determined by ELISA assay. Mixed lymphocyte culture assay was adopted to assess CD4 + T-cell differentiation. The 3ʹ UTR luciferase reporter assay was utilized to determine the miRNA target sequence. Results OVA treatment significantly up-regulated miR-146a in BMDCs in a dose- and time-dependent manner. In the OVA-treated DCs, overexpression of miR-146a (mimics transfection) down-regulated the surface markers (CD80, CD86) and increased production of anti-inflammatory cytokines TGF-β1 and IL-10 but decreased pro-inflammatory cytokine IL-12. MiR-146a overexpression promoted immature DC to induce regulatory T cells (Treg) differentiation. By contrast, transfection of miR-146a inhibitor into DC exhibited the opposite trends. Notch1 was a direct target of miR-146a, and Notch1 knock-down induced similar effects as miR-146a mimics transfection in BMDCs. Moreover, the effect of miR-146a inhibitor on OVA-induced DC was attenuated by Notch1 knock-down. Conclusion miRNA-146a promoted tolerogenic properties of DCs, at least partially, through targeting Notch1 signaling.

Abstract C129: MicroRNA-155 targets AGTR1/NF-κB /CXCR4 axis and attenuates oncogenesis in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive malignancy of all primary brain tumors. Despite the recent advances in GBM treatment, the average survival time for a patient is less than 15 months. Therefore, we sought to investigate the molecular mechanisms underlying GBM initiation, progression and identify novel drug targets for this deadly disease. Higher levels of Angiotensin II Receptor Type 1 (AGTR1) and Angiotensin II Receptor Type 2 (AGTR2) have been associated with poor patient prognosis in astrocytomas. Additionally, contribution of these receptors in proliferative and infiltrative properties of glioma cells has already been established. Moreover, overexpression of AGTR1 has been known to play a critical role in multiple human malignancies including brain. However, the underlying mechanism involved in AGTR1 upregulation in GBM is not clearly understood and remains a matter of conjecture. Here, we analysed online available RNA-seq data from two independent GBM patients’ cohorts for AGTR1 expression and observed significant overexpression of AGTR1 in glioma patients compared to normal adjacent tissue. Importantly, the overall survival probability of GBM patients with high AGTR1 expression was low as compared to patients with low AGTR1 levels. To understand the post-transcriptional regulation of AGTR1 in GBM, we employed miRNA prediction algorithms namely, TargetScan, miRanda (microRNA.org), MicroT4 (DIANA tools), RNA22 along with experimentally supported database (TarBase), and examined miRNAs that bind to 3’UTR of AGTR1. Interestingly, conserved binding of miRNA-155 was predicted by all algorithms. Further, AGTR1-3’UTR-luciferase reporter assay in HEK-293T cells also confirmed the miRNA-155 mediated post-transcriptional regulation of AGTR1. Moreover, ectopic expression of miRNA-155 in GBM cells abrogate AGTR1-mediated proliferation, invasion, foci formation and anchorage independent growth. Intriguingly, immunodeficient mice implanted with stable miRNA-155 overexpressing SNB19 cells showed a significant reduction (~95%) in tumor burden as compared to the control group. Mechanistically, we showed that miRNA-155 attenuates AGTR1-mediated epithelial-to-mesenchymal transition, angiogenesis, ERK/MAPK signaling and promotes apoptosis. Importantly, stimulation with Angiotensin II (Ang II), a bonafide ligand of AGTR1, results in activation of NF-κB signaling downstream of AGTR1, followed by an increase in expression of C-X-C chemokine receptor type 4 (CXCR4) and AGTR1. Conversely, miRNA-155 overexpression in GBM cells attenuate NF-κB signalling, leading to a notable decrease in the expression of CXCR4 and its downstream signalling accompanied with reduction in AGTR1 levels. Alternately, we treated GBM cells with IKK-16, an inhibitor of IKK complex, to abrogate the NF-κB signalling downstream of AGTR1. Interestingly, we observed a significant reduction in the AGTR1 expression along with a marked decrease in CXCR4 expression. Taken together, we demonstrate that miRNA-155 is involved in post-transcriptional regulation of AGTR1 and disrupts AGTR1/NF-κB /CXCR4 signaling axis via anticancer pleiotropic effects. The present study opens new avenues to use IKK inhibitors and possible use of miRNA-155 replacement therapy for AGTR1-positive malignancies. Citation Format: Anukriti Singh, Nidhi Srivastava, Bushra Ateeq. MicroRNA-155 targets AGTR1/NF-κB /CXCR4 axis and attenuates oncogenesis in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C129. doi:10.1158/1535-7163.TARG-19-C129

Dazl determines primordial follicle formation through the translational regulation of Tex14.

