UTR Luciferase Reporter Research Articles

Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a disease-causing mechanism

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3'-untranslated region (3'-UTR) of SPINK1 as the cause of severe infantile isolated exocrine pancreatic insufficiency. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here, we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that, owing to its particular location, Alu_Ins could independently disrupt mRNA 3' end formation and/or affect other post-transcriptional processes such as nuclear export and translation. However, employing a 3'-UTR luciferase reporter assay, Alu_Ins was found to result in only an ∼50% reduction in luciferase activity compared to wild type, which is insufficient to account for the severe pancreatic deficiency in the Alu_Ins homozygote. We then postulated that double-stranded RNA (dsRNA) structures formed between Alu elements, an upstream mechanism regulating gene expression, might be responsible. Using RepeatMasker, we identified two Alu elements within SPINK1's third intron, both oriented oppositely to Alu_Ins. Through RNAfold predictions and full-length gene expression assays, we investigated orientation-dependent interactions between these Alu repeats. We provide compelling evidence to link the detrimental effect of Alu_Ins to extensive dsRNA structures formed between Alu_Ins and pre-existing intronic Alu sequences, including the restoration of SPINK1 mRNA expression by aligning all three Alu elements in the same orientation. Given the widespread presence of Alu elements in the human genome and the potential for new Alu insertions at almost any locus, our findings have important implications for detecting and interpreting Alu insertions in disease genes.

Lithocholic acid decreases mRNA stability of nuclear receptor PPARα by upregulating miR-21 expression in hepatoma HepG2 cells

To investigate the regulatory effects of lithocholic acid (LCA) on nuclear receptor peroxisome proliferatoractivated receptor-alpha (PPARα) mRNA stability at the post-transcriptional level. PPARα 3'UTR luciferase reporter gene vectors were constructed and transfected into HepG2 cells to observe the changes in cellular luciferase activity in response to LCA treatments. Bioinformatic prediction and miRNA PCR array technique were used to identify the differentially expressed miRNAs induced by LCA and their potential binding sites on the 3'UTR. The binding sites (Mut1, Mut2 and Mut1+Mut2) were mutated to compare the changes in cellular luciferase activity following LCA treatment. Western blotting and RTqPCR were used to detect the activated signaling pathway and the expression levels of its downstream transcription factors in LCA-treated cells. The changes in PPARα protein expression level were detected in the cells following overexpression of the transcription factors. Treatment with 100 μmol/L LCA significantly reduced luciferase activity of PPARα 3'UTR1 and 3'UTR2 in HepG2 cells by more than 50% (P<0.01) and induced significant upregulation of miR-21 and miR-22, especially the former (by 2.35 folds, P<0.05). Two predicted miR-21-binding sites in the 3'UTR1 were mutated to construct Mut1, Mut2 and Mut1+Mut2 reporter gene vectors. LCA treatment down-regulated 3'UTR1 luciferase activity by 51%, while Mut1, Mut2, and Mut1+Mut2 were down-regulated by 37%, 39%, and 13%, respectively. LCA caused ERK1/2 phosphorylation and activation of the ERK1/2 signaling pathway, and treatment with 100 μmol/L LCA upregulated the expression of transcription factor early growth response 1 (EGR1) by 5.83 folds (P<0.01). Transient overexpression of EGR1 significantly decreased cellular PPARα protein levels (P<0.05). LCA reduces PPARα mRNA stability and thus decreases PPARα mRNA and protein expressions in hepatocytes by activating the ERK1/2 signaling pathway and upregulating EGR1 and miR-21, which targets 3'UTR regulatory region of PPARα mRNA.

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.

Hsa-miR-323a-3p functions as a tumor suppressor and targets STAT3 in neuroblastoma cells.