Deleted in azoospermia-like (DAZL) is a germ cell RNA-binding protein that is essential for entry and progression through meiosis. The phenotype of the Dazl knockout mouse has extensive germ cell loss because of incomplete meiosis. We have created a Dazl hypomorph model using short interfering RNA knockdown in mouse fetal ovary cultures, allowing investigation of Dazl function in germ cell maturation. Dazl hypomorph ovaries had a phenotype of impaired germ cell nest breakdown with a 66% reduction in total follicle number and an increase in the proportion of primordial follicles (PMFs), with smaller oocytes within these follicles. There was no significant early germ cell loss or meiotic delay. Immunostaining of intercellular bridge component testis-expressed protein (Tex)14 showed ∼59% reduction in foci number and size, without any change in Tex14 mRNA levels. TEX14 expression was also confirmed in the human fetal ovary across gestation. Using 3'UTR-luciferase reporter assays, translational regulation of TEX14 was demonstrated to be DAZL-dependant. Dazl is therefore essential for normal intercellular bridges within germ cell nests and their timely breakdown, with a major impact on subsequent assembly of PMFs.-Rosario, R., Crichton, J. H., Stewart, H. L., Childs, A. J., Adams, I. R., Anderson, R. A. Dazl determines primordial follicle formation through the translational regulation of Tex14.

MiR-16a Regulates Milk Fat Metabolism by Targeting Large Tumor Suppressor Kinase 1 (LATS1) in Bovine Mammary Epithelial Cells.

Milk contains a number of beneficial fatty acids including short and medium chain and unsaturated conjugated and nonconjugated fatty acids. In this study, microRNA sequencing of mammary tissue collected in early-, peak-, mid-, and late-lactation periods was performed to determine the miRNA expression profiles. miR-16a was one of the differentially expressed miRNA and was selected for in-depth functional studies pertaining to fatty acid metabolism. The mimic of miR-16a impaired fat metabolism [triacylglycerol (TAG) and cholesterol] while knock-down of miR-16a promoted fat metabolism in vitro in bovine mammary epithelial cells (BMECs). In addition, the in vitro work with BMECs also revealed that miR-16a had a negative effect on the cellular concentration of cis 9-C18:1, total C18:1, C20:1, and C22:1 and long-chain polyunsaturated fatty acids. Therefore, these data suggesting a negative effect on fatty acid metabolism extend the discovery of the key role of miR-16a in mediating adipocyte differentiation. Through a combination of bioinformatics analysis, target gene 3' UTR luciferase reporter assays, and western blotting, we identified large tumor suppressor kinase 1 (LATS1) as a target of miR-16a. Transfection of siRNA-LATS1 into BMECs led to increases in TAG, cholesterol, and cellular fatty acid concentrations, suggesting a positive role of LATS1 in mammary cell fatty acid metabolism. In summary, data suggest that miR-16a regulates biological processes associated with intracellular TAG, cholesterol, and unsaturated fatty acid synthesis through LATS1. These data provide a theoretical and experimental framework for further clarifying the regulation of lipid metabolism in mammary cells of dairy cows.

The Role of the miR-21/SPRY2 Axis in Modulating Proangiogenic Factors, Epithelial Phenotypes, and Wound Healing in Corneal Epithelial Cells.