Studies conducted in the last decades have revealed a role for the non-coding microRNAs (miRNAs) in cancer development and progression. Several miRNAs within the chromosome region 14q32, a region commonly deleted in cancers, are associated with poor clinical outcome in the childhood cancer neuroblastoma. We have previously identified miR-323a-3p from this region to be downregulated in chemotherapy treated neuroblastoma cells compared to pre-treatment cells from the same patients. Furthermore, in neuroblastoma tumors, this miRNA is downregulated in advanced stage 4 disease compared to stage 1-2. In this study, we attempt to delineate the unknown functional roles of miR-323a-3p in neuroblastoma. Synthetic miRNA mimics were used to overexpress miR-323a-3p in neuroblastoma cell lines. To investigate the functional roles of miR-323a-3p, cell viability assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction, luciferase reporter assay and western blot were conducted on the neuroblastoma cell lines Kelly, SH-SY5Y and SK-N-BE(2)-C. Ectopic expression of miR-323a-3p resulted in marked reduction of cell viability in Kelly, SH-SY5Y and SK-N-BE(2)-C by causing G1-cell cycle arrest in Kelly and SH-SY5Y and apoptosis in all the cell lines tested. Furthermore, mRNA and protein levels of signal transducer and activator of transcription 3 (STAT3) were reduced upon miR-323a-3p overexpression. A direct binding of the miR-323a-3p to the 3'UTR of STAT3 was experimentally validated by luciferase reporter assay, where miR-323a-3p reduced luminescent signal from full length STAT3 3'UTR luciferase reporter, but not from a reporter with mutation in the predicted seed sequence. miR-323a-3p inhibits growth of neuroblastoma cell lines through G1-cell cycle arrest and apoptosis, and the well-known oncogene STAT3 is a direct target of this miRNA.

MiRNA-Induced Downregulation of IPMK in Macrophages Mediates Lipopolysaccharide-Triggered TLR4 Signaling.

Inositol polyphosphate multikinase (IPMK) is a pleiotropic enzyme responsible for the production of inositol polyphosphates and phosphoinositide. IPMK in macrophages was identified as a key factor for the full activation of the Toll-like receptor 4 (TLR4) signaling pathway and inflammation by directly interacting with tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, dynamic changes of IPMK levels in lipopolysaccharide (LPS)-stimulated macrophages and their functional significance were investigated. Both the mRNA and protein levels of IPMK were acutely decreased in mouse and human macrophages when cells were stimulated with LPS for between 1 and 6 h. Analysis of the 3' untranslated region (UTR) of mouse IPMK mRNA revealed a highly conserved binding site for miR-181c. Transfection of miR-181c mimics into RAW 264.7 macrophages led to decreased IPMK 3'UTR-luciferase reporter activity and lowered endogenous IPMK levels. When the genomic deletion of a 33-bp fragment containing a putative miR-181c-binding site was introduced within the IPMK 3'UTR of RAW 264.7 macrophages (264.7Δ3'UTR), LPS-triggered downregulation of IPMK levels was prevented. LPS treatment in 264.7Δ3'UTR macrophages decreased TLR4-induced signaling and the expression of proinflammatory cytokines. In response to LPS stimulation, K63-linked ubiquitination of TRAF6 was impaired in 264.7Δ3'UTR macrophages, suggesting an action of IPMK in the suppression of TRAF6 activation. Therefore, our findings reveal that LPS-mediated suppression of IPMK regulates the full activation of TLR4 signaling and inflammation in macrophages.

MiR-23a-3p and miR-181a-5p modulate SNAP-25 expression.