PurposeSubconjunctival injection of antagomir-21 attenuates the progression of corneal neovascularization. We examined the underlying mechanism by investigating the regulation of microRNA (miR)-21 expression and the involvement of miR-21 in the homeostasis of corneal epithelial cells.MethodsCorneal epithelial cells were cultured with TGF-β1 and/or under hypoxia conditions. miR-21 expression was measured by quantitative PCR. The direct targets of miR-21 were validated by the 3′-UTR luciferase reporter assay. Alterations of proangiogenic signaling and the epithelial-mesenchymal transition (EMT) phenotype after miR-21/Sprouty2 (SPRY2) knockdown were examined by Western blotting. The effect of conditioned medium on angiogenesis was assessed using the tube formation assay. Wound healing was evaluated by the migration and scratch assays.ResultsTGF-β1 or hypoxia upregulated miR-21, and miR-21 silencing abolished TGF-β1/hypoxia-induced hypoxia inducible factor (HIF)-1α and VEGF expression. miR-21 inhibited SPRY2 by directly targeting its 3′-UTR. Simultaneous silencing of miR-21 and SPRY2 significantly upregulated p-ERK, HIF-1α, and VEGF and promoted angiogenesis. Induction of miR-21 or inhibition of SPRY2 reduced the levels of cytokeratin (CK)-3 and CK-12 and promoted EMT. Transwell and wound healing assays indicated that miR-21 promoted cell migration.ConclusionsTGF-β1 or hypoxia induced miR-21 and inhibited SPRY2, thereby enhancing proangiogenic signaling, suppressing the epithelial phenotype, and promoting wound healing in corneal epithelial cells.

Abstract 1235: Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis

Abstract The majority of breast cancer-related mortality is due to metastasis. Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is overexpressed in breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors and cytokines that implicate major tumor hallmarks including invasion and metastasis. Knocking out HuR by CRISPR/CAS9 technology inhibits cell invasion in breast cancer cells. Therefore, HuR is an emerging target for breast cancer therapy, especially the lethal metastatic breast cancer. RNA-binding proteins had previously been considered “undruggable” due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. In the study of mechanism of action, a transcription factor, FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found for the first time to be a direct mRNA target of HuR and one of the top genes that are reduced by KH-3 treatment. Exogenous introduction of FOXQ1 can rescue cell invasive capability impaired by HuR knockout and abolish the effect of KH-3 on inhibiting cell invasion in breast cancer cells. Moreover, KH-3 disrupts HuR-FOXQ1 interaction in RNP-IP, RNA pull down and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in an orthotopic xenograft model of breast cancer, but also efficiently inhibits lung metastasis and improves mouse survival in an experimental metastasis model. Our data provide a proof-of-principle that HuR inhibition by KH-3 may be developed as a promising molecular therapy for inhibiting progression and metastasis of breast cancer with high HuR. Citation Format: Xiaoqing Wu, Gulhumay Gardashova, Lan Lan, Shuang Han, Cuncong Zhong, Ragul Gowthaman, John Karanicolas, Dan A. Dixon, Danny R. Welch, Ling Li, Min Ji, Jeffrey Aubé, Liang Xu. Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis [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 1235.

MiR-409-5p negatively regulates Wnt/Beta catenin signaling pathway by targeting Lrp-8.

Signaling pathways like Wnt play a vital part in all aspects of skeletal development which include osteoblastogenesis and osteoclastogenesis. Inactivation of Wnt signaling pathway leads to bone-related disorders, whereas activation of Wnt signaling pathway can cure bone pathologies like osteoporosis. Certain microRNA(s) have been identified that commune with Wnt signaling molecules to regulate osteogenesis. In this study we reported the identification of miR-409-5p as a suppressor of osteogenesis by targeting Lrp-8 which is a positive effector of Wnt signaling. Our study showed that overexpressing miR-409-5p inhibits osteoblast differentiation whereas obstructing miR-409-5p expression by anti-miR-409 promotes osteoblast functions and matrix mineralization. Using tools like targetscan and 3'-UTR luciferase reporter assay, Lrp-8 was confirmed as a straight target of miR-409-5p. By over expressing miR-409-5p, a repression of canonical Wnt/β catenin signaling was observed. These observations were strengthened by the fact that silencing of miR-409-5p in ovariectomized estrogen deficient Balb/c mice restored the loss of trabecular bone microarchitecture and suppressed bone resorption. Thus, targeting miR-409-5p may be helpful in increasing bone density in conditions like post menopausal osteoporosis.