SNAP-25 protein is a key protein of the SNARE complex that is involved in synaptic vesicles fusion with plasma membranes and neurotransmitter release, playing a fundamental role in neural plasticity. Recently the concentration of three specific miRNAs-miR-27b-3p, miR-181a-5p and miR-23a-3p -was found to be associated with a specific SNAP-25 polymorphism (rs363050). in silico analysis showed that all the three miRNAs target SNAP-25, but the effect of the interaction between these miRNAs and the 3'UTR of SNAP-25 mRNA is currently unknown. For this reason, we verified in vitro whether miR-27b-3p, miR-181a-5p and miR-23a-3p modulate SNAP-25 gene and protein expression. Initial experiments using miRNAs-co-transfected Vero cells and SNAP-25 3'UTR luciferase reporter plasmids showed that miR-181a-5p (p≤0.01) and miR-23a-3p (p<0.05), but not miR-27b-3p, modulate the luciferase signal, indicating that these two miRNAs bind the SNAP-25 3'UTR. Results obtained using human oligodendroglial cell line (MO3.13) transfected with miR-181a-5p or miR-27b-3p confirmed that miR-181a-5p and miR-23a-3p regulate SNAP-25 gene and protein expression. Interestingly, the two miRNAs modulate in an opposite way SNAP-25, as miR-181a-5p significantly increases (p<0.0005), whereas miR-23a-3p decreases (p<0.0005) its expression. These results for the first time describe the ability of miR-181a-5p and miR-23a-3p to modulate SNAP-25 expression, suggesting their possible use as biomarkers or as therapeutical targets for diseases in which SNAP-25 expression is altered.

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.

Abstract 476: Kawasaki Disease, Metabolic Switch, And Endothelial Dysfunction

Kawasaki Disease (KD) induces endothelial dysfunction, in part, through down-regulation of miR-483. Metabolic reprogramming, including increased glycolysis and reduced oxidative phosphorylation, is associated with endothelial cell (EC) dysfunction. However, whether metabolic reprogramming is involved in KD-induced EC dysfunction is unknown. Using RNA-seq and pathway analyses, we determined that genes involved in glycolysis were upregulated by sera from acute KD patients compared with convalescent control sera. We validated by qPCR and found increased mRNA levels of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), hexokinase 1 (HK1), and hexokinase 3 (HK3), as well as the decrease in glucokinase regulator (GCKR) in cultured ECs incubated with acute KD sera. Moreover, PFKFB3 protein level was elevated in ECs cultured with acute KD sera and in the intima of coronary arteries of mice with LCWE-induced KD vasculitis. Seahorse assay revealed that KD sera increased extracellular acidification rate (ECAR) and decreased oxygen consumption rate (OCR) in ECs, indicating that serum factors in acute KD patients increased glycolysis and impaired mitochondrial function. Given the presence of a miR-483 targeting site in the 3’ untranslated region (3’UTR) of PFKFB3 mRNA, we cloned a PFKFB3-3’UTR luciferase reporter (PFKFB3-Luc) to test whether PFKFB3 is regulated by miR-483. As anticipated, miR-483 overexpression downregulated luciferase activity in ECs transfected with PFKFB3-Luc. Moreover, miR-483 overexpression decreased PFKFB3 and ECAR in ECs. In contrast, anti-miR-483 increased PFKFB3 and ECAR levels, which phenocopied acute KD-induced EC metabolic reprogramming. We conclude that glycolysis in ECs is augmented during the acute phase of KD, which promotes EC dysfunction. Mechanistically, the acute KD-associated metabolic switch in ECs is regulated, at least in part, by increased PFKFB3 as a consequence of reduced level of miR-483.

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.

BAG2 mediates coelomocyte apoptosis in Vibrio splendidus challenged sea cucumber Apostichopus japonicus

MicroRNAs (miRNAs) are closely related to the occurrence, development, and immune response of diseases. BCL2-associated athanogene 2 (BAG2) is a member of the BAG family that functions in diverse cellular processes, including cell death, differentiation, and cell division. In this study, we cloned the cDNA full-length of sea cucumber (Apostichopus japonicus) BAG2 (AjBAG2) and confirmed it is an anti-apoptotic protein in vitro and in vivo during Vibrio splendidus infection. Moreover, we identified a perfect complementarity between miR-375 and the 3′-untranslated region (UTR) sequence of AjBAG2. The miR-375 expression decreased the luciferase activity dose-dependently when co-transfected with the AjBAG2 3′-UTR-luciferase reporter containing the miR-375 target site in epithelioma papulosum cyprini (EPC) cells. This inhibition was partially recovered by a miR-375 specific inhibitor. The mRNA and protein levels of AjBAG2 were opposite to that of coelomocytes in challenged sea cucumber when treated with miR-375 mimics or inhibitors. Additionally, miR-375 expression induced coelomocytes apoptosis and blocked the anti-apoptotic activity of AjBAG2. Our data demonstrated that AjBAG2 is an anti-apoptotic protein during V. splendidus infection and this function can be inhibited by miR-375 in sea cucumbers.

Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1β and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

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.

Construction of a Full-Length 3'UTR Reporter System for Identification of Cell-Cycle Regulating MicroRNAs.

Three prime untranslated region (3'UTR) reporter constructs are widely used by the scientific community to functionally link microRNAs (miRNAs) to suppression of mRNA expression. However, full-length 3'UTR vectors are rarely employed due to labor-intensive cloning work. Instead, 3'UTR fragments containing putative miRNA binding sites are commonly utilized to mechanistically validate miRNAs. Assaying truncated 3'UTRs may falsely validate miRNAs due to altered positioning of binding sites in respect to 3'UTR length and RNA secondary structure. Here we present a detailed protocol for the construction of full-length 3'UTR luciferase reporter constructs that was used to unveil miRNAs regulating multiple cell-cycle factors.

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 (&gt;4 fold change vs. control transfection, p&lt;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-21 Overexpression Promotes Esophageal Squamous Cell Carcinoma Invasion and Migration by Repressing Tropomyosin 1

The migration and invasion of esophageal squamous cell carcinoma are associated with clinical outcomes, however, the mechanisms remain poorly understood. Here, we found that miR-21 is significantly overexpressed in ESCC, lung cancer, and bladder cancer compared with the adjacent normal tissue. MiR-21 and TPM1 expressions were analyzed by RT-qPCR and WB in 30 ESCC, 10 lung cancer, and 10 bladder cancer clinical specimens, each with matched adjacent normal tissue. Knockdown and overexpression of miR-21 as well as knockdown of TPM1 in ESCC cell lines were performed using synthetic oligonucleotides. TPM1 3′UTR luciferase reporter constructs were used to investigate targeting of TPM1 by miR-21. ESCC migration and invasion were assessed using transwell migration and invasion assays. Inhibition of miR-21 reduced migration and invasion in two ESCC cell lines, and overexpression of miR-21 promoted migration and invasion in vitro. Interestingly, TPM1 exhibited inverse patterns of expression compared with miR-21 in tissues and cell lines. Luciferase reporter assays demonstrated that TPM1 was directly regulated by miR-21. Moreover, the forced overexpression of miR-21 repressed the TPM1 expression, while silencing of miR-21 restored the TPM1 expression in ESCC cell lines. What is more, simultaneous silencing of miR-21 and TPM1 expressions did not alter the migratory and invasive characteristics demonstrating that the effects of miR-21 were mediated through TPM1. In conclusion, the aberrant overexpression of miR-21 is common in cancer and promotes the migration and invasion of ESCC through inhibiting the TPM1 expression. These results suggest that miR-21 may be a novel predictive marker and therapeutic target for treatment of ESCC.

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.

Abstract 1534: Vimentin prevents a miR-dependent negative regulation of tissue factor mRNA during epithelial-mesenchymal transitions and facilitates early metastasis

Abstract Epithelial-Mesenchymal Transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified Tissue Factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (i) vimentin silencing accelerated TF mRNA decay after actinomycin-D treatment, reflecting TF mRNA stabilization, (ii) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (iii) TF 3'-UTR-luciferase reporter vector assays implicated the 3'-UTR of TF mRNA in vimentin-dependent TF regulation, and (iv) using different TF 3'UTR luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs. Citation Format: Aline Vanwynsberghe, Marie-Emilie Francart, Christine Gilles. Vimentin prevents a miR-dependent negative regulation of tissue factor mRNA during epithelial-mesenchymal transitions and facilitates early metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1534.